Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a per R mutant. In mice, the wbdR constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against Brucella.Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus  Brucella are the etiological agents of brucellosis, a zoonosis that causes abortions and infertility in domestic livestock and wildlife and a grave and debilitating disease in humans. Eradicated in a handful of countries, this disease is endemic or increasing in many areas of Asia, Africa, and America due to changing breeding conditions and agricultural intensification, thus representing a serious problem in developing economies throughout the world , carry a S-LPS poorly recognized by cell receptors, complement and bactericidal peptides. The Brucella lipid A is a diaminoglucose disaccharide substituted with very long acyl chains that is linked to a core oligosaccharide carrying a characteristic glucosamine lateral branch  in various proportions of α-(1→2)- and α-(1→3)-linkages usually but not always with a terminal cap of at least a tetra-saccharide unit containing α-(1→3)-linked N-formyl-perosamine  and its formylation (wbkC) are indispensable for O-PS expression, gene replacement seems the simplest strategy, and potential tools are genes carried by the few Gram-negative bacteria endowed of heteropolymeric O-PS with N-acylated perosamine  and Ultraclean Microbial DNA Isolation Kit (Mo Bio Laboratories), respectively. When needed, DNA was purified from agarose gels using a Qiack Gel extraction kit (Qiagen). DNA sequencing was performed by “Servicio de Secuenciación del Centro de Investigación Médica Aplicada”  and “Unidad de Genómica del Instituto de Parasitología y Biomedicina López-Neyra” . Primers (Supplementary Table wbdR attB Fw (5′ggggacaagtttgtacaaaaaagcaggcttcATGA ATTTGTATGGTATTTTTGGT3′) and wbdR attB Rv (5′ggggaccactttgtacaagaaagctgggtcTTAAATAGATGTTGGCGA TCTT3′) were designed based on the sequence of E. coli O157:H71 and according to Gateway Cloning Technology®  instructions. Both primers were used to amplify wbdR from the start to the stop codon using E. coli O157:H7 DNA as template, and the resulting product was cloned into pDONR221 (Invitrogen) by site-specific recombination, generating pYRI-5. Then, wbdR from pYRI-5 was transferred by site-specific recombination to pRH001 (B. abortus 2308W) by conjugation  to obtain pYRI-21. The construction was verified by sequencing. Then, the EcoRI fragment of pYRI-21 containing wbdR and its promoter was subcloned into the corresponding site of pUC18R6KT-miniTn7TKm (wbdR). The miniTn7 vector carrying wbdR with its own promoter was inserted into Ba-parental chromosome II by the method of E. coli S17.1 λpir and then transferred to Brucella using an E. coli S17.1 λpir (pYRI-27)-E. coli HB101 (pRK2013)-E. coli SM10 λpir (pTNS2)-Ba-parental four-parental mating. The resulting Ba::Tn7wbdRKmR construct was examined by PCR for the correct insertion and orientation of miniTn7 between genes glmS and recG using the following primers: (i) GlmS_B (5′-GTCCTTATGGGAACGGACGT-3′) and Ptn7-R (5′-CACAGCATAACTGGACTGATT-3′) for insertion downstream glmS; (ii) Ptn7-L (5′-ATTAGCTTACGACGCTACACCC-3′) and RecG (5′-TATATTCTGGCGAGCGATCC-3′) for insertion downstream recG; and (iii) GlmS_B and RecG that only amplify the intergenic region in the absence of the mini-Tn7. The presence of only one copy of the miniTn7 was determined by Southern-blot and sequencing.To obtain a stable niTn7TKm  to obtaiwbdR construct with no Km resistance (Ba::Tn7wbdR), a non-polar kmR mutant of Ba::Tn7wbdRKmR was constructed by overlapping PCR using the Km cassette of pUC18R6KT-miniTn7TKm as template. Primers kmR-F1 (5′-AGGAAGCGGAACACGTAGAA-3′) and kmR-R2 (5′-AATCATGCGAAACGATCCTC-3′) amplified a 318-bp fragment including 312-bp upstream of the kmR start codon and codons 1 to 2 of the kmR ORF, and primers kmR-F3 (5′-gaggatcgtttcgcatgattTTCTTCTGAGCGGGACTCTG-3′) and kmR-R4 (5′-TGGTCCATATGAATATCCTCCTTA-3′) amplified a 268-bp fragment including codons 262–264 of the kmR ORF and 256 bp downstream of the kmR stop codon. Both fragments were ligated by overlapping PCR using oligonucleotides kmR-F1 and kmR-R4 for amplification, and the complementary regions between kmR-R2 and kmR-F3 for overlapping. The resulting fragment, containing the kmR deletion allele, was cloned into pCR2.1 (Invitrogen) and subcloned into the EcoRI site of the suicide plasmid pNPTS138-Cm  to generate plasmid pRCI-65. This suicide plasmid was used to delete the kmR gene of Ba::Tn7wbdRKmR using the allelic exchange by double recombination  and wbkC-R2 (5′-GCCCATGCCAATCAAGGT-3′) amplified a 393-bp fragment including codons 1–29 of the wbkC ORF (BAB1_0540), as well as 306 bp upstream of the wbkC start codon, and primers wbkC-F3 (5′-accttgattggcatgggcAGATGGTCGGAAGTCCAGATT-3′) and wbkC -R4 (5′-TCTGAACTCGGCTGGATGAC-3′) amplified a 434-bp fragment including codons 212–259 of the wbkC ORF and 287-bp downstream of the wbkC stop codon. Both fragments were ligated by overlapping PCR using oligonucleotides wbkC-F1 and wbkC-R4 for amplification, and the complementary regions between wbkC-R2 and wbkC-F3 for overlapping. The fragment containing the wbkC deletion allele was cloned into pCR2.1 and subcloned into the BamHI and the XbaI sites of the suicide plasmid pJQK . To obtain Bme::Tn7wbdR and Bme::Tn7wbdRΔwbkC the suicide plasmids pRCI-65 and pYRI-31 (see above and Supplementary Table Bme::Tn7wbdR and Ba::Tn7wbdR were grown in the presence of Km and Cm (plasmid antibiotic markers) or Km (transposon antibiotic marker), adjusted to an O.D.600 = 0.109 and CFU counted on TSA and TSA Km/Cm or TSA and TSA Km (transfer 0). A 100 μl aliquot of the culture was inoculated into 10 ml of TSB without antibiotics, the broth incubated for 48 h and CFU counted on TSA and TSA Km/Cm or on TSA and TSA Km. The process was serially repeated five times.The constructs Ba-pBrucella typing procedures  which were incubated in a Bioscreen C  with continuous shaking at 37°C. Growth was monitored at 420–580 nm every 30 min over a 65 h-period (control wells contained sterile TSB). All experiments were performed in triplicate.Colonial morphology, urease, and sensitivity to R/C phage were determined following established ocedures . S/R colocedures . AutoaggB. abortus wbdR constructs was obtained by the method of wbdR constructs, this classical precipitation step failed to yield the total of the S-LPS and precipitation was achieved with 6 volumes of methanol. Alternatively, the S-LPS remaining in the supernatant of the methanol precipitation was dialyzed and freeze-dried. These crude S-LPS were resuspended (10 mg/ml) in 175 mM NaCl, 0.05% NaN3, 0.1M Tris-HCl (pH 7.0), digested once with nucleases  and then three times with proteinase K . Finally, the S-LPS was recovered by ultracentrifugation   .g) and the supernatant was dialyzed against distilled water, lyophilized and chromatographed in acetate-pyridine buffer on Sephadex G50. Fractions were analyzed for PS by in gel immunoprecipitation in 10% NaCl, 100 mM borate (pH 8.3) (Purified LPS were used to obtain the PS by mild acid hydrolysis in 1% SDS acetate buffer 10 mM (pH 4.5) at 100°C for 2 h, a method that splits the lipid A-core linkage. After cooling at room temperature and brought to pH 7 with 0.2 M NaOH, lipid A and debris were pelleted (10 min at 5000 × (pH 8.3) .LPS were analyzed in 15% polyacrylamide gels (37.5:1 acrylamide/methylenebisacrylamide ratio) in Tris-HCl-glycine and stained by the periodate-alkaline silver method .1H NMR spectrum of the Ba-parental PS (5 mg in 0.55 mL D2O) was recorded at 25°C and the 1D and 2D NMR spectra of the Ba-pwbdR (10 mg in 0.5 mL D2O), Ba::Tn7wbdR (5 mg in 0.55 mL D2O) and Ba::Tn7wbdRΔwbkC PS (10 mg in 0.55 mL D2O) were recorded at 47°C, 25°C and 25°C, respectively. All experiments were performed on Bruker AVANCE 500 MHz or Bruker AVANCE III 700 MHz spectrometers; both equipped with 5 mm TCI Z-Gradient CryoProbes. 1H chemical shifts were referenced to internal sodium 3-trimethylsilyl--propanoate  and 13C chemical shifts were referenced to external dioxane in D2O (δC 67.40). Data processing was performed using vendor-supplied software. The assignments of the 1H and 13C resonances of the Ba-parental, Ba-pwbdR, Ba::Tn7wbdR and Ba::Tn7wbdRΔwbkC PS were obtained by analysis of 1H and 13C NMR spectra together with a multiplicity-edited1H,13C-HSQC experiment , rabbits were inoculated intravenously with 1 mg/ml of phenol-inactivated Ba-pwbdR cells. Then, similar doses were administrated intraperitoneally 2 and 4 days later and the animals bled 3 weeks later. The animals  were kept in cages with water and food ad libitum in the animal facilities of “Centro de Investigación en Farmacobiología aplicada” (University of Navarra). Rabbits were handled, bled and euthanized according to the Spanish and European recommendations , and the protocols approved by the Animal Health Care Department of University of Navarra.To obtain a serum reacting with O-PS carrying both N-acetyl-carrying O-PS  or N-formyl-carrying O-PS , the Formyl-Acetyl serum was absorbed with Ba-parental or Ba::Tn7wbdRΔwbkC cells, respectively. Absorption was performed at 4°C with continuous stirring overnight. Cells were removed , and the absorption repeated with incubation for 4 h at room temperature. As a control of absorption, Formyl-Acetyl serum was absorbed with Ba-pwbdR cells following the same procedure. Finally aliquots of Formyl-Acetyl or Acetyl serum were subsequently absorbed with cells from rough mutant BaΔper to obtain Formyl-Acetyl-OPS or Acetyl-OPS, respectively.To obtain a serum reacting only with B. abortus and Yersinia enterocolitica O:9 O-PS), and 33H8  were provided by INGENASA  and the reactivities verified by ELISA using LPS of B. abortus 2308, B. melitensis 16M and Yersinia enterocolitica O:9. Monoclonal antibody A15-6B3 (or 6B3) M; reacting with B. melitensis but not B. abortus O-PS  were electrotransferred onto nitrocellulose sheets , blocked with 3% PBS with 0.05% Tween 20 (PBS-T) overnight, and washed with PBS-T. Immune sera or monoclonal antibodies (see below) were diluted in this same buffer and, after incubation overnight at room temperature and washing the membranes with PBS-T, bound immunoglobulins were detected with peroxidase-conjugated goat anti-rabbit immunoglobulin  or peroxidase labeled protein G for mouse sera and 4-chlorine-1-napthol-H2O2. After 15 min, the color was measured in a microtiter plate reader  at 405 nm.For ELISA, 96-well ELISA plates  were coated with 2.5 μg/ml LPS resuspended in PBS overnight at 4°C. Plates were then washed extensively with PBS-T, and incubated with serial dilutions of the appropriate sera or with monoclonal antibodies at 37°C for 5 h. Then, the plates were washed extensively with PBS-T and bound antibodies were detected with peroxidase-labeled protein G , 1998. Tad libitum under P3 biosafety conditions in the facilities of “Centro de Investigación Médica Aplicada” (registration code ES31 2010000132) 2 weeks before and during the experiments. The procedures were in accordance with the current European (directive 86/609/EEC) and Spanish (RD 53/2013) legislations, supervised by the Animal Welfare Committee of the University of Navarra, and authorized by the “Gobierno de Navarra” (CEEA 045/12). To prepare inocula, TSA or TSA-Km grown bacteria were harvested, adjusted spectrophotometrically (O.D.600 = 0.170) in 10 mM in PBS and diluted in the same buffer to approximately 5 × 105 CFU/ml (exact doses were assessed retrospectively). For each bacterial strain, five mice were intraperitoneally inoculated with 0.1 mL/mouse. Eight weeks later, mice were bled and CFU numbers in spleen were determined. Statistical significance was evaluated using one-way ANOVA followed by Dunnett’s test.Female BALB/c mice  were kept in cages with water and food 4 CFU/mL in saline and dispensed in triplicate in microtiter plates (45 μL per well) containing fresh normal bovine or ovine serum (90 μL/well). After 15 and 90 min for the bovine serum or 15 and 45 min for the ovine serum at 37°C, brain heart infusion broth was dispensed (200 μL/well), mixed with the bacterial suspension and 100 μL plated on TSA.Exponentially growing bacteria were adjusted to 10Bacterial sensitivity was measured as the effect of increasing concentrations of polymyxin B and poly-l-ornithine on cell viability as described elsewhere .600 of 0.2. Measurements were performed at 25°C in a Zetamaster instrument using the PCS 1.27 software .The surface charge density was measured as the electrophoretically effective potential  as previously described . BacteriE. coli O157:H7 was selected because its O-PS contains N-acetyl-perosamine (D-Rhap4NAc)  in the native O-PS. Therefore, even though the experiments were carried out in two strains, it seems likely that wbdR can replace for wbkC in other S-Brucella species that carry N-formyl-perosamine homopolymers in the S-LPS . However, such an independence of the kind of linkage does not mean that the autochthonous O-PS biosynthetic machinery is as efficient in synthetizing the wild-type and the N-acetyl-perosamine O-PS. We observed that the degree of O-PS polymerization is reduced in the latter, and the present state of knowledge on gram-negative O-PS biosynthesis makes only possible to speculate on the causes. First, it is conceivable that the activities of the heterologous acyl-transferase, which belongs to a taxonomically distant bacterium, and the native enzymes (the glycosyltransferases for formylperosamine polymerization) are not optimal in the heterologous environment and substrates, respectively. Moreover, although to the best of our knowledge systems regulating the length of homopolymeric OPS (wzm/wzt dependent) are not known for any bacteria, it could be that such systems work differently on heterologous sugars and/or are affected by a reduced flow of precursors. The lack of stability of the Ba-pwbdR may also be explained by these hypothetical biosynthetic and export defects.The results presented in this work demonstrate that serovars . The O-Plinkages . Since wwbdRΔwbkC constructs, reactivity with monoclonal antibodies of C/Y and A>M (in B. abortus), or C/Y-A = M and M (in B. melitensis) specificity was severely hampered. This is consistent with the critical role of the formamide group in the interaction with the binding site of monoclonal antibody Yst9.1 (an anti-Y. enterocolitica O:9 antibody of C/Y reactivity) and with the observation that chemical removal of the formyl group followed by full N-acetylation reduces functional antibody affinities one thousand fold below those of the natural antigen  absent from the wild-type strains. Taking into account that the acetamido group is larger than the formamido one, it remains to be analyzed in detail whether N-acetyl-perosamine O-PS induces antibodies that can still bind N-formyl-perosamine O-PS and their affinity. Topologically, previous molecular modeling and analysis of the antibody binding sites shows that the N-formyl-perosamine formamido group is exposed on Brucella wild-type O-PS  thus replacing the formyl hydrogen atom of the 4-amido substituent of the wild-type O-PS, which accounts for the change in antibody reactivity to these O-PS.The substitution of the formamide by the acetamide group in perosamine resulted in a profound modification of the epitopic structure of the O-PS. For the  antigen . For theresidues , the resype O-PS . Modelinype O-PS  shows thBrucella O-PS of C1 access to the outer membrane proteins combined with the reduced activation of the complement cascade that depends on the structural peculiarities of its core and lipid A  carry O-PS and induce antibodies that interfere in the best diagnostic tests and thus in serological diagnosis  but also a totally new vaccine. Concerning the ancillary serological test, there is also a variety of possibilities. Classical buffered Brucella antigen agglutination and complement fixation tests use whole bacteria and, as the O-PS is surface exposed, they could be implemented with cells from a wbdR-modified strain, more likely of the wbdRΔwbkC type. Logistic difficulties posed by these tests can be circumvented using immunoenzymatic assays, and the LPS of the wbdR modified strains, or the hydrolytic polysaccharide if cross-reactivity at LPS core level poses a problem, are a clear alternative (The best available brucellosis vaccines (i.e., strains iagnosis . Althougcination . The resernative  Studies RC-Á, MI, and IM conceived and coordinated the study. GW supervised the NMR studies. EM-G, YG-R, AZ-R, JS, MZ, MI, and RC-Á performed the experiments and genomic analyses. RC-Á, GW, and IM wrote the manuscript. All authors analyzed the results and approved the final version of the manuscript.wbdR constructs. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.RC-Á, AZ-R, MI, and IM are inventors of patent EP15201717.4 covering potential uses of


In vitro studies showed Gal-1 induced invasion, the EMT phenotype and activated the non-canonical hedgehog (Hh) pathway in gastric cancer cell lines. Furthermore, our data revealed that Gal-1 modulated the non-canonical Hh pathway by increasing the transcription of glioma-associated oncogene-1 (Gli-1) via a Smoothened (SMO)-independent manner, and that upregulation of Gal-1 was strongly associated with gastric cancer metastasis. We conclude that Gal-1 promotes invasion and the EMT in gastric cancer cells via activation of the non-canonical Hh pathway, suggesting Gal-1 could represent a promising therapeutic target for the prevention and treatment of gastric cancer metastasis.Galectin-1  has been reported to be an independent prognostic indicator of poor survival in gastric cancer and overexpression of Gal-1 enhances the invasiveness of gastric cancer cells. However, the downstream mechanisms by which Gal-1 promotes invasion remains unclear. Moreover, the function of Gal-1 in the epithelial-mesenchymal transition (EMT) in gastric cancer has not yet been elucidated. In this study, we observed Gal-1 expression was upregulated and positively associated with metastasis and EMT markers in 162 human gastric cancer tissue specimens.  Gastric cancer is the fifth most common malignant tumor type and third-leading cause of cancer-related deaths worldwide . ApproxiLGALS1 gene, is a member of the carbohydrate-binding proteins family, which are characterized by their affinity for β-galactoside-containing glycans [Galectin-1 , encoded by the  glycans . Gal-1 c glycans . In the  glycans , 9. It h glycans –13, incl glycans , with po glycans . In addi glycans .It has been reported that Gal-1 promotes pancreatic carcinogenesis via activation of Hedgehog (Hh) signaling . Hh signIn this study, we investigated whether endogenous Gal-1 regulates the EMT by activating the Hh pathway in gastric cancer. We compared the expression of Gal-1 in cancer tissues and non-cancerous tissues of patients with gastric cancer and investigated the associations between Gal-1 expression and the clinicopathological features of patients with gastric cancer. Based on these clinical data, we performed in vitro experiments to assess the effects of upregulating or downregulating Gal-1 on the invasion and EMT in gastric cancer cell lines. This study suggests Gal-1 increases gastric cancer cell invasion and promotes the EMT by the activating the non-canonical Hh signaling pathway.P < 0.05). In contrast, the expression of E-cadherin was significantly lower in the gastric cancer tissues than the matched non-cancerous tissues (P < 0.05).In order to elucidate the role of Gal-1 in gastric cancer, we first performed immunohistochemistry analyses of 162 paired gastric cancer tissues and non-cancerous tissues from patients with gastric cancer. Compared with the matched non-cancerous tissues, the gastric cancer tissues exhibited significantly higher expression of Gal-1 , E-cadherin  and vimentin  in the matched primary tumors and metastatic lymph nodes. When Gal-1 immunostaining was classified as positive/negative, only five (5.15%) of the 97 cases  or three different Gal-1 shRNA sequences ; MGC-803 cells transfected with shGal1#3  efficiently reduced Gal-1 protein expression . In order to distinguish between the intracellular and extracellular activities of Gal-1 in LV-Gal-1 cells, we used β-lactose, a competitive inhibitor of Gal-1, to completely block the activity of exogenous Gal-1. Endogenous Gal-1 increased the invasion capacity of gastric cancer cells . The clinicopathological features of these patients are shown in Table The pharmacological reagent cyclopamine was provided by Selleck Chemicals . Recombinant human Gal-1  was obtained from Peprotech , and dissolved in 0.1% bovine serum albumin . Anti-galectin-1 antibody , anti-SMO antibody , anti-Gli-1 antibody (Abcam), anti-SHH antibody (Abcam), anti-E-Cadherin antibody , anti-Vimentin , anti-β-actin antibody , HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) were used in this study.Immunohistochemical staining of human paraffin-embedded gastric cancer and normal tissue sections was carried out as previously described with minor modifications . BrieflyThe human gastric cancer lines AGS (moderately-differentiated), MKN-45 (poorly-differentiated), SGC-7901 (moderately-differentiated), MKN-74 (well-differentiated) and MGC-803 (poorly-differentiated) were purchased from the Type Culture Collection of the Chinese Academy of Sciences . MGC-803 and AGS cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% FBS and the other cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone) containing 10% FBS. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Before experiments, gastric cancer cells in log phase growth in six-well plates were cultured in media containing only 1% FBS for 24 h.LGALS1 (GenBank accession numberNM_002305) was inserted into the GV248 and GV358 lentiviral vectors  to silence and upregulate the expression of Gal-1, respectively. The three shRNA sequences were as follows: Gal-1 sh1 (5'-CCGGCACCATCGTGTGCAACAGCAACTCGAGTTGCTGTTGCACACGATGGTGTTTTTG-3′); Gal-1 sh2 (5'-CCGGCCAGCCTGGAAGTGTTGCAGACTCGAGTCTGCAACACTTCCAGGCTGGTTTTTG-3′); Gal-1 sh3 (5'-CCGGGCTGCCAGATGGATACGAATTCTCGAGAATTCGTATCCATCTGGCAGCTTTTTG-3′). GV248 and GV358 lentiviral vectors were constructed to silence and upregulate the expression of LGALS1; a negative control vector containing the cytomegalovirus (CMV) promoter and expressing high levels of green fluorescent protein (GFP) was also created. The negative control was also created from GV248 and GV358. The lentiviral vectors were transfected into MGC-803 and MKN-74 cells at a multiplicity of infection (MOI) ranging from 1 to 100 in the presence of 5 μg/ml polybrene . To produce stably transfected cell lines, the cells were cultured in the presence of puromycin (Sigma-Aldrich). The cells were used for subsequent experiments after the expression of the target gene was confirmed using Western blotting.Human Total lysates of treated cells were prepared using RIPA buffer containing 1× Tris-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate. Total proteins (50 μg) from each lysate were separated by SDS/PAGE and transferred onto PVDF membranes, and then probed with the indicated antibodies using standard protocols.LGALS1, SMO, Gli-1, E-cadherin and vimentin. Total RNA was extracted using TRIzol reagent  following the manufacturer's instructions. First-strand reverse transcription was performed using the PrimeScript RT reagent Kit . The real-time PCR analyses were conducted on an iQ5 Multicolor Real-Time PCR Detection System  using SYBR Green Real-time PCR Master Mix (TaKaRa). The PCR program was 30 s at 95°C followed by 40 cycles at 95°C for 5 s, 60°C for 30 s and 72°C for 30 s. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the reference control. Fold changes in the mRNA levels of target genes were calculated relative to GAPDH. All results are reported as the average ratios of three different independent experiments. The following primers were used: Gal-1 (forward): CTGGAAGTGTTGCAGAGGTGT and (reverse) CTGGCTGATTTCAGTCAAAGG; SMO (forward) CAGGTGGATGGGGACTCTGTGAGT and (reverse) GAGTCATGACTCCTCGGATGAGG); Gli-1 (forward) GGGATGATCCCACATCCTCAGTC and (reverse) CTGGAGCAGCCCCCCCAGT; E-cadherin (forward) TCGTCACCACAAATCCAGTG and (reverse): CATTCACATCAAGCACATCC); vimentin (forward) TGAATACCAAGACCTGCTCAA) and (reverse) ATCAACCAGAGGGAGTGA ATC); and GAPDH (forward) TGACTTCAACAGCGACACCCA) and (reverse) CACCCTGTTGCTGTAGCCAAA.Real-time PCR was performed to determine the mRNA expression levels of Gli-1 and a negative control siRNA were purchased from Genechem . MKN-74 cells seeded in six-well plates were transfected with control or Gli-1 siRNA using Lipofectamine 2000  according to the manufacturer's instructions. The cells were harvested for further experiments at 24 h after transfection.The siRNA against Gastric cancer cell invasion was assessed using a chamber-based invasion assay. In brief, the upper surfaces of the 24-well Transwell inserts  were coated with 150 mg Matrigel basement membrane . The cells were re-suspended in serum-free RPMI or DMEM medium, then cell suspensions  were seeded onto the filters in 24-well chambers; 500 μl of medium containing 10% FBS was placed in the lower chambers as a chemoattractant. The cells were allowed to migrate for 24 h at 37°C. Cells remaining on the upper surface of the membrane were removed using a cotton swab. The filters were fixed with 4% paraformaldehyde, and the cells were stained with 0.05% crystal violet solution. The cells that had migrated from the upper to the lower side of the filter were counted under a light microscope in 10 randomly-selected fields at 100× magnification. Tumor cell invasion assays were performed in triplicate.2-test. Other data is presented as the mean ± standard error values. One-way ANOVA with the Least Significant Difference (LSD) post hoc test was used for multiple comparisons using SPSS version 13.0 software . P-values < 0.05 were considered significant.Protein expression levels and clinicopathological features were compared using the λ


The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD . Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies. Inclusion body myopathy with early-onset Paget’s disease and frontotemporal dementia (IBMPFD) is a rare hereditary disorder with multisystem involvement. The disease has an autosomal dominant inheritance, yet it shows adult-onset (Age 20 to 40 years). Myopathy observed in IBMPFD patients may result in disabling muscle weakness, which might eventually be life-threatening due to cardiac and respiratory muscle involvement. Paget’s disease is the cause of bone pain and fractures in the hips, the spine, the skull and in the long bones of arms and legs. In 30% of patients, brain involvement might be manifested as frontotemporal dementia, leading to learning and memory deficits, speech problems, personality changes and defects in social skills. Currently, there is no cure for IBMPFD. The only treatment modality that can be offered to patients is palliative, aiming to alleviate disease symptoms.VCP/p97 gene. Among disease-causing mutations, arginine 155 (R155) mutation was reported to be the most common one . In c. In c8].We showed that, the P137L mutant interacted with WT VCP/p97 proteins . Since tBinding of the p47 co-factor is important for VCP function in autophagic degradation. Yet, the P137L mutant was unable to bind this co-factor  and the protein formed non-functional aggregates. Under these circumstances, mutant VCP/p97 proteins seemed not to interfere with the autophagy-related function of the endogenous wild-type VCP protein. Consequently, the wild-type VCP could still retain its function in autophagosome formation, and assist in the clearence of protein aggregates by autophagy, including P137L mutant aggregates. Therefore, in the case of IBMPFD-diseased individuals carrying the P137L mutation, WT VCP/p97 that is produced by the non-mutated allele might still allow autophagic degradation to proceed in an efficient manner.in vitro cell culture conditions are valid in in vivo animal models.In IBMPFD disease,P137L mutant formed protein aggregates that did not prominently interfere with autophagic degradation activity. But, ERAD function was still perturbed and ubiquitylated proteins accumulated , resultiRecently, VCP/p97 was shown to contribute to the autophagy of mitochondria (mitophagy) , 40. YetVCP/p97 positive aggregates are not specific for IBMPFD. In a wide variety of diseases such as Parkinson’s disease, Hungtinton’s disease, Lewy Body disease, ALS and Machado-Joseph disease, VCP/p97 positive aggregates were observed –43. MoreAlthough all VCP/p97 mutations gave rise to clinical signs and symptoms of the IBMPFD disease, we showed that molecular and cellular changes caused by different VCP/p97 mutants  were not identical. Our study underlines the fact that caution should be exerted when modeling a disease based on single or few common mutations. Therefore, before designing a single common therapy approach for all patients regardless of individual genetic changes, a better understanding of the biology of various disease-associated mutant proteins should be obtained.S1 Fig(A and C) Cells expressing empty vector wild type (WT), mutant (P137L or R155C) VCP/p97 were co-transfected with RFP-GFP-LC3 fusion construct, treated with chloroquine (CQ) for 40 min and dot formation was visualized in U2OS (A) and PC-12 (C) cells. Arrow heads indicate autophagosomes whereas arrows indicate autolysosomes. (A and C) The graphs show the number of autophagosomes and autolysosomes in U2OS (B) and PC-12 (D) cells under basal and chloroquine (CQ) treated condition. Data were shown as mean ± SD of independent experiments (n = 3). * p<0.05, ** p<0.01, *** p<0.001.(PDF)Click here for additional data file.S2 Fig(A) HEK293T cells were transfected with wild type (WT) and mutant (P137L or R155C) VCP/p97 and then analyzed under confocal microscopy. (B) Mouse primary myoblast cells were cotransfected with autophagosome marker GFP-LC3 and wild type (WT) or mutant (P137L) VCP/p97 and analyzed using confocal microscopy under basal (NON-STV) or starved (STV) conditions. (C-E) Cells expressing WT or mutant (P137L or R155C) VCP/p97 were fractionated and subcellular distribution of proteins were analyzed by immunoblotting in HEK293T (C), U2OS (D) or PC-12 (E) cells. Graphs show the percentage of the insoluble fraction over total protein amount. ACTIN was used as loading control. (F) Wild type (WT) and mutant P137L VCP/p97 expressing U2OS cells transfected with a GFP-p62 construct, and association between p62 (Green) and VCP (Red) was analyzed under confocal microscopy.(PDF)Click here for additional data file.S3 FigU2OS cells were transfected with wild type (WT) or mutant (P137L) VCP/p97 in the absence or presence of 25 μM cycloheximide (CHX) for indicated time points. Western blot analysis was performed by using MYC and ACTIN antibodies. ACTIN was used as loading control. Image J software was used for the quantification of band intensities.(PDF)Click here for additional data file.S4 Fig(A)MYC-tagged wild type (WT) or mutant (P137L) were transfected to HEK293T cells. VCP proteins were precipitated using MYC-beads and immunoblots were incubated with anti-MYC or anti-VCP/p97 antibodies. ACTIN was used as loading control. (B) U2OS and (C) PC-12 cells expressing MYC-tagged wild type (WT) or mutant (P137L) were co-transfected with GFP-tagged wild type VCP/p97. Myc-tagged WT and P13L VCP proteins were immunoprecipitated using MYC-beads and blots were incubated with GFP, VCP/p97 and ACTIN antibodies. ACTIN was used as oading control.(PDF)Click here for additional data file.S5 Fig(A) WT and (B) P137L mutant VCP expressing cells were treated with 1 μg/ml Tunicamycin for indicated time points (0 to 8 hours). An anti-ubiquitin antibody was used to detect ubiquitylated total proteins. ACTIN was used as loading control.(PDF)Click here for additional data file.S6 Fig(A) HEK293T cells expressing were co-transfected wild type (WT) or mutant (P137L) VCP/p97 and autophagy marker GFP-LC3 and mitochondrial marker Mito-dsRed. 10 μM CCCP treatment for 12 h was used as mitophagy inducer and DMSO as carrier. Mitophagy was assessed as GFP-LC3-Mito-dsRed colocalization under confocal microscope. (B) Quantification of mitophagy positive cells .(PDF)Click here for additional data file.


The acetyl-CoA synthase (ACS) active site is a [4Fe4S]-[NiNi] complex (A-cluster). The dinickel site structure  was studied by X-ray absorption spectroscopy in ACS variants comprising all three protein domains or only the C-terminal domain with the A-cluster. Both variants showed two square-planar Ni(II) sites and an OH- bound at Ni(II)p in oxidized enzyme and a H2O at Ni(I)p in reduced enzyme; a Ni(I)p-CO species was induced by CO incubation and a Ni(II)-CH3- species with an additional water ligand by a methyl group donor. These findings render a direct effect of the N-terminal and middle domains on the A-cluster structure unlikely.Bacteria integrate CO Up to 1are SimX  and phas2 = 0.9) . FourierNMC and redNMC samples within error limits suggested near-stoichiometric loading with the [4Fe4S] sub-complex of the A-cluster. Apparently higher mean iron contents  in oxC and redC samples presumably reflected moderate underestimation of the protein concentration so that four Fe ions likely were also present in the truncated variant. The mean Ni/Fe ratio of ~0.4 (~0.3) in ox/redC (ox/redNMC) samples, which was slightly below the ideal value of 0.5 (for 2 Ni in the dinickel site and 4 Fe in the [4Fe4S] sub-complex), and the lower Zn/Fe ratio (~0.2) implied that maximally ~80% (~60%) of ACS proteins contained a [NiNi] site and the remainder an unoccupied binding pocket, only one Ni ion, or a [NiZn] site [TXRF analysis of metal contents in Ni-reconstituted ACS samples yielded the concentrations listed in Zn] site . This maNMCCO/CCO, the edge energy and shape were similar to redNMC/C, meaning that potential carbonyl binding to nickel  may not affect the XANES significantly. For redNMCMe/CMe, a ~0.9 eV higher edge energy compared to redNMCCO/CCO, but less pronounced edge shoulder compared to redNMC/C was observed, indicated a geometry change at nickel, possibly due to binding of a methyl group (e.g. as a fifth ligand at Nip).X-ray absorption spectra of ACS samples at the Ni K-edge are shown in NMC/C samples, the main FT peak was shifted to slightly larger distances compared to oxNMC/C, which suggested bond elongation at more reduced nickel sites. For redNMCCO/CCO, a similarly up-shifted peak and a more pronounced low-distance shoulder likely were due to a shorter Ni-C(O) bond. For redNMCMe/CMe, the smaller and somewhat broader main FT peak may suggest overlaid contributions of Ni-N/S and (longer) Ni-C(H3)/O bonds.EXAFS spectra of the ACS samples are shown in N) from the crystallographic data of the A-cluster (NMC/C quite well (Rf < 10%). The analysis revealed Ni-O/N (~2.0 Å), Ni-S (~2.2 Å), Ni-Ni (~2.9 Å), and Ni-Fe (~2.7 Å) distances (R), in good agreement with two square-planar nickel sites and a relative orientation of the [NiNi] and [4Fe4S] sub-complexes as in the crystal structure, showing a Ni-Ni distance larger than the Ni-Fe distance to the closest iron ion of the [4Fe4S] cluster  explained the relatively small amplitude contributions from metal-metal distances to the FT spectra  and [NiNi] sub-complexes in the crystal structures precludes axial ligand binding at Nip and Nid below the equatorial plane  as observed in CODH/ACS structures  cluster [p, as postulated in both mechanistic proposals [A different situation was found for methyl-treated ACS, for which binding of a methyl group in addition to the water species is suggested. Strictly speaking, the XAS data is not decisive whether the methyl (fifth) ligand is bound at Ni cluster , 48, 49.roposals , 7, 20,


Saccharomyces cerevisiae operates a mechanism that involves transporters (MALT), maltases (MALS) and regulators (MALR) collectively known as MAL genes. However, functional relevance of MAL genes during sake brewing process remains largely elusive, since sake yeast is cultured under glucose-rich condition achieved by the co-culture partner Aspergillus spp.. Here we isolated an ethyl methane sulfonate (EMS)-mutagenized sake yeast strain exhibiting enhanced maltose fermentation compared to the parental strain. The mutant carried a single nucleotide insertion that leads to the extension of the C-terminal region of a previously uncharacterized MALR gene YPR196W-2, which was renamed as MAL73. Introduction of the mutant allele MAL73L with extended C-terminal region into the parental or other sake yeast strains enhanced the growth rate when fed with maltose as the sole carbon source. In contrast, disruption of endogenous MAL73 in the sake yeasts decreased the maltose fermentation ability of sake yeast, confirming that the original MAL73 functions as a MALR. Importantly, the MAL73L-expressing strain fermented more maltose in practical condition compared to the parental strain during sake brewing process. Our data show that MAL73(L) is a novel MALR gene that regulates maltose fermentation, and has been functionally attenuated in sake yeast by single nucleotide deletion during breeding history. Since the MAL73L-expressing strain showed enhanced ability of maltose fermentation, MAL73L might also be a valuable tool for enhancing maltose fermentation in yeast in general.For maltose fermentation, budding yeast  Saccharomyces cerevisiae [Hordeum vulgare) seed. Maltose fermentation in S. cerevisiae requires a set of MAL genes , maltase (MALS), and zinc finger-type transcription factors (MALR) that positively regulate MALT and MALS genes [MALR genes , and Hu et al. [MAL13 and MAL33 do not seem to be functionally relevant in yeast under laboratory conditions [MAL genes are crucial for fermentation of maltose, as demonstrated by the observation that yeasts lacking any of these genes fail to grow in medium in which maltose is the sole carbon source [MAL loci (MAL1 to MAL4 and MAL6) have been shown to each contain up to three different genes, including: a MALx1 (where x = 1 to 4 or 6) gene encoding a maltose transporter (MALT); a MALx2 gene, encoding a maltase protein (MALS); and a MALx3 gene, a transcriptional regulator (MALR) of other MAL genes [MAL loci are located proximal to the telomeric region of the respective chromosome . In medium containing high concentrations of glucose, glucose uptake is promoted via hexose transporters (HXTs) and maltose uptake is simultaneously transcriptionally repressed, a cellular response that is known as a part of the “glucose repression” phenomenon [MAL11 gene is repressed in the presence of glucose [Generally, yeast alters its gene expression pattern in response to environmental nutritional conditions, notably including sugar and nitrogen sources. Alteration of gene expression in response to environmental sugar availability has been extensively studied using the budding yeast revisiae . MaltoseLS genes . To dateLS genes  have repnditions , 6. Thesn source . Five stAL genes –11. Thesenomenon . For exaMAL loci. The molecular mechanism(s) regulating maltose fermentation have yet to be elucidated in some commercially important yeast strains such as Japanese sake yeast [MAL genes, presumably due to artificial selection under conditions favoring malt-based fermentation. In contrast, Japanese sake yeast strains, which are utilized for fermentation in the glucose-rich environments resulting from the activity of fungal (Aspergillus oryzae) glucoamylases, show only a weak maltose fermentation ability [sake yeast K7 strain, which is a major sake yeast, has been reported [sake yeasts remains elusive. In the present study, we focused on the low maltose fermentation ability of sake yeast, and employed a genetic approach to enhance this property. Here we show that a putative MALR gene YPR196W-2, renamed as MAL73, encodes a novel hypomorphic regulatory protein whose function can be enhanced by a single nucleotide insertion that leads to the expression of MAL73L harboring an extended C-terminal region. In turn, the data also suggest that MAL73L has been functionally attenuated in sake yeast through frameshifts during breeding history. MAL73L expression in the original sake yeast strain resulted in enhanced growth on maltose medium compared to the parental strain. Moreover, MAL73L-expressing strain was capable of fermenting sake slightly faster than a control yeast strain. These results suggest that MAL73L is a genetic tool valuable for improving the maltose fermentation ability of yeast.In some yeast strains, maltose fermentation ability has been compromised or completely abolished during their evolution or via selection during domestication, as demonstrated by the loss-of-function mutation in, or absence of, one or more ke yeast . Among b ability . Althougreported , the basYeast strains used in this study are X2180-1A  and Kyokai No. 7 (K7) series strains . Cells were cultured in YPD , YPM , SD medium , SMal medium , SD+Antimycin A , 2% maltose, 2 mg/L antimycin A), or MA medium , 2% maltose, 2 mg/L antimycin A). Antimycin A is known to inhibit the mitochondrial electron transfer system, thus permitting stringent assessment of maltose fermentation ability .YPR196W-2Δ mutant was generated by using the CRISPR/Cas9 system [hygB) was amplified by using the primer pair YRP196W-2-hyg-F + YRP196W-2-hyg-R with pYC240 plasmid as the template [hygB fragment) were introduced into cells carrying Cas9 plasmid, and the resulting yeast transformants were selected using 100 μg/ml hygromycin B-containing YPD plates. After verification of the YRP196W-2 gene disruption, transformants were grown in the absence of selection for the Cas9 plasmid, and loss of the plasmid was confirmed by screening.Primers used in this study are summarized in 9 system  as descr9 system . BrieflyTDH3 promoter and terminator were amplified by using primer pairs TDH3-pro-F + TDH3-pro-R and TDH3-term-F + TDH3-term-R, respectively. This construct was designed to introduce a unique StuI site between the promoter and terminator. pJHXSB [AscI, and then these three fragments  were combined using the GeneArt Seamless Cloning and Assembly Enzyme Mix. The resulting plasmid was digested with AscI, and the fragment containing the TDH3 promoter, terminator and CEN/ARS was ligated with an AscI fragment containing the G418-resistance gene from pYC030 [The YCpG-TDH3pt vector was constructed as follows. The . pJHXSB  was digem pYC030  to yieldTEF2 promoter and terminator were amplified by using the primer pairs TEF2-pro-F + TEF2-pro-R and TEF2-term-F + TEF2-term-R, respectively. The YCpG-TDH3pt vector was digested with NotI, and then these three fragments were combined by using the GeneArt Seamless Cloning and Assembly Enzyme Mix to generate YCpG-TEF2pt. To construct the TEF2-Cas9 plasmid, Cas9 was amplified by using the primer pair Cas9-F plus Cas9-R with the CAS900A-1 plasmid (System Biosciences) as the template. The YCpG-TEF2pt vector was digested with StuI, and this linearized vector then was combined with the amplified Cas9 gene by using the GeneArt Seamless Cloning and Assembly Enzyme Mix.To construct the YCp-TEFpro-term vector, the YRP196W-2 gene was amplified by PCR with the primer pair YRP196W-2-F + YRP196W-2-R using K1801 genomic DNA of as the template. YCpG-TDH3pt was digested with NotI, and then the linearized plasmid and the amplified YRP196W-2 gene were combined by using the GeneArt Seamless Cloning and Assembly Enzyme Mix. To construct YCpG-YRP196W-2L, the YRP196W-2L gene was amplified by PCR with the primer pair YRP196W-2-F plus YRP196W-2-R using K1801M genomic DNA as the template. YCpG-TDH3pt was digested with NotI, and the linearized plasmid and the amplified YRP196W-2L gene were combined using the GeneArt Seamless Cloning and Assembly Enzyme Mix. The identity of the YPR196W-2L clone was confirmed by sequencing. To construct TDH3p-YPR196W-2, the YPR196W-2 ORF was amplified by PCR with the primer pair TDH3-YRP196W-2-F plus TDH3-YRP196W-2-R using K1801 genomic DNA as the template. YCpG-TDH3pt was digested with StuI, and the linearized plasmid and the amplified YRP196W-2 gene were combined using GeneArt Seamless Cloning and Assembly Enzyme Mix, placing the ORF under control of the TDH3 promoter.To construct YCpG-YRP196W-2, the MAL11p-lacZ, pYC-Z130 [EcoRI and HindIII, and the MAL11 promoter was amplified by PCR with the primer pair pYC-Z130-F + pYC-Z130-R using X2180-1A genomic DNA as the template. The linearized plasmid and the amplified promoter were combined using the GeneArt Seamless Cloning and Assembly Enzyme Mix.To construct pYC-Z130  was digeYPR196W-2p-lacZ, pYC-Z130 was digested with EcoRI and HindIII, and the YPR196W-2 promoter was amplified by PCR with the primer pair YPR196W-2-pro-F + YPR196W-2-pro-R using K1801 genomic DNA as the template. The linearized plasmid and the amplified promoter were combined using the GeneArt Seamless Cloning and Assembly Enzyme Mix. The G418 resistance-encoding marker was then replaced with the hygB selection marker from plasmid pYC240 [AscI restriction and ligation.To construct S. cerevisiae MAL23, AF002704.1; S. cerevisiae MAL43, M81157.1; S. cerevisiae MAL63, AF003003.1; S. cerevisiae MAL11, CP020129.1; S. cerevisiae MAL33, CP020124.1; S. cerevisiae YPR196W, CP020138.1; S. carlsbergensis MAL63, M36537.1An unrooted tree was constructed using Seaview version 4.4.2 software based on the ClustalW multiple alignment of DNA sequences, using neighbor-joining method with 1000 bootstrap counts , 22. TheMethods in Yeast Genetics .Cells carrying a 7 cells/mL and fermented at 20°C. Sugar content was measured using a refractometer and expressed in Brix unit at the indicated time point.Cells were cultured overnight in SD medium, and then cells were pelleted and resuspended in SMal medium at 1 × 10Total RNA was extracted as described by Schmitt et al. . cDNA sysake brewing tests were performed with a sake mash consisting of 72.8 g of α-preprocessed rice  derived from a single-step pre-fermentation consisting of 80 g of white rice with a polishing rate at 70% (retaining the inner 70% of the grain), 19.2 g of dried rice koji (a culture of A. oryzae on steamed rice) (Tokushima Seikiku) derived from 20 g of white rice with polishing rate at 70%, 0.1 mL lactic acid (Wako), and 174 mL of water [7 cells per g of mash; the mixture was further incubated at 15°C for 18 days (from Day 1 to Day 19) without shaking, as described previously [2 emission from mash [Small-scale of water . Each yeeviously . Progresrom mash . For sersake fermentation were measured by gas chromatography using a model GC 6890N machine  equipped with a flame ionization detector (FID) and Agilent J&W DB-624 column  [sake, was measured with a density/specific gravity meter ; readings then were converted into sake meter values using the formula: Sake meter value = 1443/S-1443, where S = Specific gravity (15/4°C). In general, larger values on this scale indicate that fermentation has progressed further.The concentrations of ethanol (and intermediates) during s, 3 μm) . Specifisake with 0.1 N NaOH. Amino acidity was measured by the formol titration method [p-toluenesulfonic acid at a flow rate of 0.7 mL/min. The column oven temperature was 40°C. Volatile aromatic compounds were measured by headspace gas chromatography [n-amyl alcohol and methyl caprate) were added to aliquots of the samples prior to heating at 50°C for 30 min. Esters and higher alcohols then were separated using a DB-WAX capillary column  after auto-injection of a headspace volume of 1 mL. The following conditions were applied: injection temperature, 200°C; oven temperature, 85°C; detector temperature, 250°C; and carrier gas (He), 2.2 mL/min.Acidity was determined by neutralization titration of 10 mL of n method . The orgtography  using anSugar content in the supernatants was determined by post-column labeling using phenylhydrazine with HPLC . HPLC wasake yeast, and the K7 genome sequence is available on a public website (http://www.bio.nite.go.jp/dogan/project/view/SC1) [sake yeast in the present study, we chose to further characterize strain K1801, based on the fact that this strain shared the K7 genetic background but exhibited a detectable  background level of maltose fermentation (K7 is a well-known iew/SC1) . Notablyiew/SC1) . Analysiiew/SC1) . To inveentation , S2 FigsTo generate mutants showing high maltose fermentation, K1801 was treated with ethane methane sulfonate (EMS) and spread on MA plates . Antimycin A is known to inhibit the mitochondrial electron transfer system, permitting more precise assessment of the maltose fermentation ability of the resulting isolates . SeveralMAL11, MAL13, MAL31, MAL32, MAL33, YPR196W and YPR196W-2 are annotated as members of the MAL gene family. Comparison of these gene sequences to those of the respective S288C homologs revealed that the K7 MAL33 gene, which encodes a MALR, harbors a single nucleotide deletion  in medium containing maltose  as that in the MAL33 gene from K7 and K1801 strains, suggesting that a MALR other than the MAL33 protein mediates the elevated maltose fermentation ability observed in K1801M.In the K7 genome sequence database, the genes deletion ; no suchM strain , S3 Fig.proteins . Notably maltose . These rtrans-activation of MAL11 observed in K1801M, we explored genetic alterations in other MALR-encoding genes in K1801M relative to the K1801 parent. As noted below, we ultimately focused on YPR196W-2. This gene is annotated in the K7 genome database, but not in the Saccharomyces cerevisiae Genome Database (SGD), and was designated YPR196W-2 because the K7 gene shows highest sequence similarity to YPR196W (MALR) among genes in the S288C genome. The Ypr196W-2p sequence was analyzed by BLAST search, and found to be structurally similar to several MALR proteins . Gibson et al. [trans-activation ability. The MALR genes in the S288C strain and the K7 strain are summarized in To clarify the increased MAL23, MAL43, and MAL63 each encode a polypeptide consisting of 470 amino acids; in contrast, the YPR196W-2 ORF is only 316 amino acids long. YPR196W-2 was the closest to MAL63  a single nucleotide deletion at a position corresponding to nucleotide position 917 of the MAL63 ORF  was consistent with the previous proposal that the K1801 strain is a triploid [YPR196W-2 occurred on one of three copies of Chromosome 7 in K1801M. Hereafter, we refer to the mutated allele of YPR196W-2 in K1801M as YPR196W-2L (Long). We also detected other single nucleotide insertions in proximity to ORF nucleotide position 917 in other maltose-assimilative mutants  using total RNA extracted from cells cultured for 6 h in YPD (containing glucose as the sole carbon source) or YPM  medium. The level of YPR196W-2 mRNA in K1801 did not differ between YPD and YPM, whereas the level of YPR196W-2L mRNA in K1801M was elevated in YPM compared to that in YPD  -178 to -164 and -174 to -160 , was introduced into the yeast along with a plasmid expressing YPR196W-2 or YPR196W-2L under the regulation of the native promoter. In YPD medium, reporter activity in yeast harboring YPR196W-2 or YPR196W-2L was statistically indistinguishable from that obtained in a strain harboring the reporter construct along with empty vector  the C-terminal domain of this MALR is required for trans-activation of MAL11, and (3) the gene expression of YPR196W-2L is responsive to maltose.These results showed that (1) Ypr196W-2Lp is able to exert autoregulatory YPR196W-2L was sufficient to confer maltose fermentation ability in the sake yeast K1801. To clarify the contribution to maltose fermentation ability of the endogenous YPR196W-2 alleles, the wild-type genes were deleted in K1801M. As it was technically difficult to selectively disrupt only the genes corresponding to the YPR196W-2 allele (given that YPR196W-2 and YPR196W-2L differ by a single nucleotide polymorphism), these allelic MALR genes were disrupted simultaneously using a CRISPR/Cas9 gene editing system in the triploid K1801M strain. Like K1801, the resulting K1801M ypr196W-2Δ strain (null) showed no maltose fermentation ability on an MA plate  that is present in K1801 provides this very weak MALR activity.Gibson et al.  previousactivity , suggestsumption . In contckground . These rin K1801 , S3 Fig.MALR gene under the control of promoters of various strengths. The maltose consumption rate of the cells harboring a construct (TDH3p-YPR196W-2) driving constitutive high-level expression of the truncated protein was lower than that obtained with YPR196W-2L under control of the endogenous YPR196W-2 promoter (YPR196W-2p-YPR196W-2L), but the TDH3p-YPR196W-2 maltose consumption rate was still higher than that obtained with YPR196W-2 under control of the endogenous YPR196W-2 promoter (YPR196W-2p-YPR196W-2)  the restored C-terminal domain in Ypr196W-2Lp is crucial for full function as a MALR, such that overexpression of the truncated protein is not sufficient to compensate for the attenuated activity of Ypr196W-2p.To assess the relationship between the levels of the MALR proteins and maltose fermentation, maltose consumption rates were determined in yeasts expressing distinct alleles of this R196W-2) . Taken tsake yeast family members are presumed to share the preponderance of their genetic background with K1801; however, maltose fermentation ability was found to differ among various members of the K7 sake yeast family  was performed with sake yeast (K1801 strain) transformed with YPR196W-2L or with control empty vector. Based on CO2 emission, yeast expressing YPR196W-2L showed slightly faster fermentation than that harboring control vector . This result presumably reflects the decreased availability of maltose (for degradation into glucose by A. oryzae) in the sake mash  lacks a thymine at nucleotide position 917 of the ORF. This single nucleotide deletion presumably occurred in the ancestral strain during differentiation and selection for sake fermentation; the resulting truncated form of Ypr196W-2p is apparently hypomorphic, resulting in decreased maltose fermentation ability. Together with the additional experiments  protein. This observation is consistent with a previous report that a truncated Mal63p, consisting of 283 amino acids, shows weaker induction of a maltase-encoding gene than the full-length (470-amino acid) Mal63p [MAL73L showed higher self-trans-activation via its own promoter than did MAL73  MALRs and/or MALR activation mechanisms in K1801.Among the tested e parent , showingMAL loci. During subsequent descent, those MAL loci presumably evolved in response to changes in natural environments and nutrient supplies, and under human selection for specialized fermentations with various plant materials. Some descendants lost entire loci from their genomes, as was the case of MAL2/4/6 in maltose fermentable yeasts [MAL genes were altered in expression or function, as was the case with MAL13 or MAL33 in S288C [sake yeast encodes a MALR  that is truncated and exhibits decreased regulatory function (compared to other known MALRs). Presumably, at some point in the past, an ancestral MALR gene suffered a single nucleotide deletion, resulting in a frameshift; this mutation introduced a premature stop codon, yielding a C-terminally truncated MALR protein. Sake fermentation characteristically employs continuous degradation of rice starch into glucose via the action of α-amylase and glucoamylase provided by the fungus A. oryzae. This process occurs in parallel with saccharification, whereby maltose is generated before immediately being further degraded into glucose monomer units , suggesting a founder effect derived from an ancestral sake yeast and/or a trace of historical human selection for the hypomorphic allele. Domestication of sake yeasts may have accompanied by genetic traits involving a specific organic compound profile in the fermented products as well as stable fermentation, since such a profile would have significantly altered the flavor and quality of sake during traditional-to-industrialized sake history. An obvious beneficial effect of the hypomorphic allele on sake quality is currently unknown, however future large-scale sake fermentations and subsequent sensory and chemical evaluations would clarify this issue.Notably, all of the S1 Figsake yeast under fermentation conditions. Cells were harvested after overnight cultivation in SD medium, transferred into SMal medium at 1 × 107 cells/mL, and further cultured at 20°C. Sugar content in the medium was measured at the indicated times.Maltose assimilation ability of (TIF)Click here for additional data file.S2 FigK1801 was bred as follows: first, K1601 was bred by the mating of a K1001 segregant with a K7 segregant. Next, K1801 was bred by the mating of a K1601 segregant and a K9 segregant.(TIF)Click here for additional data file.S3 FigEach strain was streaked on SD, SD+EtOH (vehicle control), SD+Antimycin A, SMal, SMal+EtOH (vehicle control) and MA  plates. Each plate was incubated at 30°C, and growth was monitored daily over the course of 2–6 days.(TIF)Click here for additional data file.S4 FigMAL33 (S288C) sequence was downloaded from the Saccharomyces genome database (http://www.yeastgenome.org/), and the MAL33 (K7) sequence was downloaded from the DOGAN–Saccharomyces cerevisiae K7 database (http://www.bio.nite.go.jp/dogan/project/view/SC1)The (TIF)Click here for additional data file.S5 FigYPR196W-2 homologs from other maltose-assimilative K1801-derived strains (K1801M’ and K1801M”) that were isolated through the mutagenesis screen of K1801 described in the Materials and Methods section. An asterisk is used to indicate the inserted or changed nucleotide in each strain.DNA (upper line) and amino acid (bottom line) sequences of (TIF)Click here for additional data file.S6 FigMAL-activator binding sites are indicated as MAL BD1 and MAL BD2. The consensus sequence of the MAL-activator binding site is MGCNNNNNNNNNMGS, where M is adenine or cytosine, S is guanine or cytosine, and N is any of the four deoxyribonucleotides.(TIF)Click here for additional data file.S7 FigSake mash containing yeast cells with control vector or YPR196W-2L plasmid were prepared in triplicate as described in the Materials and Methods section. Weight loss of the mash (reflecting CO2 gas emission) was measured at the same time each day. The upper panel shows total cumulative CO2 gas emission, and the bottom panel shows CO2 gas emission per day. Data are presented as the mean and standard deviation from three independent experiments.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the  Although medications and mosquito control efforts have limited the disease, malaria is still pandemic, with 198 million cases occurring in 2013, resulting in 584,000 fatalities Malaria is a mosquito-borne infectious disease caused by parasitic protozoans of the genus Plasmodium yoelii and Plasmodium berghei parasites for challenge have shown that protective immunity against pre-erythrocytic stages is mediated in part by T cells, particularly CD8+ T cells. Firstly, the major role for CD8+ T cells was shown by studies in which in vivo depletion of CD8+ T cells abrogated Spz-induced protective immunity in mice P. berghei or P. yoelii circumsporozoite protein (CSP), a major Spz antigen, confers protection against Spz challenge in naïve mice P. yoelii CSP (PyCSP), transgenic CD8+ T cells were shown to mediate protection against malaria Most vaccine efforts are directed against the pre-erythrocytic stages [sporozoites (Spz) and liver stages], and blood stages Plasmodium falciparum, PfSPZ vaccine, is very effective in inducing a high frequency of malaria-specific CD8+ T cells in the liver of nonhuman primates P. falciparum Spz by bites of infected mosquitoes, followed by chloroquine treatment induced significant malaria-specific pluripotent effector memory T-cell responses in vaccinated volunteers and protected all of them (10 out of 10) upon malaria challenge P. falciparum CSP (PfCSP) and apical membrane antigen-1 and then boosted with human adenovirus serotype 5 (Ad5) expressing the same antigens. This DNA priming/adenovirus boost immunization regimen induced sterile protection in four (27%) vaccinated subjects P. falciparum thrombospondin adhesive protein fused to multiple epitopes derived from several malaria antigens, induced sterile protection in 21% (3 out of 14) of subjects and delayed patency in 36% (5 out of 14) of subjects Beyond mouse model, Hoffman’s group has recently shown that intravenous (IV) immunization of IrSpz of In a previous study, we established a human immune system (HIS) mouse model by transducing genes encoding human HLA-A∗0201 and human cytokines using a recombinant adeno-associated virus serotype 9 (AAV9) vector 22.12, and every effort was made to minimize suffering. Human fetal liver samples were obtained via a non-profit partner . As no information was obtained that would identify the subjects from whom the samples were derived, Institutional Review Board approval for their use was not required, as previously described All animal experiments were carried out in strict accordance with the Policy on Humane Care and Use of Laboratory Animals of the United States Public Health Service. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at The Rockefeller University. Mice were euthanized using CO2.2tm1Unc Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice exhibiting features of both severe combined immunodeficiency mutations and interleukin (IL)-2 receptor gamma-chain deficiency were purchased from Jackson Laboratories  and maintained under specific pathogen-free conditions in the animal facilities at The Rockefeller University Comparative Bioscience Center.NOD.Cg2.35 HLA-A∗0201+ matched, CD34+ human hematopoietic stem cells (HSCs). CD34+ HSCs among lymphocytes derived from HLA-A∗0201+ fetal liver samples were isolated using a Human CD34 Positive Selection kit  Recombinant AAV9 (rAAV9) vectors encoding human IL-3, IL-15, GM-CSF, and HLA-A∗0201 were constructed as previously described 2.4P. falciparum CSP (AdPfCSP) was also previously constructed as described 10 AdPfCSP virus particles P. berghei sporozoite expressing full-length P. falciparum CSP (FLPfCSP/Pb sporozoite) was generated as previously described A recombinant adenovirus serotype 5 (Ad5) expressing a green fluorescent protein (GFP) alone in its transgene, AdGFP, was previously constructed 2.52. ICS assays were performed as previously described Spleens were harvested from HIS-CD8 mice 10 days after vaccination with AdPfCS or AdGFP or from naïve HIS-CD8 mice. After isolation of lymphocytes, the cells were counted and used for ICS upon stimulation with synthetic peptides corresponding to the A2-restricted CD8+ T-cell epitopes of the PfCSP  2.64 live FLPfCSP/Pb sporozoites by IV injection. After 42 h, the spleens were harvested, and splenocytes were stained with APC-labeled human HLA-A∗0201 tetramer loaded with YLNKIQNSL and PE-labeled anti-human CD8 antibody . The percentage of HLA-A∗0201-restricted, PfCSP-specific CD8+ T cells among the total human CD8+ T-cell population was determined using a BD LSR II flow cytometer .An Allophyocyanin-labeled human HLA-A∗0201 tetramer loaded with the peptide YLNKIQNSL, corresponding to the PfCSP CD8+ T-cell epitope 2.7Anopheles stephensi mosquitoes 2 weeks after an infective blood meal. Sporozoite challenge experiments were carried out as described previously 4 live sporozoites via the tail vain, and 42 h after the challenge, the parasite burden in the liver was determined by measuring parasite-specific ribosomal RNA using a 7300 Real-Time PCR system . Parasite burden was defined as the ratio of the absolute copy number of parasite ribosomal RNA to that of mouse GAPDH mRNA, and the percentage was calculated in relation to the parasite burden in naïve mice.FL-PfCSP/Pb sporozoites were obtained from salivary glands dissected from infected 2.8For the depletion of human CD8+ T cells, 0.1 mg of the rhesus IgG1 anti-human CD8 mAb MT807R1 2.9The serum level of human IgM was determined by ELISA , as we previously performed 2.10All of the statistical analyses were done using GraphPad Prism (ver. 4.03) . Bars in each figure represent geometric means. In all experiments, the values were log-transformed and then one-way ANOVA followed by a Dunnett’s test was employed to determine the differences between the groups. For a correlation analysis, linear regression analysis was applied to % of PfCSP-specific CD8+ T cells among the total human CD8+ T-cell population and % of relative parasite burden in liver, as 100% being the parasite burden in naïve and challenged mice.33.1As described previously 3.210 virus particles of AdPfCSP or AdGFP, as a control, by intramuscular (IM) injection (4 live FLPfCSP/Pb sporozoites 10 days after immunization (p = 0.006) was observed between the level of the PfCSP-specific human CD8+ T-cell response and the level of malaria protection  nization C. These 4P. yoelii or P. berghei, as a challenge model The role of CD8+ T cells in protective immunity against malaria, particularly immunity against the pre-erythrocytic stages of the parasite, has been examined in a number of mouse studies using rodent malaria parasites, such as P. berghei sporozoites expressing full-length PfCSP. The level of malaria protection was positively correlated with the level of the PfCSP-specific CD8+ T-cell response. Furthermore, depletion of human CD8+ T cells in AdPfCSP-immunized HIS-CD8 mice almost completely abolished the malaria protection, thus indicating that human CD8+ T cells mediate protective immunity against malaria, most likely against the liver stages of the parasite.We recently established a HIS-CD8 mouse model in which AdPfCSP immunization induces an HLA-A∗0201-restricted, PfCSP-specific human cytotoxic CD8+ T-cell response In our previous studies, we showed that hepatocytes in HIS-CD8 mice express HLA-A∗0201 following delivery of the gene via AAV9 and that PfCSP-specific human CD8+ T cells induced by AdPfCSP immunization recognize HLA-A∗0201+ hepatocytes presenting a peptide corresponding to the CD8+ T-cell epitope of PfCSP Grand Challenges Exploration grant (OPP1007283 GCE) from the Gates Foundation and by grants from the Mark S. Bertuch AIDS Research Fund.This work was supported by a M.T., X.L., and J.H. designed the study. X.L. performed the immunology and parasitology experiments. X.L. and J.H. generated the HIS-CD8 mice, and X.L., and J.H. maintained the HIS-CD8 mice. D.S., R.S., and A.C. provided the transgenic parasites, and M.Z. and R.F. maintained the parasites. M.T. and X.L. analyzed the data. M.T., X.L., V.N., and R.S.N. prepared the manuscript.The authors do not have any conflict of interest.


Thrombin binding aptamer (TBA), is a short DNA 15-mer that forms G-quadruplex structure and possesses anticoagulant properties. Some chemical modifications, including unlocked nucleic acids (UNA), 2′-deoxy-isoguanosine and 2′-deoxy-4-thiouridine were previously found to enhance the biological activity of TBA. In this paper, we present thermodynamic and biological characteristics of TBA variants that have been modified with novel isoguanine derivative of UNA as well as isoguanosine. Additionally, UNA-4-thiouracil and 4-thiouridine were also introduced simultaneously with isoguanine derivatives. Thermodynamic analysis indicates that the presence of isoguanosine in UNA or RNA series significantly decreases the stability of G-quadruplex structure. The highest destabilization is observed for substitution at one of the G-tetrad position. Addition of 4-thiouridine in UNA or RNA series usually decreases the unfavorable energetic cost of the presence of UNA or RNA isoguanine. Circular dichroism and thermal denaturation spectra in connection with thrombin time assay indicate that the introduction of UNA-isoguanine or isoguanosine into TBA negatively affects G-quadruplex folding and TBA anticoagulant properties. These findings demonstrate that the highly-ordered structure of TBA is essential for inhibition of thrombin activity. Anticoagulants belong to a class of compounds that influence blood coagulation by prolonging clotting time. From the medical point of view, anticoagulants prevent the formation of blood clots or inhibit the growth of formed clots, thus decreasing the risk of emboli formation in vessels of organs ,2. Anticvia two shorter TT loops, thus inhibiting enzyme activity [Anticoagulants may reduce the activity of clotting factors directly or indirectly. One of the direct thrombin inhibitors is thrombin binding aptamer (TBA), a DNA 15-mer . This olactivity ,7. Previactivity –10.via three hydrogen bonds with the strength comparable to regular G-C interactions [Isoguanosine (iG) is an unnatural nucleoside that pairs with isocytidine (iC) ractions . Interesractions ,13.4-Thiouridine (s4U) is a photoreactive uridine analogue that was found to naturally occur in bacterial and archaeal tRNAs and play a role in protecting cells from near-UV light . Moreove7 of TBA increase thermodynamic stability of G-quadruplex and improve anticoagulant potential of the aptamer [3, T7, T9 and T13 in TBA was found to inhibit thrombin-catalyzed fibrin clotting 2-fold compared to unmodified TBA [et al. performed screening of the oligonucleotide library containing all possible substitutions of 2′-deoxy-isoguanosine and indicated that its presence at positions G1, G8 or G10 in TBA improves the aptamer-thrombin interaction [It was found that certain UNA residues at position T aptamer . Similar5′GGTTGGTGTGGTTGG3ʹ) biological activity. Recently, we reported anticoagulant and antiproliferative properties of modified TBA variants containing a 4-thiouracil derivative of UNA (UNA-s4U) [U) a novel UNA derivative (R or iGR) or UNA (s4UU or iGU) series.Stimulated by the previously published results, we decided to identify potential of novel chemical modifications that alter both sugar and heterocyclic base moieties known to be favorable for TBA (UNA-s4U) . Herein,rivative , its inc1) was treated with lithium nitrite and acetic acid to yield isoguanosine (2). Treatment of compound (2) with N,N-dimethylformamide dimethyl acetal resulted in the N6 protected derivative (3) which was converted with dimethoxytrityl chloride into 5′-O-4,4′-dimethoxytrityl derivative (4). Compound (4) was treated with sodium periodate followed by sodium borohydride to produce UNA derivative (5). Selective benzoylation of (5) at O2′ was achieved by addition of benzoyl chloride at decreased temperature accordingly to the previously published protocol [6) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite gave the final UNA-isoguanine in overall yield ca. 17%. Protocol for the synthesis of UNA-isoguanine phosphoramidite is described in 1H and 13C NMR spectra were recorded on a 500 MHz (11.74 T) AVANCE III Bruker spectrometer. Thin layer chromatography was conducted on Merck silica gel 60 F254 glass plates. Silica gel column chromatography was performed using Merck TLC gel H 60.The synthesis of protected UNA-isoguanine phosphoramidite  was perfprotocol . TreatmeAll oligonucleotides were synthesized on an automated RNA/DNA synthesizer using standard phosphoramidite chemistry . The dep2EDTA, pH 7.0. Single-stranded oligonucleotide concentrations were calculated based on the absorbance measured above 80°C [-6 M. Absorbance versus temperature curves were obtained using the UV melting method at 295 nm in the temperature range of 4–90°C with a heating rate of 0.2°C/min on a JASCO V-650 spectrophotometer equipped with a thermoprogrammer. Melting curves were analyzed using the MeltWin 3.5 software by van’t Hoff analysis [i.e. plots of 1/Tm vs lnCT were fitted to the following equation:T is the total oligonucleotide strand concentration and R is the gas constant. Both methods assume a two-state model. Agreement within 15% of ΔH° obtained from both methods was indicative of the two-state approximation.Oligonucleotides were dissolved in a buffer containing 100 mM potassium chloride, 20 mM sodium cacodylate and 0.5 mM Naove 80°C  and the analysis . The the−4 M concentration of oligonucleotide are denoted by TM, and melting points for any other concentration of oligonucleotide are denoted by Tm.Melting temperatures calculated for a 102EDTA, pH 7.0, to achieve a sample concentration of 3.0 μM. All samples were denatured at 95°C for 5 min and then slowly cooled to room temperature overnight prior to data collection. The spectra were recorded in triplicate at 37°C in the 205–320 nm wavelength range. The buffer spectrum was subtracted from the sample spectra. The data analysis was performed using the Origin 8.0 software.CD spectra were recorded on a JASCO J-815 spectropolarimeter. The oligonucleotides were dissolved in a buffer containing 100 mM potassium chloride, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7.0, to achieve a sample concentration of 0.1 μM. Absorbance spectra were recorded in triplicate at 4°C and 90°C in the 220–335 nm wavelength range. The scan speed was 1000 nm/min with a 1 nm data interval. Thermal difference spectra were obtained by subtraction of the low temperature absorbance spectra from the high temperature absorbance spectra using the Origin 8.0 software. The differential spectra were normalized by dividing the data by its maximum value [The TDS measurements were performed on a JASCO V-650 spectrophotometer equipped with a thermoprogrammer. The oligonucleotides were dissolved in a buffer containing 100 mM potassium chloride, 20 mM sodium cacodylate and 0.5 mM Naum value .The thrombin time assay was performed using commercially available Dia-TT kit (DIAGON®) and coagulometer K-3002 Optic. Each reaction mixture consisted of TBA variant (0.33 μM) dissolved in 100 μl Dia-TT reagent. The reaction mixture was incubated at 37°C for 5 min and put into sample well of coagulometer K-3002 Optic. Next, the 100 μl of citrate plasma was added. Each TBA variant was analyzed using plasma samples derived from five healthy volunteers. The anticoagulant effect was obtained by subtraction of time needed for plasma clotting in the presence of the aptamers from time needed for clotting of poor plasma.’1, G8 and G10 were selected according to a previous report indicating these positions as the most favorable for anticoagulant properties of TBA modified with 2′-deoxy-isoguanosine [TBA variants containing single and multiple substitution of modified residues were synthesized and analyzed to determine the influence of RNA-iG or UNA-iG on the G-quadruplex thermodynamic stability, folding topology and biological properties. The positions of modification G1 and G10, involved in G-quartet formation, significantly destabilize G-quadruplex structure (1) by UNA-iG or RNA-iG decreases G-quadruplex thermodynamic stability in a comparable manner . In contrast, the presence of UNA-iG within internal position G10 of the G-quartet destabilizes G-quadruplex more than RNA-iG . Despite previous reports suggesting that mixed G-quartets containing both isoguanosine and guanosine can be formed [anti-conformation of N-glyosidic bond relative to syn-oriented 2′-deoxyguanosines at G1 and G10. Nevertheless, in the future further structural studies are needed to confirm the above theory.Thermodynamic analysis showed that the presence of UNA-iG or RNA-iG at positions Gtructure . Modifice formed  in this 8 by UNA-iG or RNA-iG also destabilizes G-quadruplex structure, but the effect is reduced in comparison to positions G1 and G10 that are involved in G-quartet formation. The presence of UNA-iG at G8 decreases G-quadruplex thermodynamic stability by 1.95 kcal/mol, whereas RNA-iG causes destabilization mounting to 1.23 kcal/mol. According to some structural analysis of unmodified TBA, the carbonyl group of 2′-deoxyguanosine at G8 may form a hydrogen bond with the 2′-deoxyguanosine at G-quartet [8 might result from H-bond disruption between the nucleobase at G8 and the G-quadruplex core. This is most likely due to opposite carbonyl and amino group arrangement in isoguanosine relative to guanosine. Notably, UNA-iG at position G8 decreases TBA thermodynamic stability more than RNA-iG at the same position. The increased flexibility of UNA-iG at G8 compared to RNA-iG might additionally hinder a more favorable nucleobase orientation contributing to a higher destabilization for the UNA than RNA modification. A similar trend was observed for G10 but not for the G1 position. This is probably due to the internal (G8 and G10) vs. terminal (G1) location, which is less and more tolerant towards structural changes, respectively [The substitution of TBA loop at position G-quartet –28. Therectively ,30.1, G8 and G10 positions caused significant destabilization that prevented thermodynamic analysis of most multi-modified G-quadruplex variants . Unfortunately, we were unable to analyze the thermodynamics of TBA variants with substitutions at positions T13 and G1 by UNA-s4U and UNA-iG or RNA-s4U and RNA-iG due to extremely large destabilization.The presence of RNA-s4U at positions T G1, T12 . On the 8 by UNA-iG or RNA-iG and T3, T7, T9 or T12 by UNA-s4U or RNA-s4U usually caused an increase in G-quadruplex thermodynamic stability compared to TBA modified with single UNA-iG or RNA-iG at G8 . Interestingly, introduction of UNA-s4U at position T7, T9 or T13 and UNA-iG at G10 changed intramolecular to intermolecular folding of TBA  are often used to supplement circular dichroism studies. This is a fast and cost-effective method to analyze differences in UV spectra. Absorbance values of nucleic acid structures below and above the melting point correspond to folded and unfolded nucleic acid state, respectively . The subThrombin time assay is a commonly known test to diagnose blood coagulation disorders but it is also routinely used for analysis of anticoagulant potential of different compounds. Thrombin time (TT) is the time needed to form a clot within plasma samples after adding an excess of thrombin. The differences in thrombin time of plasma with and without anticoagulant can be named anticoagulation effect (AE). Despite the fact that previous report indicated that the presence of 2′-deoxy-isoguanosine improves TBA-thrombin interactions , our stu1, G8 or G10 significantly decreases thermodynamic stability of G-quadruplex structure. CD and TDS analyses indicate that modified monomer substitution is unfavorable for G-quadruplex folding. These substitutions promoted a significant decrease in the anticoagulant properties of TBA variants compared to unmodified TBA. This study enhances our understanding of how UNA-iG and RNA-iG affect the thermodynamics and biological function of TBA. This study also shows influence of novel isoguanine derivative of UNA on the thermodynamic stability and folding topology of G-quadruplexes in general.The studies described herein, clearly demonstrate that the presence of isoguanosine in both UNA and RNA series is detrimental for G-quadruplex formation and biological function of TBA. Substitution at positions GS1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The MAM serves as bridges between ER and mitochondria regulating multiple functions such as Ca2+ transfer, energy exchange, lipid synthesis and transports, and protein folding that are pivotal to cell survival and defense. Recently, emerging evidences indicate that the MAM is critical in maintaining neuronal homeostasis. Thus, given the specific localization of the Sig-1R at the MAM, we highlight and propose that the direct or indirect regulations of the Sig-1R on mitochondrial functions may relate to neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). In addition, the promising use of Sig-1R ligands to rescue mitochondrial dysfunction-induced neurodegeneration is addressed.The sigma-1 receptor (Sig-1R) is a chaperone that resides mainly at the mitochondrion-associated endoplasmic reticulum (ER) membrane  and acts as a dynamic pluripotent modulator in living systems. At the MAM, the Sig-1R is known to play a role in regulating the Ca Emerginulations –105. Def2O2  and decreased cytochrome c oxidase activities in an animal model [2+ caused by Aβ25–35 through the activation of Sig-1R. Afobazole reduced NO production, prevented upregulation of the proapoptotic protein Bax, activated caspase-3, and inhibited the downregulation of Bcl-2 induced by Aβ25–35 [Swe/Lon mouse brain prior to the plaque formations, while decreased Sig-1R protein levels were observed in the human cortical postmortem brain tissue [2+ shuttling from ER into mitochondria. Aβ also increased the expression of MAM-associated proteins such as IP3R3 and increased ER-mitochondria contacts in hippocampal neurons. Similar results were found in PET scan studies, in which Sig-1R expressions were lower in the brain of early AD patients [2+ overload in the mitochondria [SIGMAR1 and APOE may interact to influence the severity of AD [2+ concentrations between ER and mitochondria [2+ channels, IP3Rs and Ca2+-dependent mitochondrial transport proteins were reduced as well. Overexpression of this mutant altered the ER-mitochondrion tethering and associated transport in the neuronal cell [The major symptoms of AD include selective cognitive decline and memory loss, which are now accepted as being caused by the Aβ plaques and the tau neurofibrillary tangles. Aβ is generated from the serial enzymatic digestion of amyloid precursor protein (APP) which has been found to accumulate in the mitochondrial import channel in AD brains . Aβ alsoal model . Aβ affeal model , 110. Real model . Afobazo Aβ25–35 . Up-regun tissue . The Sigpatients . On the patients . Proteinpatients . In a brchondria . Brain vty of AD . Furtherty of AD , suggestty of AD . Increasty of AD . Overexpchondria . In the nal cell . Tau pronal cell , 125. HoParkinson’s disease is a slowly progressing disorder, causing impaired motor functions such as bradykinesia or tremor, and other non-motor complications. The pathological characteristic of PD is the deposit of Lewy bodies composed of α-synuclein, ubiquitin and neurofilaments . α-Synuc2+, nitric oxide (NO) and ROS concentrations [2+ homeostasis by enhancing the ER-mitochondria associations [2+ transferring between the two organelles, and the inhibition of ATP production. Neither expression of WT/mutant nor silencing of α-synuclein changed the protein expression of Sig-1R, indicating that α-synuclein may not influence the translational level of Sig-1R [Sig-1R expressions were lower in putamen of PD patients as demonstrated by PET studies . Dopamintrations , 128. Mociations  and was ciations . Intriguciations . Vesiclef Sig-1R . Howeverf Sig-1R , 129–131f Sig-1R , and itsf Sig-1R . Parkin f Sig-1R , 142. A HTT, and is clinically characterized by progressive retardation in motor, cognition and psychiatric states [HD is an inherited disorder in an autosomal dominant pattern due to an elongated CAG repeat in the Huntingtin (Htt) gene, c states . HD mutac states . Mitochoc states . Overexpc states . Furtherc states .κB-p65, and activating NF-κB signaling without changing mitochondrial Ca2+ concentration. A partial co-localization of Sig-1R with aggregates of cytoplasmic mutant Htt was observed, indicating that the Sig-1R may play some unknown roles in the Htt aggregates such as being hijacked by the aggregates with a loss of its function. Similar results were observed in that Sig-1Rs translocated and colocalized with the mutant Htt in the nucleus [2+ levels were not affected by mutant Htt proteins in this model, another report indicated that the interaction of type I IP3R with BiP was reduced in the HD mouse model that was accompany by impaired Ca2+ releasing activity of type I IP3R [2+ levels in medium spiny neurons in the co-culture system [Expression of N-terminal Htt proteins with expanded polyglutamine activates ER stress, increases BiP protein expression, and causes cell death in neuronal cells. Compound that inhibits ER stress such as salubrinal could rescue the cell death and eliminate protein aggregations resulting from mutant Htt proteins . A simil nucleus . Althouge I IP3R . Moreovee I IP3R . Pridopie I IP3R . Furthere system . Althoug2+ concentrations and increased mitochondrial volumes [The clinical hallmark of ALS is the presence of upper and lower motor neuron dysfunction as seen in the limbs that can further manifested as muscular atrophy in other regions . Mitocho volumes . Several volumes –159, FUS volumes , TDP-43  volumes , OPTN [1 volumes  and C9Or volumes . SOD1 sc volumes . SOD1 mu volumes . Motor n2+ scavenging and ER stress inhibition in motor neurons [SIGMAR1 was identified in the inherited juvenile ALS. The mutant variant of Sig-1R showed reduced stability and was incapable of binding to IP3R3s. The mutant SOD1 was also detected at the MAM where the mutant was observed in neurons but not in astrocytes or other cell types of the SOD1 mouse model. Furthermore, deficiency of Sig-1Rs accelerated the onset of SOD-1-mediated ALS in mouse model. Deficiency of Sig-1R or accumulation of mutant SOD1 could induce the collapse of the MAM, leading to the mislocalization of IP3R3s, the activation of calpain, and the dysfunction of mitochondria. Administration of the Sig-1R agonist PRE-084 restored the Sig-1R-IP3R3 interaction and prevented the Sig-1R aggregation [SIGMAR1 was identified in patients from the frontotemporal lobar degeneration-motor neuron disease (FTLD-MND) pedigree [SIGMAR1 mutation carriers showed cytoplasmic inclusions of TDP-43 or FUS. Overexpression of Sig-1R increased the mislocalization of TDP-43 and FUS from the nucleus to the cytoplasm while Sig-1R antagonists reduced the cytoplasmic to nuclear TDP-43 ratio. The mutation of the SIGMAR1 (p.E102Q) has also been found in the ALS patients [Sig-1R proteins were reduced in the lumbar spinal cord of ALS. They were also accumulated in enlarged C-terminals and ER structures of alpha motor neurons. The disrupted Sig-1R localization was also observed in SOD1 transgenic mice . A Sig-1 neurons . The col neurons . Watanabregation . TDP-43 regation , and theregation . Moreoveregation . The asspedigree . Brains patients . Overexppatients , 171. Thpatients . Those f2+ concentration and signaling [2+ signaling increased from the ER into mitochondria as well as the ATP production while higher concentrations of DMT induced the translocation of Sig-1Rs from the MAM to other cellular compartments, and inhibited ion channel’s activities [In addition to the synthetic agonists and antagonists listed above, the endogenous ligands of Sig-1Rs include the steroids  , 173, haignaling . DMT is ignaling . Mice inignaling , indicattivities . DMT protivities . Later stivities , 187. Sitivities  which retivities , 189. Id2+ gradient between ER and mitochondria through the MAM. Recently, the crystal structure of Sig-1R proposing a trimeric architecture with a single transmembrane domain in each protomer, with one side facing the ER lumen and the other side facing the surface of the ER in cells [The function of Sig-1R is activated when cells are under stress. The Sig-1R chaperone protein exerts pluripotent properties that can exist in the nuclear envelope, the nucleoplasmic reticulum, the MAM, the ER, and potentially the plasma membrane . The maiin cells . This diin cells . The reg


The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher’s exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation. South Africa is experiencing one of the world’s most devastating HIV-1 epidemics, with an estimated 18.9% of the adult population infected with HIV-1 at the end of 2016 . While tMost HIV-1 drug resistance studies have focused on HIV-1 subtype B, the dominant subtype in developed countries ,5, but aData indicating whether similar mutations develop under NRTI drug selection pressure in the RNase H domain of HIV-1 non-B subtype viruses is limited ,8. GivenHIV-1 subtype C sequences spanning the entire RNase H region (RT codons 431–560) were obtained from 112 ART-experienced and 28 ART-naive patients enrolled in the South African Resistance Cohort Study (SARCS), McCord Hospital, and the Paediatric Tropism Study, King Edward VIII Hospital, Durban, South Africa, respectively. The SARCS study was a prospective study assessing the prevalence of drug resistance after virologic failure in patients on combined antiretroviral therapy (cART) between January 2005 and August 2006  and the http://www.phrap.org/phredphrapconsed.html) and aligned against HXB2 reference strain [http://www.bioafrica.net/GDElinux/GDEmicrobial). HIV-1 subtype was determined by the REGA HIV-1 subtyping tool version 3 [Viral RNA was isolated from 140 µL of plasma using the QIAamp RNA kit  according to the manufacturer’s instructions. A 469 base pair product was amplified by nested PCR, using primers and PCR conditions as previously described . PCR proe strain . The aliersion 3 ,18. A maersion 3 . In orden = 359) to minimize the epidemiological bias. In addition, a control dataset of subtype B sequences from ART-naive patients (n = 1782) spanning the RNase H domain were obtained from the Los Alamos database. Major NRTI resistance mutations were defined according to the Stanford HIV-1 drug resistance database [p-Values were adjusted using a False discovery rate step-down procedure to reduce false positive results [Treatment-related mutations in RNase H were identified by comparing mutation prevalence between viral sequences from ART-experienced patients and sequences from ART-naive patients. Additional subtype C sequences spanning the RNase H domain were obtained from the Los Alamos database, limiting to ART-naive patients from South Africa  of the ART-naive sequences . The anap < 0.001). In contrast, mutations E432D, A446S and P537Q occurred at a lower frequency in the treatment-experienced versus naive sequences.We searched for treatment-associated mutations by comparing the prevalence of amino acids at each position between sequences from 387 ART-naive and 112 NRTI-experienced patients. The most frequently observed reverse transcriptase (RT) drug resistance mutations in NRTI-experienced patients were M184I/V (64.2%), D67N (25.9%), K70R (19.6%), K219Q/E (17.9%) and T215Y/F (13.4%). Other known NRTI-elicited mutations were less prevalent (<1%). A total of 9 mutations in RNase H were found positively selected in ART-experienced patients compared to ART-naive patients, these included respectively A435L (5.4% vs. 1.0%), S468A (5.4% vs. 0.5%), T470S (39.3% vs. 26.1%), L484I (11.6% vs. 3.1%), A508S (6.3% vs. 1.0%), Q509L (4.5% vs. 0.7%), L517I (14.3% vs. 4.6%), Q524E (29% vs. 15.5%) and E529D (20.5% vs. 2.3%) . Only E5We explored associations between the presence of natural polymorphisms, the emergence of mutations and NRTI treatment experience using Bayesian network learning. The role and effects of mutations in the RNase H domain in contributing to NRTI failure have not been fully elucidated, particularly in non-B subtypes, with most available data describing subtype B sequences from developed countries. Here, treatment-related mutations were identified by comparing mutation frequencies in subtype C NRTI treatment-experienced sequences to those in subtype C ART naive sequences. Our findings indicate that several previously identified NRTI drug resistance mutations  of subtype B ,12 repreAs expected, most of the amino acids in the active site and primer grip site were conserved, as mutations in these regions impact on the functioning of RNase H ,9. PreviNone of the resistance-associated mutations in RNase H  identified for subtype B ,13. In cResistance pathways to NRTI treatment were further investigated using Bayesian Network Learning. The presence of RNase H mutation E529D was directly dependent on whether or not the patient received an NRTI, supporting its role in the development of NRTI resistance. Recent work by Barral et al. (2016) also observed a prevalence increase of E529D following treatment administration in subtype C patients in Brazil , furtherThis study identified differences in the mutational response of RT to NRTI treatment between subtype B and South African subtype C, with novel mutation E529D, in particular, strongly associated with treatment experience. Results obtained from this study strongly support the need for additional research on the role of the RNase H region in the development of HIV-1 drug resistance, using larger association studies and drug susceptibility assays. Findings from subtype B studies should be re-evaluated to exclude a confounding effect of epidemiology on the identification of mutations.