Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins . Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of  Mycobacterium tuberculosis (M. tb) genetics and the host's immunological response 8.8 million incident tuberculosis (TB) cases and 1.45 million TB-related deaths were reported in 2010, with the highest TB rates occurring in sub-Saharan Africa M. tb infection M. tb-specific and Major histocompatibility complex (MHC) class I restricted CD8+ T-cells requires the intracellular processing of M. tb antigens and subsequent presentation of epitopes by MHC class I cell-peptide complexes on the cell surface of antigen presenting cells. The immunogenicity of an M. tb epitope is influenced by several factors: i) the epitope must be generated and transported to the endoplasmic reticulum, by overcoming limitations in; cross-presentation CD8+ T-cells contribute to the cellular immune defense against TB M. tb epitopes, it is vital to correlate peptide MHC-binding to the actual detection of antigen-specific T-cells without in vitro manipulation (i.e. using in vitro expansion). This can be achieved in an unbiased way using MHC class I multimers loaded with molecularly defined pathogen-derived epitopes In order to determine immunogenicity of selected candidate M. tb; the host-pathogen interplay dictates the outcome of infection. Different clustering of MHC alleles, human leukocyte antigens (HLA), are associated with geographic locations or ethnicity M. tb have previously been identified M. tb peptide presentation on three levels: 1) peptide-MHC association, 2) TCR-recognition and 3) phenotype and cytotoxicity of CD8+ antigen-specific T-cells directed against different M. tb target antigens.A marked heterogeneity characterizes the human immune-response against M. tb T-cell epitopes, restricted by a limited number of MHC class I alleles have been identified M. tb epitopes restricted by a more diverse MHC class I allelic repertoire that reflects ethnical populations in Africa. In many regions of sub-Saharan Africa including South Africa, the most common HLA-A molecules are A*02, A*23, A*30 and A*68, the most common HLA-B alleles are B*15, B*42, B*53 and B*58 and the most common HLA*C alleles are C*04, C*06, C*07 and C*17 M. tb epitopes have previously been defined Up until now, only a few M. tb genome is relatively stable, however an increasing number of strain-to strain differences, i.e. insertions, deletions and point mutations in the M. tb genome have been observed M. tb epitopes may also be responsible for the observation that mixed M. tb infections occur M. tb strains have been sequenced and most genetic variations appear to reside in non-essential M. tb genes. This appears to be true for many M. tb proteins, with the exception of the small immunogenic protein Rv0288. This protein has the following identified naturally occurring single amino acid exchanges: A10T, G13D, S27N and A71S Not only the MHC class I alleles show diversity, also the pathogen shows variations. It was previously thought that the M. tb-derived antigens in this study, since M. tb proteins can be differentially expressed or secreted during the course of infection. The immunodominant secreted TB proteins Ag85B (Rv1886c) and ESAT-6 (Rv3875) are part of several new TB vaccine candidates, M. tb and a few other mycobacterial species (e.g. M. marinum and M. kansasii) M. tb, expressed in the Mycobacterium bovis derived vaccine strain bacillus Calmette-Guérin (BCG) as well as in mycobacteria other than tuberculosis. The non-secreted M. tb proteins, glycosyl transferase (1) (Rv2958c), glycosyl transferase (2) (Rv2957) and cyclopropane-fatty-acid synthase (CFA synthase) (Rv0447c) are predominantly expressed in slow growing bacteria. They have previously been identified from our group as targets for humoral and cell-mediated immune responses M. tb antigens are involved in cellular metabolism We chose to include 6 different M. tb peptides to some of the most frequent MHC alleles in South Africa, their impact on T-cell recognition and T-cell homing/differentiation markers using a broad panel of M. tb MHC class I-peptide multimers.We report in this study the detailed mapping of Escherichia coli Bl21 DE3 pLys  as inclusion bodies, they were purified, solubilized and folded to correct trimeric structure in a pH 8.0 Tris-EDTA-arginine buffer  together with allele-specific candidate peptides . The peptides were: A*02:01 – FLPSDFFPSV (HBV CORE), A*24:02 – RYLKDQQLL (HIV env), A*30:01 – KTKDIVNGL (F-actin capping protein beta), A*30:02 – KIQNFRVYY (HIV-integrase), A*68:01 – KTGGPIYKR (Influenza virus nucleoprotein), B*07:02 – TPRVTGGGAM (CMV pp65), B*58:01 – IAMESIVIW (HIV RT) and C*07:01 – KYFDEHYEY (CDC28 protein). Correctly folded trimeric p-MHC-complexes were then concentrated, biotinylated using the enzyme BirA  and affinity purified using an avidin column .The heavy and light chains of HLA-A*02:01, A*24:02, A*30:01, A*30:02, A*68:01, B*07:02, B*58:01 and C*07:01 were cloned into bacterial expression vectors (pET24d+ and pHN1). The MHC molecules were produced as previously described β2-microglobulin. Positive binding was detected using a fluorescent labeled conformation-dependent antibody -conjugated anti-HLA-A, -B and –C ). The binding of each candidate peptide was compared to the binding of an appropriate control peptide . For selected peptides further characterization of the binding were made using affinity and off-rate assays. Affinity was measured by incubating titrated peptide concentrations (10−4 to 10−9 M) on the MHC coated 96-well plates in a manner similar to the binding assay. An ED50 value, i.e. the peptide quantity needed to achieve 50% binding saturation was calculated. By incubating the candidate peptide for different time points , the stability of the p-MHC complexes could be assessed as a t1/2 value, which is defined as the time point when 50% of the initial peptide concentration has dissociated from the HLA-peptide molecule complexes. The binding, affinity, and off-rate-values were calculated using the iTopia™ System Software . Sigmoidal dose response curves were generated using Prism® 4.0 (GraphPad).96-well plates coated with biotinylated-bovine serum albumin (BSA) and avidin  were used to immobilize 0.5 µg/ml biotinylated monomers. Overlapping nonameric peptides  covering the three TB proteins TB10.4 (Rv0288), Ag85B (Rv1886c) and ESAT-6 (Rv3875) were used in binding, affinity and off-rate studies as well as variant peptides from Rv0288. The duplicated experiments were performed as previously described Based on the data generated in the peptide binding assays, previously reported data M. tb were enrolled at the University of Stellenbosch, Cape Town and at the Pretoria Academic Hospital in South Africa. Demographic data of the patients are provided in 27 patients diagnosed with active pulmonary TB (prior to any treatment), diagnosed with at least two positive sputum smears for acid-fast bacilli or positive sputum culture for Freshly thawed PBMCs were incubated with MHC class I multimers for 30 min at 37°C. Multimer positive events were recorded in the CD3+CD8+CD4− compartment using the following antibodies: anti-CD3-phycoerytin (PE)/Texas red (ECD) (clone: UCHT1) , anti-CD8α- allophycocyanin (APC)/Cy7 (clone: SK1)  and anti-CD4-Pacific orange (clone: S3.5) . The multimers were fluorescently labeled with streptavidin-PE, streptavidin-APC and fluorescein isothiocyanate (FITC). Cells in the CD3+CD8−CD4+ compartment were excluded from enumeration of CD3+CD8+multimer+ events. All cellular analyses were performed using a FACS Gallios flow-cytometer . Only multimer responses at least at least three times higher than the negative control and for which we could detect more than 50 events were further analyzed.Phenotypic analysis of antigen-specific cells was performed using the following Abs: anti-CD45RA-PerCP/Cy5.5 (Clone HI100)  and anti-CCR7-PE/Cy7 (Clone 3D12) . Anti-CD107a-Pacific blue (PB) (clone: H4A3)  was used as a degranulation marker and analysis of IL-7Rα  was performed using anti-CD127 APC/Alexa-700 (clone: R34.34) .Statistical significance between different T-cell populations was evaluated using Prism® 4.0  using a Student's two-sided t-test.M. tb proteins Rv1886c (Ag85B) (n = 260) and Rv3875 (ESAT-6) (n = 87) were used to study individual peptide binding to 5 HLA-A MHC class I molecules , 2 HLA-B alleles (B*07:02 and B*58:01) and 1 HLA-C allele (C*07:01). Peptides from Rv0288 (TB10.4) (n = 88) were used to study peptide binding to previously non-reported MHC class I alleles . Peptides were regarded as positive binders if the binding exceeded >40% of the respective positive control peptide. In total, we could identify 361 MHC class I binding epitopes in the three proteins , of which 92% were previously not reported. 68 epitopes were associated with A*02:01, 43 associated with A*24:02, 8 with A*30:01, 99 with A*30:02, 43 with A*68:01, 25 with B*07:02, 45 with B*58:01 and 30 with C*07:01. In total, A*30:02 had the highest binding frequency (27%) followed by A*02:01 (19%), A*24:02 (12%), A*68:01 (12%), B*58:01 (12%), C*07:01 (8%), B*07:02 (7%) and A*30:01 (2%).Overlapping peptides covering the amino acid sequences of the For Rv3875, we could identify 40 binding epitopes, of which more than half were associated with HLA-A*30:02 (n = 21), followed by A*02:01 (n = 11), A*68:01 (n = 3), A*24:02 (n = 2), B*58:01 (n = 2) and C*07:01 (n = 1). We were not able to identify any epitopes from Rv3875 binding to the alleles A*30:01 and B*07:02. In total, 34 peptides (39%) bound to at least one HLA allele and most of the binding peptides were located in the middle of the protein .M. tb protein Rv1886c, 292 epitopes could be identified associating with the following alleles: A*02:01 (n = 57), A*24:02 (n = 41), A*30:01 (n = 8), A*30:02 (n = 78), A*68:01 (n = 29), B*07:02 (n = 25), B*58:01 (n = 29) and C*07:01 (n = 25). The epitopes were scattered throughout the amino acid sequence with some clustering of promiscuous epitopes in the middle. 155 peptides (60%) bound to at least one MHC class I allele; 75 epitopes were found to be promiscuous, i.e. binding to more than one MHC class I allele. Some were highly promiscuous and cross-binding to 7 out of 8 MHC class I alleles  or 6 out of 8  (For the much larger *58:01]) .M. tb epitopes to 24 novel epitopes which were constructed including one or two amino acid substitutions , B*58:01 (n = 14) and C*07:01 (n = 4) . We alsoitutions . The var1/2) of the peptide-MHC complex. The affinity ranged between 1 μM and 5 nM with the majority of the peptides exhibiting an affinity in the lower end of the interval. The dissociation rate ranged between 0.2 h and 4.9 h; both extremes applied for HLA-A*02:01 . The nature of peptide binding to an MHC class I molecule can be divided into affinity, i.e. the binding strength between the peptide and the MHC molecule, and the dissociation rate, i.e. the off-rate (t-A*02:01 .M. tb candidate epitopes. The affinity ranged between 400 μM and 5 nM with the majority between 9 μM and 10 nM. Some of the peptides showed a very rapid dissociation rate (<0.1 h), while some formed a stable MHC class I-peptide complex for up to 8 hours. We identified a high range of intra-allelic differences  as well as inter-allelic differences . The peptides bound generally in a more stable fashion to the HLA-A alleles as compared to the B and C alleles , with the exception of HLA-A*30:01 which exhibited an off-rate of less than 1 h. Also, peptides binding to alleles with a limited set of binding epitopes tended to dissociate more rapidly  not being previously reported, 2) high binding compared to the positive control and 3) promiscuity . In total, we analyzed affinity and dissociation rate from 121 novel  rapidly .We identified in total 29 novel epitopes from the protein Rv0288 binding to the alleles HLA-A*6801, B*58:01 and C*07:01 and analyzed the binding characteristics for the entire peptide set. The affinity ranged between 700 μM and 30 nM, and the off-rate between 0.1 h and 6 h. We could not see any specific inter- or intra-allelic differences regarding affinity but we could detect a very rapid dissociation rate of less than 20 minutes regarding all candidate epitopes binding to C*07:01 .SMSSTHEA (Rv028870–78) to A*30:02 and MLDHAGDMA (Rv028811–19) to A*02:01.Alterations in individual amino acids lead to an increased or decreased binding strength in an almost equal number of cases. However we were only able to measure affinity in 20 out of 53 cases due to that too low MHC-peptide binding makes it impossible to reliably study affinity and off-rate. This was the case for all the variant epitopes restricted by A*30:01, A*68:01, B*07:02 and C*07:01. In most cases, where the peptide variation resulted in increased affinity, it increased the total peptide binding. In cases where the MHC class I-peptide stability increased, the increase was only marginal. In general, the amino acid exchange lead to decreased MHC binding that could be explained by either a decrease in 1) binding strength and/or 2) stability of the MHC class I/peptide complex. However, a few outliers were identified for which we could see an increase in both parameters (binding strength and affinity) while the total peptide binding decreased, e.g. HM. tb proteins ESAT-6 (Rv3875), Ag85B (Rv1886c) and TB10.4 (Rv0288) as well as 12 multimers from other (non-secreted) M. tb antigens  with the intention to evaluate the frequency, phenotype and functionality of antigen-specific T-cells. MHC-peptide multimers were used to enumerate CD8+ T-cell responses in blood from 27 patients diagnosed with active pulmonary TB in South Africa. Examples of staining and the gating strategy are provided in A*02, A*24, A*30 A*68, B*07, B*58 and C*07 belong to the most prevalent MHC class I alleles in a South African population. Therefore, we constructed 33 different multimers from the VLAGSVDEL, A3001-Rv1886cVANNTRLWV and A3002-Rv1886cVANNTRLWV). The second group constituted epitopes for which we could detect specific T-cells only in some patients . The third group contained epitopes that were recognized in blood from all patients   . Based oMYNYPAM) .ELNNALQNL. On the other hand, Rv2957 derived epitopes were not recognized in blood from HLA-A*02 and A*68 positive individuals at least the single epitope included in this study derived from this protein MYNYPAM(LG), we were also able to examine the nature of antigen-specific T-cell recognition of ‘MHC-promiscuous epitopes’. We could not detect significant differences in the detection of antigen-specific T-cells, neither inter-allelic, i.e. if the same epitope IMYNYPAML was presented via the different HLA alleles , nor intra-allelic when the almost identical epitopes was restricted by the same allele, e.g. A*24 (IMYNYPAML), MYNYPAMLG [0.3–0.4%]), and A*30:02 (QIMYNYPAM and IMYNYPAML [0.3–0.4%]). These data may suggest non-allele-specific T-cell recognition of ‘promiscuous epitopes’ like (I)MYNYPAML(G) (Since we managed to produce 6 different multimers presenting the YPAML(G) , at leasM. tb antigen-specific cells. The phenotype of CD8+ T-cells can be defined by the cell-surface markers CD45RA and CCR7 Since we detected differences in epitope/antigen recognition based on the restricting MHC class I allele and in the numbers of antigen-specific T-cells, we proceeded to analyze the phenotype of M. tb specific) T-cells were found, to a higher extent, in the terminally differentiated effector compartment (p = 0.02) as compared to the other HLA-A restricted antigen-specific T-cells. A similar trend could be seen for the allele HLA-C*07  (M. tb antigen and ranged from 5% (B*07) to 50% (A*68). M. tb epitopes could be divided into 4 groups according to the frequency of the antigen-specific CD8+ T-cells expressing CD107a. To the first epitope group belonged T-cells with more than 70% expressing CD107a, i.e. predominantly A*68 restricted epitopes. The second group consisted of epitopes recognized by cells expressing CD107a on 40–70% of the cells, i.e. mostly A*30-restricted epitopes. The third group consisted of epitopes recognized by cells expressing CD107a on 20–40% of the antigen-specific T-cells, i.e. mostly A*30 and A*24 restricted epitopes, and the rest belonged to the forth group. Generally, the HLA-A*30 and A*68 restricted antigen-specific T-cells expressed higher frequencies of CD107a , which correlates with the cytotoxic capacity of CD8+ T-cells. We compared the frequency of total CD8+ T-cells and the  0.0005) . The incf CD107a .M. tb epitopes to CD8+ T-cells, except B*07 . We were able to detect a significantly increased expression of this cell-surface marker on antigen-specific T-cells (81%) as compared to the total CD8+ T-cells (69%) (p = 0.001) . This waept B*07 . Based oof CD127 . This waof CD127  Also herM. tb target epitope (from H37Rv) and epitopes containing one or two naturally occurring substitutions for the alleles A*02 and A*24. The two epitopes binding to HLA-A*02 (AML(G/D)HAGDM and ML(G/D)HAGDMA) had the mutations in the 3rd or the 4th amino acid potentially being part of the TCR recognition site, while the epitope associating with A*24 (MYNYP(A/T)ML(G/D)) contained two mutations in the 6th and 9th position (of which the first position might affect TCR recognition). Although the association of these variant M. tb epitopes to its corresponding MHC molecule had either no effect, or showed decreased MHC binding, multimers containing these M. tb variant epitopes showed a tendency of detecting a higher frequency of antigen-specific T-cells as compared to multimers presenting the wild-type epitopes (A2D)HAGDMAMLHAGDMAML(G/ 0.13/0.25% and A24T)ML(G/D)MYNYP(A/ 0.44/0.51%)  . Next, w epitope .M. tb epitope-specific recognition frequencies, it was possible to determine that among the 7 epitopes, which showed the highest average antigen-specific recognition (>0.4%), the affinity varied considerably (between 6 µM and 8 nM), as well as the off-rate (0.5 h–27 h). In addition, we could detect allele-specific characteristics such as very high affinity regarding all the HLA-A*30 restricted epitopes and the opposite was found to be true regarding the B*07 restricted epitopes. However, this did not correlate with the frequency of antigen-specific CD8+ T-cells (in PBMCs from patients with active TB). The highest frequency of M. tb-specific T-cells for almost all epitopes was restricted by HLA-A*24 , we were able to detect allele-specific differences which potentially affect CD8+ T-cell immune-recognition. Regarding the p-MHC associations, we were able to confirm a broad peptide-binding repertoire for some alleles  and a more restricted peptide-binding repertoire for other alleles (e.g. A*30:01 and B*07:02). This is in line with previously reported data (yet with different peptide ligands) M. tb epitopes included in this study. Affinities (ED50) between 100 µM and 10 nM have previously been identified using a similar experimental design and our findings in this study are in line with these data The stability and off-rate are factors that are biologically important; they determine the time frame in which a certain epitope would be available for T-cell priming and for recognition in the effector phase of the immune response. The allele-specific differences in the p-MHC dissociation rate probably originate from intrinsic structural features of the allotype. This would imply that not only a limited number of epitopes would be available for presentation by these alleles but also that the time-frame in which the presentation could occur would be shorter. This scenario might entail a disadvantage for immune-recognition in individuals with certain allotypes, yet a fast peptide off-rate seems to be able to be counter-balanced by a high affinity, as shown for a number of M. tb proteins of inter-allelic binding such as Rv1886c [(KL)VANNTRL(WV) and (L)AAYHPQQF(I)] and Rv0288 [(QI)MYNYPAM(LG)], adding more alleles to the already identified hotspot in this protein We identified highly promiscuous ‘hot-spots’ in M. tb. epitopes seem to be HLA-B restricted M. tb infection, since the mapping using 48 different multimers . This seems to be more in line with the low frequency of disease-reactive T-cells in the peripheral circulation that previously has been seen in the context of cancer M. tb-specific or BCG-reactive T-cells in both adults and children belonging to ‘naïve’/precursor (CD45RA+CCR7+)/(CD45RA+CD28+) M. tb antigen-specific T-cells resides in the (CD45RA+CCR7+) precursor population – in contrast to the phenotype of total CD8+ T-cells which showed equal number of naïve, effector memory and terminally differentiated cells. It has previously been speculated for HIV infection, where this phenomenon also has been observed, that the antigen-specific T-cells with cell-surface expression of both CD45RA and CCR7 might represent effector memory cells that reverted back to a ‘naïve’ like phenotype without losing this subset's ability of proliferation upon antigen-stimulation Deciphering the phenotype of antigen-specific CD8+ T-cells is important in the context of memory formation as well as for immune effector functions M. tb epitope and thus due to the nature of the target protein, since the promiscuous epitopes were similarly recognized by inter-allelic restricted T-cells. However, when we compared several different epitopes, restricted by the same allele, allele-specific patterns of immune-recognition emerged while the antigen-specific patterns were remarkably constant. This was in contrast to the levels of vaccine-induced antigen-specific T-cells which were found to be higher targeting epitopes originating from Rv1886c (compared to Rv0288) in one previous study only a single epitope/protein was compared.We detected allele-specific variations, rather than antigen-specific, regarding frequencies and the differentiation/maturation profile (based on CD45RA/CCR7) of antigen-specific T-cells. First, we hypothesized that this finding was due to differences in the presented M. tb antigen specific T-cells with high CD107a expression ex vivo (indicating cytotoxic function).To our knowledge, no correlations between the T-cell profile and the restricting allele have been shown before in TB. One example has been reported in HIV infection where some alleles like B*57 and B*58, that have been associated with ‘elite controllers’, associated with higher numbers of specific cytokine producing cells M. tb previously has been shown to express low sequence variability in most of its epitopes, it was speculated that some immune-recognition might be beneficial for the bacteria, for instance in the establishment of a latent infection or by contributing to transmission bM. tb epitopes.We were able to detect biologically relevant differences concerning the naturally occurring amino-acid substitutions in the protein Rv0288. Since M. tb-specific responses in African with TB infection. We could detect a low frequency of a broad repertoire of co-dominant M. tb epitopes in the peripheral circulation of patients with active TB. The study of host-pathogen effects on immune-recognition in M. tb derived CD8+ T-cell epitopes revealed 1) MHC class I allele specific differences in the broadness of the peptide-binding repertoire, 2) associations of the frequency and phenotype of M. tb antigen-specific T-cells with the MHC class I restricting alleles and 3) differences between variant M. tb epitopes concerning peptide binding and T-cell recognition. This study provides a first view of the nature of the anti-M. tb specific CD8+ response in patients with an African background. It is our hope that these findings, as well as these novel tools will aid future research on M. tb in Africa.In conclusion, we constructed a broad panel of novel tools to be able to study Figure S1Example of staining using the cell surface markers CD3, CD4, CD8, multimer, CD45RA, CCR7, CD107a and CD12 (PAT 1284 and PAT 3282) in African TB patients. Numbers show the frequencies of cell positive for the specific marker/markers. The gating strategy was the following: Based on forward and side-scatter, lymphocytes were detected. From the lymphocytes, T-cells were enumerated using the CD3 marker. These cells were then divided into CD4+ T-cells and CD8+ T-cells based on expression of CD4 and CD8. In the CD8+ T-cells, multimer-specific cells as well as cells expressing the cell surface markers CD45RA, CCR7, CD107a and CD127 were detected. Finally, in the multimer positive cells the frequencies of cells expressing the markers CD45RA, CCR7, CD107a and CD127 were detected as well.(TIF)Click here for additional data file.Figure S2Examples of positive and negative multimer staining. The multimer specific cells were detected in the CD3+CD8+ population using negative multimers and epitope specific multimers presenting the following epitopes: FIYAGSLSA (HLA-A*02:01), ELNNALQNL (A*24:02), QIMYNYPAM (A*30:01 and A*30:02), HAMSSTHEA (A*68:01), IPKLVANNT (B*07:02), QTYKWETFL (B*58:01) and ANNTRLWVY (C*07:01). Numbers are indicating frequencies of multimer-specific cells.(TIF)Click here for additional data file.Figure S3The antigen-specific recognition of the HLA-A*02:01 (A) and A*30 (A*30:01 – open symbols and A*30:02 – closed symbols) (B) restricted multimers, each dot represents the individual staining in one patient, the colors represents the M. tb protein and the derivative epitope .(TIF)Click here for additional data file.Figure S4Frequency of total CD8+ and antigen-specific T-cells belonging to the (A) naïve compartment  and (B) central memory compartment  divided per restricting MHC class I allele . Total CD8+ and antigen-specific T-cells belonging to the (C) naïve compartment , central memory compartment (D) , (E) effector memory compartment  and (F) terminally differentiated compartment  divided per immunogenic TB protein . Each dot represents an individual tetramer in one individual TB patient.(TIF)Click here for additional data file.Figure S5Frequency of total CD8+ T-cell belonging to the (A) naïve compartment , (B) central memory compartment , (C) effector memory compartment  and (D) terminally differentiated compartment  specific for the ‘super-epitope’ (QI)MYNYPAM(LG) . Each dot represents an individual tetramer in one individual TB patient.(TIF)Click here for additional data file.Figure S6Frequency of total CD8+ and antigen-specific T-cells expressing (A) CD107a and (B) CD127 divided per epitope derived protein  and antigens expressed primarily on slow growing bacteria . CD8+ T-cells expressing (C) CD107a and (D) CD127 specific for the ‘super-epitope’ (QI)MYNYPAM(LG) . Each dot represents an individual tetramer in one individual TB patient. Each dot represents an individual tetramer in one individual TB patient.(TIF)Click here for additional data file.Table S1Demographic data of the included patients.(PDF)Click here for additional data file.Table S2MHC class I binding affinity and off-rate data for peptide-epitopes derived from Rv1886c (Ag85B).(PDF)Click here for additional data file.Table S3Frequency of epitope-specific T cells identified by multimer staining.(PDF)Click here for additional data file.


Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents.Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors.The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine. Cocaine dependence is difficult to treat also because it is often associated with the abuse of other psychoactive compounds via blockade of adenosine A2A receptors located on striatal medium spiny neurons (MSNs) via the retrograde release of endocannabinoids (eCBs). Accordingly, the endocannabinoid system finely interacts with striatal glutamatergic and dopaminergic transmission Cocaine self-administration raise even more concerns when relevant doses of other commonly assumed psychoactive compounds, such as caffeine-containing beverages, are used together with this drug. In fact, caffeine is a well-known psychoactive drug displaying multiple effects on the central nervous system and specifically antagonizing adenosine receptors Thus, the aim of the present study is to characterize the synaptic interaction between cocaine and A2A-R antagonists in distinct striatal neuronal subtypes and to explore the possibility that this interaction influences motor activity providing a possible model to explain how the concomitant use of caffeine-containing beverages exacerbates behavioral and motor alterations induced by cocaine.A-R antagonist BMI (10 µM), evoked EPSPs and EPSCs during intracellular and patch-clamp recordings, respectively -expressing MSNs of the indirect pathway project to the medial globus pallidusThe classical model of basal ganglia functioning suggests that the ability of DA to modulate motor control is due to the segregation of D1 and D2 DA receptors in two distinct groups of striatal MSNs. According to this model, D1 receptor (D1-R)-expressing MSNs of the direct pathway project to the Thus, we investigated whether the blockade of DAT by cocaine in conjunction to A2A-R antagonism could affect glutamatergic synaptic transmission in the entire MSN population or in a single subset of neurons. Striatal MSNs obtained from mice expressing BAC-EGFP under the control of D1-R promoter (D1-EGFP) or D2-R promoter (D2-EGFP) were visualized with an infrared and fluorescence-equipped microscope. Only neurons that displayed a marked fluorescence were approached for patch-clamp recordings and underwent subsequent standard electrophysiological characterization. We bath applied 10 µM cocaine plus 1 µM ZM, in the continuous presence of 10 µM BMI, after recording a stable EPSC baseline both in D1-EGFP- and in D2-EGFP-MSNs. In these conditions the EPSC amplitude was reduced by 39.9±6.6% in D1-EGFP-MSNs (n = 5) and by 34.5±6.8% in D2-EGFP-MSNs (n = 6), A,B.To verify that the observed pharmacological effect induced by cocaine and A2A-R antagonists was expressed in all MSNs, in a subset of experiments, we combined electrophysiological recordings and immunohistochemical identification of the neurons. Thus, we patch-clamped MSNs using a biocytin-containing pipette in order to allow subsequent immunohistochemical identification of substance P-positive or A2A-positive MSNs, E. As shper se the spontaneous release of excitatory neurotransmitter, in fact, neither the amplitude nor the frequency of sEPSCs were different from that measured in pre-drug conditions , the amplitude and the frequency of sEPSCs were firstly recorded in the presence of cocaine , D2-R agonist quinpirole , or A2A-R inhibitor ZM241385 . These drugs did not affect  P>0.05, B. On th P>0.05, A,B. In  P>0.05, A,B. Fur P>0.05, A,B.vs AM, n = 5, P>0.05; quinpirole plus ZM plus AM vs AM, n = 5, P>0.05, As observed for the modulation of the evoked synaptic transmission, when the experiment was performed in the presence of the CB1-R antagonist AM251 (3 µM), the reduction of the frequency of spontaneous EPSC in the presence of cocaine or quinpirole, co-applied with the A2A-R antagonist ZM, did not occur (cocaine plus ZM plus AM We recently reported that choline acetyl transferase (ChAT)-positive striatal large aspiny (LA) interneurons co-express D2- and A2A-Rs and are deeply involved in the D2/A2A-R-dependent modulation of striatal excitatory synaptic transmission since D2-R agonist and A2A-R antagonist reduced the firing rate of these interneurons that potentially control the synaptic transmission of the entire population of striatal MSNs LA interneurons were patch-clamped in current-clamp mode and, as shown in The block of DA transporter and the subsequent increase of endogenous DA levels together with A2A adenosine signaling might thus regulate the firing discharge of cholinergic interneurons toward MSNs and subsequently regulate striatal excitatory synaptic transmission by an eCB-mediated mechanism In physiological conditions D2-R agonists, in combination with A2A-Rs antagonists, were able to reduce striatal cholinergic transmission and, in turn to inhibit glutamate release toward MSNs acting on postsynaptic M1-like muscarinic receptors To unveil if reduced glutamatergic synaptic excitatory transmission in both D2- and D1-R-expressing MSNs, produced by concomitant exposure to cocaine and A2A-Rs antagonists, might have direct behavioral consequences, we analyzed motor effects produced by the co-administration of these drugs.First, we evaluated motor response of mice to different doses of cocaine  in order to find the concentration that was ineffective to stimulate locomotor activity A. Amongpost-hoc analysis). Interestingly, treatment with the selective CB1-R antagonist AM251 (5 mg/kg) abolished the motor stimulation induced by co-administration of cocaine and CSC . To confirm this observation, we tested another selective A2A-R antagonist, such as ZM241385 -caffeine and ZM241385. In this study we provided for the first time electrophysiological evidence of a significant inhibition of striatal excitatory synaptic transmission exerted by cocaine in combination with A2A-R antagonists. In a previous our publication Cocaine and A2A-R inhibition of synaptic response also required postsynaptic intracellular calcium changes and the activation of CB1-Rs since intracellular BAPTA or bath application of a CB1-R antagonist significantly opposed this inhibitory response. Thus, according to previous published works Interestingly, as revealed by immunofluorescence analysis of patch-clamped MSNs from BAC-EGFP mice and from MSNs expressing substance P or A2A-Rs we found that the combined effect of cocaine and A2A-R antagonism was not segregated to specific neuronal striatal subpopulations. In fact, we report that in the presence of cocaine plus the A2A-R antagonist ZM, the inhibition of the EPSC amplitude was similar in D1-EGFP- and SP-expressing neurons as well as in D2-EGFP and A2A-R-expressing cells, identified as D1-R or D2-R expressing MSNs, respectively. According to these data it has been shown that in most of the recordings from striatal MSNs, activation of D2-Rs inhibits striatal glutamate release by a retrograde eCB signaling These findings are in line with results from our previous work in which we have shown that LA interneurons of the intrastriatal network may exert a major modulatory role of the synaptic excitatory response of both D2-R- and D1-R-expressing MSNs. This action was achieved by regulating intracellular calcium via M1 muscarinic receptors, and we showed that cholinergic firing tone was reduced by a the D2-R agonist quinpirole and a A2A-R antagonist Furthermore, we found that a sub-threshold dose of cocaine that does not affect the firing frequency of LA neurons, is sufficient to produce a robust effect in reducing the firing frequency when applied in combination with the caffeine derivative CSC that specifically blocks A2A-Rs. These findings support the important role of intrastriatal cholinergic in the regulation of motor functions.2+ channels We can hypothesize that the synergistic action of cocaine and A2A-R antagonists in inhibiting the firing rate of cholinergic interneurons would result in the reduction of endogenous acetylcholine release and the consequent reduced activation of M1 muscarinic receptors on MSNs. The established effect of M1 receptor inhibition would be the opening of L-type CaElectrophysiological effects of combined exposure to cocaine with either CSC or ZM reflected in changes of the motor function. In fact, while cocaine administration per se increased spontaneous motor activity, as previously reported It has been already suggested that eCBs signaling mediates psychomotor activation by A2A-R antagonists drd1a-EGFP) or D2-R promoter (drd2-EGFP) were used for electrophysiological experiments. BAC-EGFP mice were originally generated by the GENSAT (Gene Expression Nervous System Atlas) program at the Rockefeller University All the experiments were conducted in conformity with the European Communities Council Directive of November 1986 (86/609/ECC) in accordance with a protocol approved by the Animal Care and Use Committee at the University of Perugia and every effort was made to minimize animal suffering. Two to three months-old male Wistar rats (Harlan) and 5–6 weeks old male C57Bl/6J mice carrying BAC that express enhanced green fluorescent protein (BAC-EGFP) under the control of D1-R promoter (2–5% CO2 gas mixture. The composition of the Kreb’s solution was (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 25 NaHCO3. Drugs were bath-applied by switching the solution to one containing known concentrations of drugs. Powders were dissolved in water or DMSO and aliquoted. Drugs were applied by dissolving them to the desired final concentration in the external Kreb’s solution Coronal slices including the cortex and the striatum were cut from rats  or from BAC-EGFP mice  brain using a vibratome. A single slice was transferred to a recording chamber and submerged in a continuously flowing Kreb’s solution  bubbled with a 95% O+-gluconate, 0.1 CaCl2, 2 MgCl2, 0.1 EGTA, 10 HEPES, 0.3 Na-GTP and 2 Mg-ATP, adjusted to pH 7.3 with KOH. When a BAPTA-containing internal solution was used, 20 mM BAPTA was added to the standard solution and K+-gluconate was lowered to 125 mM. Some electrodes were filled with the standard solution plus biocytin.Whole-cell voltage-clamp recordings  were performed with borosilicate pipettes (4–7 MΩ) filled with a standard internal solution containing (in mM): 145 K+-gluconate, 0.1 CaCl2, 2 MgCl2, 1 EGTA, 10 HEPES, 0.3 Na-GTP and 2 Mg-ATP, adjusted to pH 7.3 with KOH. These cells presented a pronounced h-current (Ih) and a typical sag potential in response to hyperpolarizing steps of current A bipolar electrode, connected to a stimulation unit (Grass Telefactor), was located in the white matter between cortex and striatum to stimulate glutamatergic fibers (0.1 Hz) and evoke excitatory postsynaptic potentials (EPSPs) and currents (EPSCs). Current traces for spontaneous EPSCs were recorded in the continuous presence of 10 µM BMI (sEPSCs) or BMI plus 1 µM tetrodotoxin to isolate miniature EPSCs (mEPSCs). Five minute episodes were acquired during the pre-drug condition and 20 minutes after of drug application. Cholinergic large aspiny (LA) interneurons, were localized under IR-DIC visualization by their large soma and subsequently identified by their electrophysiological properties. They were recorded in whole-cell current-clamp mode using an internal solution containing (in mM): 120 KQuantitative data are expressed as a percentage of EPSP and EPSC amplitudes or firing frequency with respect to the relative control values, the latter representing the mean of responses recorded during a stable period (10–15 min). The spontaneous events were detected using Clampfit 10 (Molecular Devices) and confirmed visually for accuracy. Amplitude and frequency were measured in epochs of 5 minutes.Brain slices that were used for electrophysiological recordings were processed to verify the presence of cells filled with biocytin. Thus, they where post-fixed overnight at +4°C with 4% paraformaldehyde in saline solution and then cryo-protected in phosphate buffer (PB 0.1 M) with sodium azide 0.02% for 48 hours at 4°C. Sections were incubated with streptavidin-Cy3 diluted 1∶600 in PB-TX-100 0.3% for 2 hours at room temperature and quickly observed, under an epi-illumination ﬂuorescence microscope (Zeiss Axioskop 2).A first set of brain slices was preincubated with a primary antibody guinea pig anti-SP or rabbit polyclonal anti-A2A to label MSNs of the “direct” or “indirect” basal ganglia pathway, respectively In order to label biocytin-filled cholinergic interneurons, a second set of slices, used for electrophysiological recordings, was also processed to detect the presence of choline acetyl transferase. Slices were incubated with a primary antibody rabbit anti-choline acetyl transferase  to label the cholinergic interneurons. The primary antisera were used at a 1∶400 concentration in 0.1 M PB containing Triton X-100, 0.3% and sodium azide, 0.02% for 24 h a room temperature and 48 h at 4°C. Sections were then rinsed three times for 15 min at room temperature, and subsequently incubated with a goat anti-rabbit Cy2-conjugated secondary antibody  for 2 hours at room temperature. Tissue was mounted on gelatin-coated slides, coverslipped with GEL-MOUNT and examined under an epi-illumination fluorescence microscope (Zeiss Axioskop 2), and a CLSM (Zeiss LSM 510) was used to acquire all the images.Locomotor activity was analyzed as previously described post-hoc analysis, when required.Mice were first habituated to the experimental cage for 60 min and then injected with the drugs reported above. CSC, AM251, ZM241385 and L-sulpiride were dissolved in vehicle  whereas cocaine was prepared in saline. Recording of locomotor activity started 15 min after last injection and was detected over test session 45 min by a computerized video tracking system . Total distance traveled was analyzed by ANOVA, followed by Fisher’s t-test were used. Values are mean ± SE; “n” is the number of recorded neurons. The significance level was established at P<0.05 (*), P<0.01 (**) and P<0.001 (***).Off-line analysis was performed using Clampfit 10 (Molecular Devices) and GraphPad Prism 5.0 (GraphPad Software Inc.) software. Two-way ANOVA or Student’s AM251 (AM), pirenzepine dihydrochloride, (-)-quinpirole hydrocholoride, (L)-sulpiride, tetrodotoxin citrate (TTX), ZM241385 (ZM) were from Tocris-Cookson; (-)-bicuculline methiodide (BMI), cocaine, 8-(3-Chlorostyryl)-caffeine (CSC), streptavidin-Cy3 were from Sigma-Aldrich. Anti-SP was from abcam; anti-A2A was from Alexis; alexa fluor 488 and alexa fluor 647 were from Invitrogen, Molecular probes.


The macrophage migration inhibitory factor (MIF)-homologue D- dopachrome tautomerase (D-DT) recently has been described to have similar functions as MIF. However, the role of D-DT, as opposed to MIF, in tumor biology remains unknown. We hypothesized that D-DT could represent a target for therapeutic interventions in cancer. We analyzed the production of D-DT in the murine melanoma model B16F10 and the murine breast cancer model 4T1 by western blot and ELISA. D-DT was released by tumor cells both in vitro and in vivo. RT-PCR revealed the expression of the D-DT receptor CD74 on both tumor cell lines. Tumor bearing mice had higher serum levels of D-DT compared to healthy controls. Remarkably, knock-down of D-DT by siRNA reduced proliferation of B16F10 cells in BrDU-assay and rendered them more prone to apoptosis induction, as shown by flow cytometry. In vivo neutralization of D-DT by antibodies reduced tumor progression in the B16F10 subcutaneous syngeneic tumor model. In summary, we could show that D-DT and its receptor are expressed in the murine tumors B16F10 and 4T1. Knock-down of D-DT through siRNA or blocking by antibodies reduced proliferation of B16F10 tumor cells. This qualifies D-DT for further evaluation as a therapeutic target. The macrophage migration inhibitory factor (MIF) was one of the first cytokines to be described and has since been implicated in many diseases including infectious diseases and cancer . D-DopacAnalysis of clinical samples further showed that D-DT, like MIF, is elevated in sera of patients suffering from ovarian cancer and a correlation between D-DT levels and disease progression has been reported .In cancer cells, MIF seem to play both an autocrine and paracrine role for tumor cell survival and invasiveness , 9. In aBecause MIF and D-DT have similar biological functions, we hypothesized that D-DT neutralizing therapeutic strategies may have a similar impact on tumor biology as demonstrated for MIF. We analyzed two aggressive murine tumor models for D-DT secretion in vitro and in vivo. We provide first evidence that mice are a suitable model for the analysis of D-DT in cancer. Furthermore, we demonstrate that the depletion of D-DT via siRNA or neutralization via antibodies results in reduced tumor growth. Therefore these strategies may be developed as a treatment modality against cancer.To address if D-DT could be used as a target in cancer models, we first analyzed if D-DT was produced by two murine cancer cell lines (the melanoma cell line B16F10 and the breast cancer cell line 4T1). D-DT expression in these cell lines was revealed by Western blot Figure . Both ceMIF mediates cell proliferation and protects tumor cells from apoptosis , 11. We Finally, we tested if D-DT neutralization impacts on tumor growth in vivo. We purified polyclonal D-DT antibodies from immunized rabbit serum by affinity chromatography using the recombinant antigen. Affinity-purified D-DT antibodies or polyclonal rabbit IgG were administered at a dose of 250 µg per mice every second day from tumor injection (n = 4 mice per group). While anti-D-DT antibody injections only resulted in a trend towards longer survival , treatment significantly slowed tumor progression Figure .This report is the first to show expression and secretion of D-DT in the B16F10 melanoma and 4T1 breast cancer model. We also show for the first time that D-DT has a proliferative and anti-apoptotic effect on B16F10 melanoma cells and that targeting of D-DT through siRNA or polyclonal D-DT antibodies may slow tumor progression in this model.MIF expression has been demonstrated in a wide variety of tumors . In contDue to the overlapping biological functions of MIF and D-DT, we hypothesized that D-DT may have proliferative and anti-apoptotic effects. The in vitro and in vivo findings confirmed this hypothesis. The findings have important implications, since they constitute the first step in evaluating D-DT as a potential therapeutic target for cancer therapy. Due to the wide spectrum of expression of D-DT in many cancer types analyzed so far, targeting of D-DT may form an attractive option.In summary, we demonstrate that D-DT may be a suitable target for cancer therapy . PossiblThe murine melanoma cell line B16F10 was cultured in DMEM with 10% fetal bovine serum , 1% penicillin and streptomycin (PS) and 1% L-glutamine . The murine breast cancer cell line 4T1 was provided by M. Wartenberg  and cultured in RPMI 1640 with 10% FBS, 1% PS and 1% L-glutamine.Wild type C57BL/6RJ and Balb/cJ mice were purchased from Janvier . B16F10 and 4T1 tumors were induced by injecting 5 x 105 or 106 tumor cells per mice, respectively. 250 µg of affinity-purified polyclonal rabbit D-DT antibody or polyclonal rabbit IgG were injected i.p. every second day. All animal studies were approved by the local regulatory agency (Regierung von Oberbayern).D-DT ELISA was constructed with in-house antibodies and was performed as described . In briePolyclonal rabbit D-DT antibody was generated and used for Western blot as described , 13.Reverse transcription and polymerase chain reaction was performed as described  using thsiRNA lipofection was performed as described  with theAnalysis of apoptosis was performed as described . StaurosFor statistics GraphPad Prism software, version 5.0b was used. All t-tests were two-tailed and p-values < 0.05 were considered significant. Differences between experimental conditions were analyzed using the unpaired Student's t-test. For in vivo experiments, differences between groups were analyzed using two-way ANOVA with correction for multiple testing.


CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins.We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay.This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology. Transcription factors are important and well-studied regulators of gene expression. Accordingly, cells need to tightly control transcription factors at multiple levels, and by multiple mechanisms, to correctly coordinate biological processes. Such control is achieved by the orchestrated action of, for example, protein phosphorylation, proteolytic processing, protein-protein interactions or subcellular localization e.g., .in vivo, which is highly prevalent across phyla, and its degree correlates with organism complexity Intrinsic protein disorder reflects the lack of a well-defined 3-dimensional structure CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) proteins comprise a family of transcription factors that are critical for metazoan development. As the effector components of the Notch receptor signalling pathway, they are context-dependent activators or repressors of target genes required for various cell differentiation-related decisions Saccharomyces cerevisiae. In all fungal genomes that harbour CSL genes, at least one representative from each fungal class was identified. The domain organization of fungal CSL proteins resembles that of their metazoan counterparts, with the notable exception of N-terminal extensions that are generally missing in animals The Notch pathway is confined to metazoa Schizosaccharomyces pombe, as a model. We found that Cbf11  and Cbf12  play antagonistic roles in several cellular processes, including cell adhesion and the coordination of cell and nuclear division, the former being an important virulence trait in pathogenic fungi In order to gain insight into the similarities and differences between the metazoan and fungal CSL proteins, we have employed the fission yeast, In this study, we conduct a more in-depth analysis of CSL protein sequences. Specifically, we explore the potential functional role(s) of the fungi-specific long N-terminal tails. We show here that these regions are likely disordered and enriched in regulatory motifs. Furthermore, in a case study of Cbf12, we demonstrate that the N-terminus negatively affects the Cbf12-dependent DNA binding activity.To obtain a catalogue of the available fungal CSL family members, we have searched multiple publicly available nucleotide and protein sequence databases as described Cryptococcus neoformans class F1 protein).The annotated gene structures for all fungal candidates were inspected manually and corrected as described We have obtained a set of 33 fungal CSL sequences (including 15 novel sequences from 7 species), with 16 sequences belonging to class F1 and 17 sequences belonging to class F2. In addition, the 11 CSL family members from 8 metazoan species used previously Multiple sequence alignments were carried out using the ClustalX 2.0.12 algorithm with default settings An unrooted phylogenetic tree was constructed from ClustalX-aligned protein sequences using the MEGA 4.0 package All algorithms were run with default settings. Protein regions of low sequence complexity (LCRs) were detected using the GBA algorithm Data handling was done using a set of in-house Python and R scripts. Statistical tests were performed in R. Unless stated otherwise, one-sided Wilcoxon signed-rank test with continuity correction was used at the 0.05 significance level. As the metazoan (class M) CSL N-terminal regions are in general very short, thus possibly biasing any metrics derived from them, and as this study is mostly focused on the two fungal CSL classes, the p-values for the class M N-termini statistics are not reported in the text.The plasmids and strains used for thiamine-repressible expression of N-terminally HisMyc-tagged Cbf11 and full-length Cbf12 were reported previously +s cbf12-3HA::natRh) was constructed in a wild-type background (JB32: +sh) by standard PCR-mediated one-step gene tagging using the pFA6a-3HA-natMX6 plasmid as template CGGATCCCCGGGTTAATTAA-3′5′-CAGTGGAATTATCTCCCATTTTATTATTTCAATACGAGACACTCTTTCATTCTGGATATAAGTGGCCTTTGGAAAGTCAC) consisted of 80 nt complementary to the 3′ end of the cbf12 open reading frame (stop codon not included) and 20 nt complementary to the 5′ end of the 3HA-natMX6 tagging cassette (underlined). The reverse primer (MP56: GAATTCGAGCTCGTTTAAAC-3′5′-GTTGTAGTAATAAATAAACACAGTAGTGCGAAAGGATATGGCAAATATGTGTAGTTGACAATAAAACCATTTTTTAAAGA) contained 80 nt complementary to the genomic sequence starting 80 nt downstream of the cbf12 open reading frame (the 80 nt gap was introduced to obtain a primer with a higher melting temperature) and 20 nt complementary to the 3′ end of the 3HA-natMX6 tagging cassette (underlined). The primers were used to PCR-amplify the tagging cassette; the PCR product was gel-purified, transformed into S. pombe cells and nourseothricin-resistant clones, in which the cassette had been integrated by homologous recombination, were selected as described The fission yeast knock-in strain expressing C-terminally triple HA-tagged Cbf12 from its endogenous chromosomal locus (JB817: − ura4-D18 leu1-32 ade6-M216h). The MP43 forward primer and a reverse primer complementary to the region immediately downstream  of the cbf12 open reading frame (MP44: GAATTCGAGCTCGTTTAAAC-3′5′-AAAACAAAAAGAGTAAAAATAAATATACTAATCCCTTGCAAAAACTTTTCAATAATAAAAAAGTAGTAAAGACAAATAAT) were used for the amplification of the tagging cassette from the pFA6-CTAP4-natMX6 plasmid − ura4-D18 leu1-32 ade6-M216 cbf12-CTAP4::natRh) was then crossed with the wild type JB32 strain to obtain a final strain without the auxotrophic markers (JB796: +s cbf12-CTAP4::natRh).The strain expressing C-terminally double TAP-tagged Cbf12 from its endogenous chromosomal locus was constructed analogously in an auxotrophic background  and kept at −80°C. Protein extracts were prepared by breaking the cells with glass beads in Lysis buffer 1 ; pH 7.6 ) or Lysis buffer 2 , 1 mM PMSF, with or w/o Phosphatase inhibitor cocktails 1 and 2 (Sigma)). Extracts were treated with the λ phosphatase (New England Biolabs) for 30 min at 30°C where required.Cells were harvested by centrifugation, washed with STOP buffer  or a 7.5% Tris-glycine gel, transferred on a nitrocellulose membrane and probed with either the mouse monoclonal anti-HA  or anti-His antibody , as appropriate. A goat-anti-mouse HRP-conjugated secondary antibody  was used for chemiluminescent detection of the tagged CSL proteins.4OH, 0.5 mM EDTA), precipitated with 25% TCA, washed once with ice-cold acetone containing 0.05 N HCl and once with acetone only.Thirteen litres of the JB796 strain culture were grown to mid-log phase in rich YES medium. Cells were harvested by centrifugation, washed once with water and then frozen in liquid nitrogen as noodles. Cells were broken using the RM200 mortar (Retsch), lysate was prepared in Lysis buffer 2 (see above) and clarified by ultracentrifugation at 100,000 g for 40 min. Standard tandem affinity purification S. pombe database  was searched with the Myrimatch algorithm S. pombe database and reversed and concatenated all sequences to allow estimation of false discovery rates . Myrimatch search parameters were as follows: strict tryptic cleavage; modification of methionine , S/T/Y  and cysteine  were allowed; precursor ions were required to be within 0.6 m/z of the peptide monoisotopic mass; fragment ions were required to fall within 0.5 m/z of the expected monoisotopic mass. IDPicker 3PO4) neutral loss peak at the MS2 level and (2) b and y ion intensities >20% of the neutral loss peak (3) contained two or more sequential fragments (b and/or y) bracketing the phosphorylation site(s). Phosphorylation sites were assigned based on the presence of sequential fragment ions surrounding the modification; if these ions were missing, the phosphorylation site(s) were assigned to multiple sites ambiguously.Purified proteins were denatured, reduced with Tris 2-carboxyethyl phosphine, alkylated with iodoacetamide, and digested overnight at 37°C with Trypsin Gold (Promega) or Chymotrypsin (Princeton Separations) after diluting to 2 M urea with 50 mM Tris pH 8.5. The resulting peptides were subjected to 2D LC-MS/MS (MudPIT) on a Thermo LTQ as previously detailed GTGGGAAAAAACATTTGTT-3′5′-CGATTACATCC and ‘c1450.16c’: TTCCCACGGATGTAATCGT-3′5′-ACAAATGTTTT derived from fission yeast ste6 and SPCC1450.16c gene promoters, respectively; the CSL binding site is underlined), or a mutated CSL site . Our final fungal set contained 33 unique CSL proteins ; three fungal species were only represented by a single CSL protein, because the other paralog did not pass our sequence quality control criteria. For Malassezia globosa, only a single CSL protein (class F1) was found in the GenBank database. For comparison, 11 selected metazoan CSL proteins from 8 species ranging from C. elegans to human, were also used in this study  CSLs revealed that these proteins have a unique fold consisting of two Rel-like domains (RHR-N and RHR-C) with an intervening beta-trefoil domain. These domains are further flanked by short N- and C-terminal extensions of low sequence conservation and unknown fold As noted earlier, the proteins in both fungal classes are typically much longer than their mammalian counterparts. This property is largely attributable to the presence of a long insertion in the BTD of most class F2 proteins, and, even more importantly, to the presence of unusually long extensions at the class F1 and F2 N-termini The amino acid sequence of the N-terminal regions is poorly conserved  and is hC. elegans LAG1 crystal structure As mentioned above, Pfam analysis The importance of intrinsically disordered regions has been recognized for processes such as signal transduction and transcription Protein phosphorylation is a post-translational modification well-known for the regulation of DNA-binding factors Another hallmark of the CSL N-termini identified by the analysis above was a strong enrichment in prolines (p<0.0001). PEST motifs, amino acid sequences rich in proline, glutamic acid, serine and threonine, are condition-specific protein degradation signals The bioinformatic analyses conducted so far generated several hypotheses that could be tested experimentally. We exploited the data and resources available for the fission yeast CSL proteins Cbf11 and Cbf12, members of class F1 and F2, respectively, to validate some of the predictions To obtain more refined information on Cbf12 phosphorylation, we conducted large-scale purification of TAP-tagged Cbf12 and subjected the purified protein to analysis by mass spectrometry. We achieved 82.7% overall coverage of the Cbf12 sequence with the average coverage depth being 51.1-fold, 44.7 -fold and 30.5-fold for the N-terminal, core and C-terminal region, respectively. In this high-coverage dataset we have identified 16, 3 and 0 phosphorylated sites comprising 3.5%, 0.8% and 0% of the N-terminal, core and C-terminal region , respectThe gel in We report in this study that fungal CSL proteins contain large regions of computationally predicted intrinsic disorder in their extended N-termini, and that these regions are enriched in two types of regulatory elements: phosphorylation sites and PEST motifs. We also provide experimental evidence that Cbf12, the fission yeast class F2 CSL protein, is phosphorylated with the majority of phosphorylated sites being located in the N-terminal region of Cbf12. Moreover, our data suggest that Cbf12 undergoes regulated proteolysis, and that the removal of its N-terminal tail enables the protein to bind to DNA, a property not observed for the full-length protein.Despite their low sequence complexity and low degree of evolutionary conservation, the long N-termini of fungal CSL proteins show conserved features, which suggest that these regions are important for CSL function. Many fungi with CSL proteins are simple organisms with short generation times and small, streamlined genomes These N-terminal tails are rich in potential kinase target sites and are likely disordered, which is expected to broaden the spectrum of proteins that can access them Δcbf11 or Δcbf11 Δcbf12 cells under multiple experimental conditions, which suggests that no other S. pombe protein can bind to the ‘RBP’ probe . The presence of the N-terminus thus seems to inhibit the ability of Cbf12 to interact with DNA, perhaps by imposing a sterical obstruction, or by affecting the binding site conformation or contacts with interaction partners. The situation is obviously more complex in a wild-type background where both full-length and truncated Cbf12 isoforms are found together , but their roles in these processes seem to be antagonistic. The relative levels of both Cbf11 and Cbf12 need to be finely tuned . The Cbftogether , and othIt will be interesting to see how the data from fungal systems apply to the CSL family members of class M. To our knowledge, there is little evidence for post-translational modifications of metazoan CSL proteins. It has been found that the human CBF1 is acetylated on K266 and phosphorylated on S230, which are located in the DNA-binding BTD. However, both pieces of evidence come from large-scale proteomics studies with limited biological conclusions for CBF1 Interestingly, a prominent role for intrinsic disorder has recently been suggested for class M RHR-C domain, which apparently undergoes coupled folding/binding during the interaction with Notch ankyrin repeats C. neoformans may cause meningitis in immunocompromised individuals Malassezia globosa has been associated with skin diseases Rhizopus oryzae is an opportunistic human pathogen causing potentially fatal mucormycoses U. maydis; Postia placenta; Phanerochaete chrysosporium; The data and conclusions on class F1 and F2 CSL proteins presented here should be useful even if the proposed regulatory mechanism proves to be fungi-specific. Some of the species discussed in this paper are relevant to human disease. For example, Table S1Summary of CSL sequences used (HTML).(HTML)Click here for additional data file.Table S2Cbf12 phosphopeptides identified by mass spectrometry (DOC).(DOC)Click here for additional data file.Text S1Sequences of CSL proteins used (TXT).(TXT)Click here for additional data file.Text S2New and corrected CSL cDNA sequences (RTF).(RTF)Click here for additional data file.Text S3Multiple sequence alignment used for CSL sequence partitioning (TXT).(TXT)Click here for additional data file.


Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01E and RTS,S/AS02D. Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2+ CD4+ T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01E and RTS,S/AS02D induced adaptive immune responses including antibodies, circulating memory B cells and CD4+ T cells directed against P. falciparum CS protein.The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against ClinicalTrials.govNCT00307021 Plasmodium falciparum  infection and disease with an efficacious adjuvanted protein vaccine is an efficient and feasible strategy to reduce the burden of malaria in endemic regions. The candidate vaccine RTS,S targets the circumsporozoite protein (CS) in the pre-erythrocytic stage of the parasite and is based on a recombinant fusion protein (RTS) comprising the CS central tandem repeats and the C-terminal regions of CS fused to the N-terminal of hepatitis B surface antigen P. falciparum challenge as compared to RTS,S/AS02 Vaccination against Plasmodium infection in small animal studies + T cell responses + T cell responses in children aged 18 months to 4 years, vaccinated with AS01E or AS02D based formulations. While the rationale for investigating CD4+ T cell responses is based on their potential role in protection against infection in adults Both antibodies and T cell pre-erythrocytic responses have been shown to confer protection against The supporting CONSORT checklist for this trial is available as supporting information; see The ethics committee of the International Foundation of the Albert Schweitzer Hospital of Lambaréné and the Western Institutional Review Board (USA) approved the study protocol. The trial was undertaken following the International Conference on Harmonisation of Good Clinical Practice guidelines. GSK Biologicals, Rixensart, Belgium, monitored the trial. In addition, a local safety monitor and a data and safety monitoring board closely reviewed the conduct and results of the trial.E and RTS,S/AS02D formulations administered intramuscularly at 0, 1 and 2 months in children aged 18 months to 4 years in Lambaréné, Gabon. The primary endpoint of the trial was safety. The immunological analyses presented here were exploratory endpoints. A total of 180 eligible children were randomly assigned to a treatment group on the day of first vaccination. Because of limited volumes of blood, each group was randomly divided into two subgroups for evaluation of memory B cell responses using Enzyme Linked Immunospot Assay (ELISPOT) or T cell responses by detection of intracellular cytokine expression using whole blood. Blood samples were collected in lithium-heparin tubes before vaccination, one month post doses 2 and 3 and 12 month post dose 3 (study month 14). Peripheral blood mononuclear cells (PBMCs) for use in the memory B cells ELISPOT assay were isolated using Ficoll-Paque PLUS .This trial has been described in detail in a previous publication Blood samples were collected before vaccination, one month post doses 2 and 3 and 12 months post dose 3. In all participants, serum antibodies to the NANP repeat region of CS (B cell epitope) were measured by a standard, validated enzyme-linked immunosorbent assay (ELISA) using plates adsorbed with the recombinant antigen R32LR that contains the sequence [NVDP(NANP)15]2LR, at a GSK validated laboratory . Titres were calculated using a reference standard curve with a 4 parameter logistic fitting algorithm and expressed in EU/ml, with cut-off for seropositivity set at 0.5 EU/ml 6 cells/ml for 7 days in RPMI 1640 containing 1 mM sodium pyruvate, non-essential amino acids, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercapto-ethanol and 10% Fetal Calf serum in the presence of 2.5 µg/ml CpG 2006  and 10 ng/ml IL-15  at 37°C, 5% CO2 humidify atmosphere in 24 well culture plates. Cultured cells were then counted and transferred onto 96 well ELISA plates coated with 100 µl of 10 µg/ml R32LR or with 5 µg/ml of γ chain specific goat anti-human IgG  for 3 hours at 37°C and saturated with PBS containing 3% BSA. CS-antigen specific immune responses were measured in triplicates over a period of three hours. A starting concentration of 106 cells/well followed by two three-fold serial dilutions  was used for non-coated and antigen-coated wells . For the IgG positive control, threefold serial dilutions were done with 27,000 cells/well as the highest density, followed by a series of three three-fold dilutions . Plates were washed and incubated with a biotin-labeled goat anti-human IgG  followed by streptavidin-alkaline phosphatase . After a final washing step, wells were covered with a 0.6% agarose solution in 1 M 2-amino-2-methyl-1-propanol, 1.58 mM MgCl2, 0.01% Triton X-405 and 2.3 mM 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine, pH 10.25. Plates were then incubated for 3 hours at 37°C for spot development, digitally photographed and counted manually using ImageJ software . The frequency of B lymphocytes expressing R32LR specific IgG was expressed as a number of antigen-specific cells per total IgG expressing cells after substraction of background responses. Samples which had less than 2000 IgG+ spots per million PBMCs were not considered for analysis.The assay used for detection of circulating antigen specific B cells is based on a published method + and CD8+ T cells expressing at least CD40L, IFN- γ, IL-2 or TNF-α. A child was considered to be a responder if, upon CS antigen whole blood stimulation, the frequency of CS-specific cytokine expressing T cells was greater than or equal to the geometric mean + 3 standard deviations of cytokine expressing T cells in unstimulated samples.Intracellular cytokine staining (ICS) was used to assess cell-mediated immune responses, using a previously described method Immunogenicity analyses presented here were performed on the According To Protocol cohort for immunogenicity which included all subjects that received 3 doses of study vaccine according to protocol and for whom valid immunogenicity measurements were available.The frequency of CS-specific B cells and T cells were summarized by descriptive statistics and tabulated by study group and timepoint. Between groups comparisons were performed by Wilcoxon Rank Sum tests . Within group comparisons were performed by Wilcoxon Signed Ranks test.Spearman's rank correlation co-efficient (R) was used to assess the correlation between the log of anti-CS IgG level and log of the frequency of circulating memory B cells.No adjustment for multiplicity of analysis was done.E and RTS,S/AS02D in this study have been reported previously E and RTS,S/AS02D (GMT 196 EU/ml 95% CI: 144–265 and 174 EU/ml 95% CI: 130–233 respectively) and all subjects remained seropositive for anti-CS antibodies one year after the last vaccine administration (GMT 15 EU/ml 95% CI: 10–24 and 19 EU/ml 95% CI: 12–31 respectively at month 14 of the study).Vaccination with RTS,S has been shown to induce persistent antibody responses to CS in malaria exposed Gambian adults E and RTS,S/AS02D induced a memory B cell response in children, the presence of circulating memory B cells producing IgG specific for the P. falciparum CS protein was evaluated in this study using in vitro maturated memory B cells to detect antibody secreting cells by ELISPOT. Anti-CS antibody secreting cells were detected only in a few subjects, in low numbers, before the first vaccine administration , the frequency of CS-specific circulating memory B cells had decreased in both groups but was still significantly higher than pre-vaccination values.To investigate whether vaccination with RTS,S/AS01stration . At one E and RTS,S/AS02D were observed in hepatitis B vaccinated and unvaccinated children. For example, at month 3 (one month post dose 3), the median (Q1–Q3) number of CS-specific memory B cells in children in the RTS,S/AS01E group previously vaccinated (N = 16) or not (N = 10) with hepatitis B vaccine was, respectively 1617 (594–3718) and 1496 (525–4657); in children in the RTS,S/AS02D group hepatitis B vaccinated (N = 16) or not (N = 16) was 1496 (525–4657) and 2071 (1075–3163), respectively.As, in this study, prior exposure to hepatitis B vaccination was associated with higher levels of CS-specific antibodies Correlations between antigen-specific antibody levels and memory B cells have been reported in adults P. falciparum controlled challenge trials E or AS02D before vaccination and one month after the second and third vaccination. The detection of the intracellular cytokine expression following overnight stimulation with SEB (positive control), the CS peptide pool or medium (negative control) was performed by flow cytometry. Gating strategy and representative Scatterplots are presented in T cell responses to the CS antigen have been reported to be associated with protection in + T cells expressing IL-2 were detected upon in vitro stimulation of whole blood in children vaccinated with RTS,S/AS01E or RTS,S/AS02D . No CS-specific CD8+ T cell responses were detected one month after vaccination (data not shown). Considering the low level of response seen at peak (one month post dose 3) for IL-2+ CD4+ T cells, ICS analysis at 12 months post dose 3 was not performed. For information, in children who had T cell responses assessed, the induction of anti-CS antibodies (data not shown) showed a similar pattern to what has been described for children in the B cell subset.Low frequencies of CS- specific CD4,S/AS02D . In bothP. falciparum CS-specific IgG+ memory B cells and IL-2+ CD4+ T cells detected upon short-term in vitro restimulation, after vaccination of young children with AS01E and AS02D based formulations of the RTS,S pre-erythrocytic vaccine candidate. Previous work has shown that in infants receiving RTS,S/AS02D, a significant IL-2 production was detected in the supernatant of blood lymphocytes upon long-term in vitro restimulation with a CS-derived peptide pool This study investigated the adaptive B and T cell immune responses induced by the RTS,S antigen formulated with two different adjuvant systems in 18 month to 4 year old children living in a malaria endemic region. This is the first study reporting significant amounts of E and RTS,S/AS02D vaccinated children. Similar to the association between the levels of antibodies to serogroup C meningococcal polysaccharide and the levels of circulating memory B cells in infants vaccinated with the MenC glycoconjugate vaccine This study demonstrates the induction one month after doses 2 and 3 as well as persistence at month 12 after last immunization of CS- specific circulating memory B cell and CS-specific antibodies in RTS,S/AS01E than with RTS,S/AS02D. This may suggest a stronger priming and induction of large size germinal centers by RTS,S/AS01E as we showed also a trend towards higher anti-CS antibody titers in the recipients of RTS,S/AS01E in the same trial E and RTS,S/AS02D groups had a significant level of memory B cells after the second vaccine dose and maintained a significant level of CS-specific antibodies one year after the last vaccination. The higher frequency of CS-specific memory B cells induced with the RTS,S/AS01E after the second dose may be associated with the higher immunogenicity and efficacy of this vaccine observed in studies conducted in animals and naïve adults After two vaccine administrations, a higher frequency of CS-specific memory B cells was induced with the RTS,S/AS01In contrast to antibody responses + T cell responses in children is based on the observation that CS-specific CD4+ T cells are associated with increased protection in adults + T cell responses to the CS antigen in children vaccinated with RTS,S/AS01E or RTS,S/AS02D, using a whole blood assay, IL-2 expression by CD4+ T cells was the only significant response detected. CD40L expression was not observed. While the children in this study had detectable CS-specific IL-2+ CD4+ T cells, these responses were lower than those seen, using a PBMC based assay, in malaria naïve adults vaccinated with RTS,S/AS01 or RTS,S/AS02 + T cells were not detected at one month post dose 2 and 3, in agreement with the absence of a detectable CD8+ T cell response to vaccination with RTS,S/AS01 or RTS,S/AS02 in adults The rationale for investigating CD4+ T cells producing at least 2 immune markers amongst IL-2, TNF-α, IFN-γ and CD40L was significantly higher in protected subjects as compared to non-protected subjects Production of IL-2 has been linked to the establishment of memory T cell responses. It has been suggested that IL-2 and IFN-γ producing T cells after vaccination of adults are associated with the induction of memory T cell responses to pre-erythrocytic malaria antigens Helper T cells promote antibody class switch + T cells may act as a growth factor for follicular helper T cells FH) are now recognized as the subset of helper T cells that regulate the multiple stages of the B cell compartment FH migrate to the follicular regions of lymphoid organs to engage in cell-to-cell contacts with antigen-primed B cells. CD40L, a marker for activated antigen-specific CD4+ T cells is a co-stimulatory molecule which is vital in the delivery of TFH cell help for the development of antigen-specific memory B cells + T cells observed in this study, we found that all children had detectable and persistent levels of anti-CS serum IgG after vaccination as well as CS-specific memory B cells up to one year after last vaccination.IL-2 produced by antigen-specific CD4E and RTS,S/AS02D induces significant CS-specific long term humoral immunity as well as CS-specific IL-2 producing CD4+ T cells in young children. While CD40L was undetectable, the induction and persistence of CS-specific memory B cells in all recipient suggest that both RTS,S/AS01E and RTS,S/AS02D vaccines engaged CD4+ T helper (TFH) in the development of CS-specific B cells. These mechanisms may occur with a greater amplitude following the vaccination with RTS,S/AS01E as it generated higher frequency of CS-specific memory B cells and higher level of CS-specific antibodies than RTS,S/AS02D and could explain the finding that the AS01E adjuvant improved the RTS,S vaccine efficacy as shown in the sporozoite challenge model + CD4+ T cells are linked to the memory T cell compartment and may be involved in RTS,S/AS01E and RTS,S/AS02D vaccine-induced long term protection. Further immunological investigations in RTS,S studies, including efficacy studies, will contribute to a better understanding of vaccine-induced protection against malaria.Together, these data show that vaccination with RTS,S/AS01Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.


In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11  xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.The xylanases from T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.Because of its high catalytic activity and good thermostability,  O- and/or 3-O-, by 4-O-methyl-glucopyranosyl uronic acid in the position of 2-O, and/or by acetyl groups in the positions of 2-O and/or 3-O -β-D-Xylp-(1→4)-β-D-Xylp -(1→4)-D-Xyl, XU4m2XX) as the shortest acidic fragment from birchwood glucuronoxylan -β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-D-Xyl, U4m2XX) [α-L-Araf-(1→3)]-β-D-Xylp-(1→4)-D-Xylp-(1→4)-D-Xyl, XA3XX) as the shortest arabinoxylan oligosaccharides (AXOS) from oat spelt arabinoxylan, whereas arabinoxylobiose (α-L-Araf-(1→3)-β-D-Xylp-(1→ 4)-D-Xylp, A3X) was the shortest AXOS liberated by Ta Xyn10A  on the hydrolysis of hydrothermally pretreated, washed wheat straw on the basis of formation of reducing sugars Figure . Ta Xyn1n stover . Howevern stover . The Ta T. aurantiacus was more efficient than the N. flexuosa xylanase in hydrolysis of isolated xylans and lignocellulosic biomass. The mode of action of the xylanases from T. aurantiacus and N. flexuosa on xylan was typical of enzymes belonging to the GH10 and GH11 families, respectively. Because of its high catalytic activity and good thermostability, the T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas the N. flexuosa xylanase has better potential as an enzyme for the production of XOSs.The two purified recombinant xylanases were shown to have good thermal stability at 70°C. As compared on the protein basis, the xylanase from AX: arabinoxylobiose; AXOS: arabinoxylan oligosaccharide; CBM: cellulose-binding module; DM: dry matter; GH: glycosyl hydrolase family; HPAEC-PAD: high-performance anion exchange chromatography coupled with pulsed amperometric detection; TLC: thin-layer chromatography; XAX: arabinoxylotriose; XAXX: arabinoxylotetraose; XOS: xylooligosaccharide; UXX: aldotetrauronic acid; XUXX: aldopentauronic acid.The authors declare that they have no competing interests.JZ designed and coordinated the experimental work, analyzed the results and drafted the manuscript. MS, TP and MTa reviewed the paper. MTe designed the work, analyzed the results and reviewed the paper. LV conceived and coordinated the overall study and helped to analyze the results and finalize the paper. All authors read and approved the final manuscript.


In functional experiments, miR-34a mimic suppressed cell growth, migration and invasion, meanwhile it increased cellular apoptosis and caspase activity in HCC cells. miR-34a mimic also reduced phospho-ERK1/2 and phospho-stat5 signaling. In addition, miR-34a mimic enhanced the effect of cell proliferation inhibition and caspase activity induction of agents targeting c-MET . In conclusion, miR-34a may act as a tumor suppressor miRNA of HCC. The strategies to increase miR-34a level might be a critical targeted therapy for HCC in future.Aberrant expression of microRNA-34a (miR-34a) has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in hepatocellular carcinoma (HCC) has not been completely clarified. In the current study, we have investigated the clinical significance and the  On the other hand, the miRNAs can have oncogenic or tumor suppressor activities, so miRNAs are emerging as vital targets for cancer molecular therapies P<0.01). The expression of miR-34a in the tissues in clinical TNM III and IV stages was significantly lower than that in I and II stages. Furthermore, in the group with metastasis, miR-34a expression was down-regulated compared to the group without metastasis (P<0.05). When studied the relationship between miR-34a expression and other clinicopathological parameters, we found that miR-34a level was correlated with the status of portal vein tumor embolus. miR-34a level was lower in the cases with portal vein tumor embolus than those without . Thus, different internal references were used in the current study. The primers for miR-34a, RNU6B, RNU48 and let-7a were included in TaqMan® MicroRNA Assays . Sequence of miRNA and references using in current study are : miR-34a (Applied Biosystems Cat. No. 4427975-000425) : UGGCAGUGUCUUAGCUGGUUGU; RNU6B (Applied Biosystems Cat. No. 4427975- 001093): CGCAAGGAUGACACGCAAAUUCGUGAAGCGUUCCAUAUUUUU; RNU48 (Applied Biosystems Cat. No. 4427975- 001006): GAUGACCCCAGGUAACUCUGAGUGUGUCGCUGAUGCCAUCACCGCAGCGCUCUGACC; let-7a (Applied Biosystems Cat. No. 4427975- 000377): UGAGGUAGUAGGUUGUAUAGUU. The reverse primers were also used in the reverse transcription step with TaqMan® MicroRNA Reverse Transcription Kit  in a total volume of 10 µl. Real-time qPCR for miRNA was performed with Applied Biosystems PCR7900. The miR-34a abundance in each sample was normalized to its references. The miR-34a expression in FFPE experiment was calculated with the formula 2−Δcq, and the change ratio of miR-34a in the in vitro experiments was: (1-1/2ΔΔCq)×100% The total RNA including miRNA was isolated from tumor sections using the miRNeasy FFPE Kit  according to the manufacturer's instructions with modifications by changing the incubation time after mixing with proteinase K to 36 h at 55°C, meanwhile, adding proteinase K every 12 h to maintain its concentration. RNA concentration was detected by Nanodrop 2000 . For in vitro experiments were performed in triplicate. HCC cells were seeded in a 24-well plate (2.5×104 cells per well) or a 96-well plate (2.5×103 cells per well) and incubated at 37°C for 24 h. The cells were then transfected with miR-34a inhibitor, miRNA inhibitor negative control, miR-34a mimic and miRNA mimic negative control, respectively  at a final concentration of 200 nmol/L for 96 h using combiMAGnetofection  in accordance with manufacturer's procedure. The c-MET specific siRNAs as an “on target smartpool” and their scrambled controls  were transfected into HCC cells with the same method as above. The sequences of the siRNAs are summarized in The human HCC-derived cell lines HepB3 (HB-8064), HepG2 (HB-8065) and SNU449 (CRL-2234) were purchased from the American Type Culture Collection . They were cultured as previously described Cell viability, cell proliferation, apoptosis and nuclear morphology and caspase-3/7 activity were performed as described previously Western blot were performed as described previously t-test were used to analyze significance between paired or unpaired groups. Statistical significance was determined at a P<0.05 level. In the analysis of additivity and synergism, the theoretical zero-interaction (exactly additive) dose-response curve for each miR-34a mimic+other agent combination was calculated by applying Bliss independence criterion SPSS19.0  was performed for statistical analysis. Results were representative of three independent experiments unless stated otherwise. Values were presented as the mean ± standard deviation (SD). One-way Analysis of Variance (ANOVA) test was used to analyze significance between groups. The Least Significant Difference (LSD) method of multiple comparisons with parental and control group was applied when the probability for ANOVA was statistically significant. Student's paired or unpaired Figure S1Combinational effect of c-MET siRNA and miR-34a mimic in HepG2 cells. c-MET siRNA  was combined with miR-34a mimic  and cell proliferation was detected by MTS assay. Biosoft CalcuSyn program was used to calculate (A): Median-effect plot, (B) Dose-effect curve, (C) CI. This indicates no synergistic effect, (D) Algebraic estimate.(TIF)Click here for additional data file.Figure S2Combinational effect of su11274 and miR-34a mimic in HepG2 cells. su11274  was combined with miR-34a mimic  and cell proliferation was detected by MTS assay. Biosoft CalcuSyn program was used to calculate (A): Median-effect plot, (B) Dose-effect curve, (C) CI. This indicates no synergistic effect, (D) Algebraic estimate.(TIF)Click here for additional data file.