Porcine enterovirus G (EV-G) is a member of the family Picornavirdae, genus Enterovirus. To date, eleven EV-G types (EV-G1 through EV-G11) have been identified in pigs from Asia and Europe however they have never been reported in North America. In this study, we isolated and characterized the complete genome of NP/2013/USA, an EV-G from a porcine diarrhea sample from the United States. The complete genome consists of 7,390 nucleotides excluding the 3′ poly(A) tail, and has an open reading frame that encodes a 2,169 amino acid polyprotein. NP/2013/USA was most similar at the nucleotide (84%) and amino acid (95%) level to the HM131607, an EV-G1 type isolated from China in 2012. Picornaviridae, genus Enterovirus and were originally divided into 13 serotypes (PEV-1 to -13) Teschovirus, Sapelovirus, and Enterovirus with porcine species porcine teschovirus (PTV), porcine sapelovirus (PSV) and porcine enterovirus B (PEV-B) Porcine enteroviruses (PEV) are members of the family PEV infections are generally asymptomatic. However, occasionally PTV and PSV have been associated with a wide variety of clinical conditions such as polioencephalomyelitis, enteric disease, and pneumonia EV-Gs are small, non-enveloped viruses with a positive-sense single stranded RNA genome. The genome is approximately 7400–7500 nucleotides in length. It has one open reading frame (ORF), which encodes the viral polyprotein and is flanked by 5′ and 3′ untranslated regions (UTR). The 5′-UTR vary in length between 700 and 825 nucleotides while the 3′-UTR is considerably shorter To date there are only seven whole genomes sequences of EV-G from samples collected from Asia and Europe. EV-G has never been reported in the United States EV-G used in this study was isolated from a sample submitted to Newport Laboratories for routine diagnostic testing. The sample was obtained from a naturally infected animal in the field, by qualified veterinarians, as a part of normal veterinary care and diagnostic testing procedures.EV-G was isolated from intestinal homogenate submitted to Newport Laboratories in August, 2013, from commercial 4-week old pig with diarrhea for diagnostic testing.2 incubator and inspected daily for cytopathic effect (CPE). If CPE was noted, media was collected and analyzed for common enteric viruses  by quantitative reverse-transcription polymerase chain reaction (RT-PCR) (data not shown).Rhesus monkey kidney cells (Marc-145) (ATCC CRL-12219) were used for virus isolation. The Marc-145 cells were propagated in DMEM  with either plasmocin (25 mg/l)  or normocin (100 mg/l) (Invivogen). Confluent cells were washed 2–3 times with replacement media (DMEM (Pellgro) with trypsin (EDTA) at 5 µg/mL and either plasmocin (25 mg/l)  or normocin (100 mg/l) (Invivogen). The diagnostic sample, intestinal homogenate, was centrifuged at 5000×g for 10 min to remove debris. Supernatant was used to inoculate confluent, washed Marc-145 cells. The cultures were incubated at 37°C in a CORNA was extracted from Marc-145 cells with 100% CPE. 1 ml of cell culture supernatant was centrifuged at 1500×g for 10 minutes. RNA was extracted from 140 µl of the supernatant using a QIAamp Viral RNA kit  according to manufacturer’s instructions. RNA was eluted in 35 µl of the supplied elution buffer.5′-TTGAGGATCCTCCGGCCCCTGAATGCG-3′) and 37-R primers (5′-ATCTAAGCTTGTCACCATAAGCAGCCA-3′) were used for EV-G. 1222-F (5′-GTGGCGACAGGGTACAGAAGAG-3′) and 1223-R (5′-GGCCAGCCGCGACCCTGTCAG-3′) primers were used for PTV and PSV. Amplified products were visualized after separation on 1.5 % agarose gels (TBE) containing ethidium bromide.PTV, PSV and EV-G were detected by one step RT-PCR reactions using primers sets published in 2O.Marc-145 cells that showed 100% CPE following virus infection were used for RNA extraction. 20 ml of cell culture supernatant was filtered using the 0.2 µm bottle top filters . The filtrate was centrifuged at 50,000×g for 2 hrs. Supernatant was discarded and the pellet was suspended in 1000 µl of water. Samples were concentrated to a final 100 µl volume using Amicon ultra centrifugal filters  . Cellular DNA and RNA were removed by incubation with DNase I (25 units)  and RNase A (25 units)  at 37°C for 1 hr. RNA was extracted using Trizol LS Reagent  according to manufacturer’s instructions. The pellet containing RNA was resuspended in 20 µl of sterile H10 µg of total RNA was depleted of ribosomal RNA using GeneRead rRNA depletion kit (Qiagen) and RNA sequencing libraries were generated using the Ion Total RNA-seq kit v2  according to manufacturer’s instructions. Sequencing was carried out using Ion Personal Genome Machine (PGM) sequencing platform  as previously described Newport Laboratories received an intestinal homogenate sample from a pig with diarrhea for diagnostic testing. Samples were positive for group A rotavirus (Ct = 33) and negative for groups B and C rotavirus by one step RT-PCR (data not shown). Subsequently, isolation of group A rotavirus was attempted on MARC-145, however, was unsuccessful as determined by one step RT-PCR despite CPE suggestive of virus propagation. Samples were next tested for PTV, Sapelovirus (PEV-8), and EV-G by one step RT-PCR (data not shown). The samples were positive only for EV-G. Further deep sequencing identified viral reads mapping soley to EV-G.The NP/2013/USA genome was sequenced with an Ion Torrent Personal Genome Machine. The complete genome contained 7,390 nucleotides (nt) excluding the 3′ poly(A) tail. The 5′-UTR consisted of 811 nucleotides and the 3′-UTR was comprised of 72 nucleotides. A single large open reading frame was identified from nucleotides 812 to 7318 and encoded a hypothetical 2,169 amino acid polyprotein. The genome had 28.3% A, 24.9% T, 24% G, and 22.9% C which is similar to the other EV-G isolates. The genome organization of NP/2013/USA was identical to the CPE group III strains, now classified as EV-G To elucidate the genetic relatedness of NP/2013/USA with PTV, PSV, and EV-G types, a phylogenetic tree was constructed using the maximum likelihood algorithm and bootstrapped using 1000 replicates using the complete genome sequence. NP/2013/USA segregated into the same clade as other EV-G and was clearly distinct from PTV and PSV . NP/2013NP/2013/USA showed overall nucleotide similarities of 84, 81, 80, 80, 75, 77, and 77% respectively to HM131607 (EV-G1), JQ818253 (EV-G6), AF363453 (EV-G1), Y14459 (EV-G1), AF363455 (EV-G2), HQ702854 (EV-G3), and JN807387 (EV-G4) strains respectively , and 95,http://www.picornaviridae.com). The high amino acid homology of NP/2013/USA to EV-G1 suggests that it should be classified as type G1 , JQ818253 (EV-G6), AF363453 (EV-G1), Y14459 (EV-G1), AF363455 (EV-G2), HQ702854 (EV-G3), and JN807387 (EV-G4) strains respectively , and VP1 type G1 .Enteroviruses utilize recombination as a mechanism for evolution, with genetic diversification principally in genes encoding structural proteins EV-G infection was detected at higher frequencies in younger pigs than in adults from Vietnam, Czech Republic, China, Hungary, Italy, and Spain In conclusion, the present study was the first to identify EV-G in pigs in the United States. NP/2013/USA’s nucleotide and protein sequence was most homologous to HM131607, an EV-G1 isolate from China in 2012. It is unclear whether our finding of EV-G in U.S. pigs represents a recent introduction of this virus to the U.S. or whether EV-G has circulated in U.S. herds undetected for some time. Previous work has failed to find EV-G in U.S. pigs Disclaimer: This document is provided for scientific purposes only. Any reference to a brand or trademark herein is for informational purposes only and is not intended for a commercial purpose or to dilute the rights of the respective owner(s) of the brand(s) or trademark(s).


N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is  Plants may be a viable alternative to producing complex recombinant proteins over conventional cell culture-based systems with the potential for providing both cost effectiveness and scalability1246An ultimate goal of plant-made biopharmaceuticals research is to produce proteins at high levels. This is often achieved by targeting proteins through the secretory pathway81011Nicotiana species8Until recently, little was known about ER stress, UPR and ERAD in plants. Recent reviews summarized two UPR arms in plants: one induces activation of two membrane-associated transcription factors (basic-region leucine zipper [bZIP]17 and bZIP28) and the other involves the RNA-splicing factor IRE1 and its target RNA (bZIP60)13Nicotiana benthamiana has become one of the most widely used hosts due to its capacity to support various transient overexpression systems based on viral and non-viral vectors11617N. benthamiana could be of significant importance in plant-made biopharmaceuticals production systems.Among different plant species used for recombinant protein production, Agrobacterium-mediated delivery of a tobamovirus replicon vector (the magnICON system) in N. benthamiana. One of our primary objectives was to investigate the role of N-glycosylation in ER stress upon transient CTB production in N. benthamiana. Other reports on CTB production in transgenic plants such as potato202324N. benthamiana is N-glycosylated at Asn426N. benthamiana using the magnICON system. While N4S-CTB-KDEL accumulated at a high level and retained molecular integrity and oral immunogenicityN-glycosylated, non-ER-retained CTB (gCTB) did not cause such tissue damage and provided an exceptionally high yield, significantly greater than that of N4S-CTB-KDEL. These intriguing findings prompted us to investigate various stress markers in N. benthamiana overexpressing gCTB and N4S-CTB. Given the outstanding producibility, we purified and characterized gCTB using biochemical, biophysical and immunological experimentation towards possible vaccine development. These studies suggest gCTB as a potential alternative to the bacterial CTB used in an internationally licensed oral cholera vaccine. Moreover, the data reported herein contribute to our understanding of the stress response caused by transient overproduction of foreign proteins in N. benthamiana, and also indicate potential utilities of N-glycosylation for the development of plant-made glycoprotein pharmaceuticals.In this report, we studied the production of a potent oral immunogen, cholera toxin B subunit (CTB) using N. benthamiana leaf material using Agrobacterium-mediated delivery (agroinfiltration) of the magnICON tobamovirus replicon vectora priori expectation of obtaining a high production yield. To revisit the role of ER retention in CTB biosynthesis and accumulation in planta, we performed site-directed mutagenesis to remove the codons coding for the C-terminal KDEL extension. The resulting variant, N4S-CTB, was expressed in N. benthamiana using the magnICON vector. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-denaturing conditions of crude leaf extracts, 5 days post vector inoculation (dpi), revealed no visible amount of N4S-CTB as compared to N4S-CTB-KDEL, which showed a clear band at around 60 kDa corresponding to the GM1-ganglioside receptor binding, pentameric form . We hypothesized that N-glycosylation might facilitate folding and assembly of CTB via the help of lectin chaperones such as calnexin and calreticulin1112N-Glycosylation of the protein was evident from the increased size compared with N4S-CTB-KDEL on an SDS-PAGE gel , which lost an N-glycosylation site at Asn4 but gained one at Asn21 (N4S-CTB-K23T); and another Asn4→Ser mutant, which gained an N-glycosylation site at the C-terminal Asn106 by adding a C-terminal extension of a Val-Thr-Lys-Ala-Leu-Leu sequence following the Asn residue . These proteins were expressed in N. benthamiana leaves using the magnICON vector. We found that both N4S-CTB variants accumulated at relatively high levels at 5 dpi, with 1.0 g/kg for the former and 1.19 g/kg for the latter variants . Accumulation levels of GM1-ganglioside-binding N4S-CTB and gCTB in leaf tissue at 3 dpi were 0.067 and 0.38 g/kg, respectively. Hence, poor accumulation of N4S-CTB had already taken place at an early stage of protein expression, which appeared to be accompanied by tissue damage. These findings suggested that inefficient folding and/or assembly of the non-glycosylated protein might have caused plant stress. Thus, we measured the expression of representative stress markers, i.e., PDI, BiP, bZIP60, SKP1, 26S proteasome and pathogenesis-related protein 1a (PR1a), by reverse-transcription quantitative real time (RT-q)PCR in leaf tissues-expressing N4S-CTB and gCTB at 2 dpi. The results demonstrated that N4S-CTB-expressing plants displayed a statistically significant increase in transcript levels of PDI, BiP, bZIP60, SKP1 and PR1a compared with control plants infected with empty vector , whereas gCTB-expressing plants showed no increase in expression of these genes , suggesting the potential contribution of HR along with ER stress to the tissue damage induced by the non-glycosylated protein expression.Unlike other genes examined, the expression of N. benthamiana stimulated our interest in investigating whether the protein may be developed as a novel vaccine antigen. Thus, we purified gCTB to characterize its molecular properties relevant for vaccine use. We employed the extraction and purification protocols previously developed for N4S-CTB-KDELThe finding that GM1-ganglioside receptor-bindable gCTB was produced at remarkably high levels in Vibrio cholerae-derived commercial CTB ; CTB and gCTB showed 50% inhibitory concentrations (IC50) of 1.8 and 2.4 nM, respectively. Surface plasmon resonance was performed to determine the equilibrium dissociation constants (KD) of these molecules .First, we investigated the GM1-ganglioside binding affinity of gCTB because it is the most essential attribute for vaccine efficacy. A competitive GM1-ELISA  revealedolecules . EmployiTm) to assess potential structural instability associated with N-glycosylation of CTB. Using differential scanning fluorimetry (DSF), CTB and gCTB showed Tm values of 73.93 ± 0.23 and 70.47 ± 0.31°C, respectively  and mass spectrometry (MS). Eighteen distinctive fractions of 2-aminopyridine (PA) derivatives were identified in reversed-phase (RP)-HPLC -linked Xyl, one or two terminal N-acetylglucosamine (GlcNAc) and/or a terminal β-linked galactose -HPLC , each ofalactose . This glV. cholerae-derived CTB was used as a control in these experiments, where 3 µg of gCTB or CTB were orally administered to mice followed by an oral boost using the same dosage 2 weeks after the initial immunization, following a standard regimen used for the Dukoral® cholera vaccinet-test) serum anti-holotoxin IgG levels, with average titers of 152,500 ± 48,369 and 225,000 ± 67,905, respectively , a vaccine antigen against cholera, accumulated to high levels in e damage , S1. Up e damage , which woduction  and littoduction , it is cN. benthamiana. Given that virus-based transient production of recombinant proteins in N. benthamiana is most progressive, information leading to the mechanism of failed UPR, ERAD and PCD can lead to healthier plant conditions and increase protein accumulation. In RT-qPCR analysis -Xyl not found in mammals, and have α-Fuc attached to the core N-acetylglucosamine (GlcNAc) instead of α-Fuc found in mammals. In addition, β-galactose and α-Fuc are found at the terminal GlcNAc in plants in lieu of β-galactose in mammalsN. benthamiana showed a distinct profile comprised of approximately 40% β-Xyl but only 0.8% Fuc   see . KifunenCTB sequence (GenBank accession no. AY475128) with an 18-nucleotide extension (TCCGAGAAGGATGAACTC) at the 3′ end that encodes the C-terminal SEKDEL sequence27V. cholerae secretory signal from the original CTB gene, using pNM47 as a template. The PCR product was sub-cloned into pICH11599 to generate pNM134. Site directed mutagenesis was performed using the Quikchange II Site-Directed Mutagenesis Kit  according to the manufacturer's instructions, with pNM134 as template and primers that mutated the nucleotide A at position 74 (GenBank accession no. AY475128) to G, creating pNM156 for the Asn4→Ser CTB variant, N4S-CTB-KDELde novo synthesized based on the CTB sequence . The two variants were sub-cloned into pICH11599 to generate pNM307 and pNM326, respectively.The magnICON tobamovirus replicon system54N. benthamiana leaves using the vacuum agroinfiltration methodCTB proteins were expressed with the rice α-amylase signal peptide based on our previous findingsN4S-CTB-KDEL, gCTB, N4S-CTB-K23T, and N4S-CTB-VTKALL were purified from leaf extracts as previously describedLeaf material was extracted in 1x SDS sample buffer with a 2:1 buffer to tissue ratio using a Precellys homogenizer, then centrifuged twice  generating crude leaf extracts. A polystyrene plate was coated with goat anti-CTB antibodies , blocked with PBSTM and crude leaf extracts were added. Ubiquitinated CTB proteins were probed with rabbit anti-ubiquitin antibodies  followed by HRP-conjugated goat anti-rabbit antibodies . Finally, ubiquitinated CTB-antibody complexes were detected using TMB substrate  and reading absorbance at 450 nm.Glycan profiling was performed as previously described26KD of GM1 ganglioside to CTB was measured using a Biacore X100 2.0 instrument, the Tm and acid stability of CTB were determined by differential scanning fluorimetry (DSF), and acid stability was also determined by GM1-ELISA as previously describedCompetitive GM1-ELISA was performed as described previously260/280 and OD260/230 ratios greater than 2. RNA was immediately stored at -80°C and used within 1 month. For reverse transcription, first strand cDNA was synthesized using the RNA PCR Kit (AMV) Version 3.0 kit , step A only. The reaction (10 μl) was performed at 42°C for 1 h using 500 ng RNA, AMV reverse transcriptase and the provided random 9-mers. cDNA was diluted 1:10, stored, in aliquots, at -20°C and used within 2 months. RT-qPCR was performed on an iQ5 Multicolor Real-Time PCR Detection System  using iQ SYBR Green Supermix (Bio-Rad). The provided Bio-Rad iQ protocol was followed for a final reaction volume of 20 µL, including master mix, primers (100 nM) and 5 µL of cDNA. 18S ribosomal RNA5918S was evaluated using two established methods, NormFinder18S, F-Box and PP2A, respectively. BestKeeper analysis is shown in 18S to be the highest ranked reference gene.RNA was extracted from 100 mg of fresh plant leaf material , 48 h post-infection, by disrupting the tissue using liquid nitrogen and a mortar and pestle followed by QIAShredder and RNeasy Mini Kits . The provided protocol for Plants and Fungi was used, utilizing the optional on-column DNase digestion, to eliminate genomic DNA. RNA samples were eluted in 50 µL RNase-free water and nanodropped  to determine concentration. All RNA samples were pure as determined by having ODad libitum. All experimental procedures were approved by the University of Louisville's Institutional Animal Care and Use Committee. Mice were acclimated for approximately one week prior to the initiation of the studies. Mouse immunization, sample collection and quantification of mucosal and systemic Igs were performed as previously describedEight week-old female C57BL/6J mice were purchased from The Jackson Laboratory in Bar Harbor . Four animals were housed in each cage in a temperature- and humidity-controlled room with alternating light/dark cycles of 12 h, with access to Laboratory Autoclavable Rodent Diet 5010  and water t-test unless otherwise stated, using the GraphPad Prism 5.0 software. Differences were considered statistically significant if P < 0.05.Statistical significance was analyzed by one-way ANOVA with Bonferroni's multiple comparison test or student's N.M. and K.T.H. conceived of and designed the study. K.T.H., J.C.K., J.M.J., B.N., L.J.M., A.S.H. and H.K. performed experiments. K.T.H., J.C.K., A.S.H., H.K., K.F. and N.M. analyzed data. K.T.H., J.C.K. and N.M. wrote the manuscript. All authors reviewed the manuscript.Supplementary figures and legends


Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis.via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process.When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K in vitro.Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria  Panax, including Panax ginseng, Panax quinquefolium, and Panax notoginseng -), corresponding to a molecular weight of 1108 Daltons. The fragment ions at m/z 945 ([M-H2O-H]-), m/z 783 ([M-2H2O-H]-), and m/z 621 ([M-3H2O-H]-) in the CID spectrum corresponded to loss of one, two, and three glucose molecules from Rb1, respectively.Rb1 was eluted at 33.2 min and showed its pseudo-molecular ion at The retention times and mass spectral profiles of M1 (35.1 min), M3 (38.2 min), and M5 (44.4 min) were identical to those of Rd, F2, and Cpd K, respectively. The retention time and mass spectral profile of M2 (35.6 min) were identical to those of G-XVII, which was formed from Rb1 by hydrolytic removal of one glucose molecule from the C-3 position of Rb1. Thus, M1–M3 and M5 were unambiguously identified as ginsenosides Rd, G-XVII, F2, and Cpd K, respectively.M4 was eluted at 39.7 min. The MS spectral profile of M4 was similar to those of M3 (ginsenoside F2) and ginsenoside Rg3 Table , supportin vitro resulted in the formations of gypenosides as well as metabolites of the Rd pathway.Taken together, Rb1 biotransformation by human gut bacteria in vitro anaerobic incubation system showed good linearity  over the concentration ranges tested . Because the positional isomers Rd and G-XVII showed similar UV responses, as evidenced by the very close constant coefficients   were observed in Rb1 metabolism and each pathway by human gut bacteria , great inter-individual variations were observed in terms of the remaining Rb1 and the amounts of metabolites formed r2 > 0.998, P < 0.001) over the concentration ranges tested  and one C-3 glucose to generate G-XVII (minor); Rd lost another C-20 glucose to generate F2 and then one C-3 glucose to generate Cpd K; G-XVII further lost one C-20 glucose to form F2 (major), one C-3 glucose to generate G-LXXV (minor), or both; both were finally metabolized to Cpd K. However, under acidic conditions, the degradation of Rb1 was initiated by simultaneous loss of two glucose molecules from both the C-3 and C-20 positions to generate F2 (major) or Rg3 (minor). The metabolic pathway of Rb1 in human gut bacteria shown in Figure The via Rb1 → Rd → F2 → Cpd K (Rd pathway) [via Rb1 → G-XVII → G-LXXV/F2 → Cpd K has been reported in rats [Fusobacterium sp. and Bifidobacterium longum sp. [Ginsenosides generally have poor oral bioavailability because of their low membrane permeability, active biliary excretion, biotransformation, and so on . Their dpathway) ,37. Anot in rats , single  in rats -22, and ngum sp. ,39.via both G-XVII → G-LXXV and G-XVII → F2 was also evidenced, for the first time, by the observation that G-XVII produced G-LXXV, F2, and Cpd K, while Rd only generated F2 and Cpd K, in human gut bacteria in vitro.In this study with application of both LC-MS and LC-DAD techniques, G-XVII and three metabolites  were identified from the Rd pathway and G-LXXV was tentatively assigned. The existence of both the Rd and gypenoside pathways of Rb1 metabolism in human gut bacteria was confirmed. The conversion of Rb1 to Cpd K Eubacterium sp., Streptococcus sp., and Bifidobacterium sp., which were more potent in hydrolyzing gentiobiose than sophorose, metabolized Rb1 to Cpd K via Rd rather than G-XVII [Fusobacterium K-60, which hydrolyzed sophorose more potently than gentiobiose, metabolized Rb1 to Cpd K via G-XVII. Bifidobacteria are probiotics and belong to the predominant gut bacteria, while streptococci and fusobacteria are pathogenic bacteria and are in the minority in healthy humans [The gypenoside pathway was demonstrated to be the minor pathway of Rb1 biotransformation in human gut bacteria by comparing the maximum levels of Rd and F2 with those of their respective positional isomers G-XVII and G-LXXV determined from a time-course study. Moreover, the maximum levels of the gypenoside products were reached more rapidly than the products of the Rd pathway, indicating that the gypenoside pathway was a transient intermediate process. In contrast, the conversion rate of Rd to Cpd K was faster than that of G-XVII to Cpd K, as judged by the times required to reach the maximum level of Cpd K when Rd and G-XVII were separately incubated with human gut bacteria. These findings suggested that the removal of the remote glucose molecule at the C-20 position by human gut bacteria was faster than that of the glucose molecule from the C-3 position, while further cleavage of the second glucose from the C-3 position is a much more difficult process, leading to the formation of F2 much more rapidly than the formation of G-LXXV from G-XVII. n G-XVII . Howevery humans . This main vitro [in vivo [In this study, rapid degradation of Rb1 was observed in SGF and its products including F2, Rg3, Cpd K, and Rh2 were detected, consistent with the findings obtained from previous in vitro  and in v[in vivo  studies.Great inter-individual variations in Rb1 metabolism by human gut bacteria were observed with both the gypenoside and Rd pathways owing to the great individual variations in the species and amounts of the gut bacteria. These variations showed no differences between sexes and ages.in vivo in the human body [in vivo.Thus far, only the Rd pathway has been reported man body ,26. The in vitro.Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria S)-protopanaxadiol; G-XVII: Gypenoside XVII; G-LXXV: Gypenoside LXXV; Cpd K: Compound K; HPLC-UV: High-performance liquid chromatography with a UV detector; MS: Mass spectrometry; ESI: Electrospray ionization; BHI: Brain heart infusion; SGF: Simulated gastric fluid; QC: Quality control; DAD: Diode array detector; LC/MSD: Liquid chromatograph/mass selective detector; IS: Internal standard; SD: Standard deviation.DMSO: Dimethyl sulfoxide; PBS: Phosphate-buffered saline; PPD: 20(The authors declare that they have no competing interests.RY conceived the study. JPCL, YTW, and RY designed the study. WIL carried out the ginsenoside Rb1 biotransformation by pooled and individual human gut bacteria. HS carried out the experiments on metabolism by human gut bacteria and acidic stability. JQR collected the human fecal samples and prepared bacterial suspensions. SLL and JPCL performed the statistical analysis. WIL, HS, and RY wrote the manuscript. All authors read and approved the final manuscript.Intraday and interday variability for the assays of ginsenoside Rb1, Rd, F2 and compound Kin incubates with human gut bacteria.Click here for file


Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years. Structure determination of membrane proteins by either nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography is complicated by the need to solubilize membrane proteins in native-like environments Membrane proteins have important biological functions and are the targets of over 50% of all modern medicinal drugs. However, only a small number of membrane protein structures have been solved up to now (Hydrogen/Deuterium (H/D) exchange has long been used to probe protein structures Here we demonstrate that the solvent protection of single residues in liposome-embedded transmembrane proteins can be studied using solution-state NMR spectroscopy. Using a dedicated H/D exchange protocol, the information of the membrane-embedded state is transferred to the denatured state and analyzed using multidimensional NMR. The method is applied to the 349-residue protein Tom40 that forms the protein translocation pore in the outer mitochondrial membrane and has resisted structural analysis for many years neurospora crassa (ncTom40) was expressed, refolded and purified as previously described 15N- and 13C-labelled protein was expressed in minimal medium with 15NH4Cl as nitrogen source and 13C6-D-glucose as carbon source. Amino acid-selective labelled ncTom40 protein was expressed according to a recently published protocol 2O. Back-exchange was monitored in a dissolution buffer containing 4 M guanidinium thiocyanate (GdnSCN), 0.4% formic acid, pD 2.5.Tom40 from 15N-labelled samples  experiments were recorded at 295K and 278K, while 7D HNCO(CA)CBCANH and 5D CBCACONH were measured at 310K  samples . Back-ex13C-13C proton-driven spin diffusion (PDSD) correlation experiments and +15°C for INEPT-type experiments. Initial cross-polarization time for PDSD was set to 600 µs for 1H-13C transfer. 13C-13C mixing was accomplished by PDSD for 15 ms to obtain intra-residual correlations. The 13C-13C INEPT-TOBSY Solid-state NMR experiments were conducted using 4 mm triple-resonance magic-angle spinning (MAS) probeheads at a static magnetic field of 18.8 T and using 8.33 kHz MAS. Sample temperatures were +5°C for 13C/15N-labeled ncTom40 was prepared recombinantly and subjected to refolding in detergent. Circular dichroism spectra of our ncTom40 preparation in decylmaltoside  liposomes. In a two-dimensional  solid-state NMR spectrum of liposome-embedded ncTom40 a large number of cross-peaks was observed  spectra are recorded  the dead time before starting the NMR experiment is zero and (ii) that the intrinsic exchange rates of different residues are identical. To overcome these limitations we further analysed NMR signal intensities during back-exchange in 75% Dn buffer . When amn buffer . In contn buffer . Notably2O buffer, more residues could be analysed when compared to dissolution in 100% D2O  or the presence of chemical exchange CD spectroscopy and solid-state NMR spectroscopy supports a β-barrel structure of ncTom40 . In addiWe demonstrated that the solvent accessibility of single residues in large transmembrane proteins that are embedded in the near native environment of liposomes can be probed using a combination of H/D exchange and solution-state NMR spectroscopy. The method is applicable to highly challenging systems such as Tom40, which has resisted structural analysis by X-ray crystallography and NMR spectroscopy for many years.Figure S11H,15N]-HSQC spectrum of uniformly 15N-labeled ncTom40 reconstituted into lauryldimethylamineoxide.Two-dimensional -HSQC spectrum of uniformly 15N-labeled ncTom40 in dissolution buffer containing 4M GdnSCN, 0.4% formic acid.Two-dimensional -HSQC spectra of ncTom40 with amino acid selective 15N-labeling.[ Shown are the spectra at the end of back-exchange, i.e. time point 15 hr, in dissolution buffer containing 75% D2O. Spectra were recorded at 278K, to slow down back-exchange. In all spectra, the contour level is set to five times the noise level as estimated by Sparky. Variations in overall signal intensity are due to differences in protein concentration.(TIF)Click here for additional data file.Table S115N-labeled samples of ncTom40 and their contribution to the H/D exchange experiments.Amino acid selective (DOC)Click here for additional data file.


Peanut, a high-oil crop with about 50% oil content, is either crushed for oil or used as edible products. Fatty acid composition determines the oil quality which has high relevance to consumer health, flavor, and shelf life of commercial products. In addition to the major fatty acids, oleic acid (C18:1) and linoleic acid (C18:2) accounting for about 80% of peanut oil, the six other fatty acids namely palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0) are accounted for the rest 20%. To determine the genetic basis and to improve further understanding on effect of FAD2 genes on these fatty acids, two recombinant inbred line (RIL) populations namely S-population (high oleic line ‘SunOleic 97R’ × low oleic line ‘NC94022’) and T-population  were developed. Genetic maps with 206 and 378 marker loci for the S- and the T-population, respectively were used for quantitative trait locus (QTL) analysis. As a result, a total of 164 main-effect (M-QTLs) and 27 epistatic (E-QTLs) QTLs associated with the minor fatty acids were identified with 0.16% to 40.56% phenotypic variation explained (PVE). Thirty four major QTLs (>10% of PVE) mapped on five linkage groups and 28 clusters containing more than three QTLs were also identified. These results suggest that the major QTLs with large additive effects would play an important role in controlling composition of these minor fatty acids in addition to the oleic and linoleic acids in peanut oil. The interrelationship among these fatty acids should be considered while breeding for improved peanut genotypes with good oil quality and desired fatty acid composition. Arachis hypogaea L., 2n = 4x = 40) is a major oil crop along with soybean and cotton. China, India, and the United States are the leading producers contributing about 70% of the world peanut crop. Peanut seeds are used for extracting cooking oil by crushing, consumed as fresh/boiled/roasted seeds, confectionary preparations, flour and butter. Peanut seeds contain around 25% protein, 50% oil and many useful secondary plant metabolites such as flavonoid, folic acid, tocopherols and resveratrol [Peanut or groundnut .A monounsaturated fatty acid (MUFA), such as oleic acid (C18:1), is about 47% in normal and up to 80% in high oleic peanut lines and is associated with several human health benefits such as decreasing the risk of cardiovascular disease (CVD) by reducing the levels of serum low-density lipoproteins (LDL) cholesterol and maintaining the levels of high-density lipoproteins (HDL). The MUFA also helps in hampering the development of adrenoleukodystrophy (ALD) and reversing inhibitory effects of insulin production , 4, 5. Ttrans fatty acid in food manufacturing [The oleic acid C18:1) and linoleic acid (C18:2) accounts for about 80% of peanut oil while the minor saturated fatty acids (SFA) such as palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0) account for the remaining 20% fatty acid . Althoug:1 and liacturing . Furtheracturing . Interesacturing .FAD2A and FAD2B [FAD2 genes over SFAs is not known. Nevertheless, three recent studies using germplasm collection as well as biparental populations indicated that there was a positive correlation between oleic acid (C18:1) with gadoleic acid (C20:1) and lignoceric acid (C24:0); and a negative correlation of oleic (C18:1) with stearic acid (C18:0), palmitic acid (C16:0), linoleic acid (C18:2), arachidic acid (C20:0) and behenic acid (C22:0) [Efforts for improving the peanut oil quality have yielded the identification of a high oleic acid peanut lines (F435-2-1 and F435-2-2) comparable to that in olive oil . The hignd FAD2B . However (C22:0) , 16.FAD2 genes revealed that it encodes microsomal oleoyl-PC desaturase or Δ12 fatty acid desaturase [FAD2 genes with peanut oil quality and suggested that FAD2B contributed higher phenotypic variance for oleic (C18:1) and linoleic (C18:2) acids than the FAD2A alleles. However, so far no quantitative trait locus (QTL) study has been undertaken for the minor fatty acids except oleic (C18:1) and linoleic (C18:2) acids in peanuts.In recent years, although the understanding of the fatty acid biosynthetic pathway in crop plants has improved rapidly , 18, butsaturase . Furthersaturase , 22, 23.saturase  reportedFAD2A and FAD2B mutant alleles on these fatty acids.In this study, two segregating populations with two years phenotypic data were used to identify QTLs controlling oil quality traits in peanut. The results obtained could improve our current understanding and provide evidences on the following aspects: (i) correlations among different saturated and unsaturated fatty acids, (ii) QTLs controlling these fatty acids and (iii) identification of consistent QTLs for these fatty acids, and (iv) effect of Phenotyping data for two seasons were generated for all the six SFAs  along with UFAs (oleic and linoleic acid) on all four parents and the complete set of RILs of the S-  and T-poMulti-season phenotypic data together with genotypic data generated on two mapping populations were used for conducting QTL analysis. The QTL analysis using QTLCartographer and QTLNetwork resulted in identification of 164 main effect QTLs (M-QTLs) and 27 epistatic QTLs (E-QTLs) for the six fatty acid traits in both populations Tables  and 4. TOf the total 164 M-QTLs identified, 35 M-QTLs were major QTLs. In the case of the S-population, three major QTLs were identified using QTLCartographer  while onFAD2B) was located on the linkage group b09_S in the S-population  and a09_T (T_mqPA_a09-2 between marker GNB377-FAD2A) explaining 12.44% and 22.58% PV, respectively , a total of 11 and 19 M-QTLs were identified using QTLCartographer in the S- and T-population with PVE up to 22.04% and 37.37%, respectively . Only onion Figs . Similar:0, a totely Figs . Furtherely Figs  Tables. ely Figs  Tables.For stearic acid (18:0), six and 15 M-QTLs were detected using QTLCartographer in the S- and T-population, respectively . NeitherFor arachidic acid (20:0), a total of 6 and 14 M-QTLs were detected in the S- and T-populations using QTLCartographer. No major QTL was detected in the S-population and the QTL with the highest PVE was ‘S mqAA b09’ with 6.43% PVE located on the linkage group b09_S ; Fig 4. FAD2A), b04_T (PM15-IPAHM451) and b07_T (GM1254-GM2744) , QTLCartographer identified a total of 20 M-QTLs in the T-population with PVE ranging from 2.98% (T_mqGA_b07-4) to 26.13% (T_mqGA_b04-1) . Of thesb04_T PM1-IPAHM45144) Figs . The par44) Figs . The QTL44) Figs . All the44) Figs ; Fig 6.For behenic acid (22:0), the QTLCartographer identified a total of five minor M-QTLs in the S-population . SimilarFor lignoceric acid (24:0), total five and 13 M-QTLs were identified in the S- and T-populations, respectively using QTLCartographer . In the The QTLNetwork detected a total of 27 E-QTLs i.e., nine in the S-population and 18 in the T-population with only two-locus interactions . The PVEFAD2A and FAD2B genes playing important role in controlling the two unsaturated fatty acids i.e., oleic acid (C18:1) and linoleic acid (C18:2) in the peanut oil, it would be interesting to compare the QTLs associated with the five SFAs and a UFA (gadoleic acid) (C20:1) with oleic and linoleic acid. In order to draw such comparison, QTLs identified by Pandey et al. [FAD2A and FAD2B genes while the T-population segregates only for FAD2A gene. Therefore, an effort was made to compare the QTLs obtained for oleic acid and linoleic acid in the S- and T-population with the QTLs obtained for these minor fatty acids  including clustering and localization in the same location  in the cooking peanut oil is healthier for human consumption as it reduces the risk of cardiovascular disease (CVD) by lowering low-density lipoproteins (LDL) levels in blood in addition to providing longer shelf life and oxidative stability to the peanut products , 4, 5. AFAD2A and FAD2B) while the T-population segregates only for FAD2A. As these two mutant genes are known to affect the oleic acid and linoleic acid content, correlation study revealed that these two major fatty acids also affect the minor fatty acids. A positive correlation was observed for oleic acid with gadoleic acid and lignoceric acid while a negative correlation with stearic, palmitic, linoleic, arachidic and behenic acids  which forms the palmitic acid (C16:0). The addition of acetyl residues makes stearic acid (C18:0). Although there is no change to the palmitic acid but the formation of stearic acid gave rise to two directions. The first direction upon addition of acetyl residue reactions forms arachidic acid (C20:0), followed by behenic acid (C22:0) and then lignoceric acid (C24:0). The second direction upon desaturation of stearic acid (C18:0) produces oleic acid (18:1) which possess one double bond. Further, the oleic acid (C18:1) can either produce linoleic acid  or gadoleic acid  [FAD2 genes encode for microsomal oleoyl-PC desaturase or Δ12 fatty acid desaturase [FAD2 basically blocks this pathway. Plants however cannot afford to completely eliminate the synthesis of linoleic acid (C18:2) as linoleic acid further forms linolenic acid (C18:3) which plays an essential role in photosynthesis [FAD2 genes might trigger the plant to mobilize its resources to the production of more linoleic acid, thereby resulting in the accumulation of oleic acid (C18:1) while burning up of the source, in this case palmitic acid (C16:0) (The correlation between different fatty acids can be better understood if current understanding of biosynthetic pathway of fatty acid metabolism in plants is taken under perspective . In planesidues) , 18, 19.ic acid C:0, folloynthesis  and pollynthesis . Therefo (C16:0) . This phThe identification of QTLs controlling oil-quality traits should contribute to better understanding of oil synthesis as well as use of linked markers will facilitate oil quality improvement through genomics-assisted breeding. It is noteworthy here that this is the first study to report the identification of QTLs for the minor fatty acids in peanut. In the present study, two genetic software were employed for identification of main-effect QTLs (M-QTLs) and one for epistatic QTL interaction (E-QTL). Thus the total phenotypic variation for a complex trait is the result of multiple QTLs as well as their interactions. QTL analysis resulted in the identification of a total of 191 QTLs (164 M-QTLs and 27 E-QTLs) for the six fatty acids in the S- and T-population Tables  and 4 acFAD2B mutant allele in the T-population which had shown control over several fatty acids in the S-population such as oleic acid, linoleic acid and palmitic acid. In addition to the above possible explanation, genetic background might have also played an important role in the formation of these fatty acids. Similarly, when the total numbers of M-QTLs in both S- and T-population for these minor fatty acids were taken into account, it revealed altogether a different picture. A large number of M-QTLs were identified for palmitic acid (35) and gadoleic acid (35), followed by stearic acid (26), arachidic acid (23), behenic acid (24) and lignoceric acid (21). The higher number of QTLs for each minor fatty acid indicates that many other genetic factors are also involved in fatty acid metabolism. However, E-QTLs from these two populations indicated that the highest E-QTLs were associated with arachidic acid (7), gadoleic acid (5), palmitic acid (4), stearic acid (4), behenic acid (4) and lignoceric acid (3). Therefore, there were several epistatic QTLs which could also control these traits, such as in soybean [The number of QTLs identified in the T-population (118 M-QTLs and 18 E-QTLs) was more than double that of in the S-population (46 M-QTLs and nine E-QTLs) indicating that genetic control of minor fatty acids is more complex in the T-population than in the S-population. The above difference also might be because of absence of  soybean . A total soybean , and theFAD2 genes play a critical role in modifying the overall composition of fatty acids in peanut oil and the effect of these genes have been well established [FAD2 genes. In order to extract above mentioned information, the M-QTLs and E-QTLs identified for oleic acid and linoleic acid in the same two populations by Pandey et al. [FAD2A and FAD2B and the T-population has only one i.e., FAD2A.ablished . Furthery et al.  were ploy et al. , 24. In FAD2A, ‘SunOleic 97R’ increased oleic acid and gadoleic acid traits, in contrast ‘NC94022’ increased palmitic acid, linoleic acid and behenic acid. This indicates that the contrasting alleles from ‘SunOleic 97R’ decreased palmitic acid, linoleic acid and behenic acid. This can be correlated with the fact that ‘SunOleic 97R’ as a parental line has high oleic acid content due to the presence of FAD2A mutant allele. Similarly, in the T-population, seven clusters were identified possessing co-localized QTLs for oleic acid and/or linoleic acid. However, only one cluster (CL-T2) had higher trait alleles from one parent, i.e. ‘Tifrunner’ while remaining six clusters had higher alleles from different parents for different traits. Additionally, 11 other clusters were identified to co-host QTLs for one and/or all six fatty acids. Out of these 11 clusters, six clusters had higher alleles from one parent while the remaining five clusters had contrasting allele effects for higher traits. The identification of QTL clusters is a common phenomenon where several related traits contributing the complex traits or part of same metabolism/pathway were studied. For example such clusters for drought tolerance related traits were identified where several component and related traits to drought tolerance were phenotyped and used for QTL analysis [FAD2A genes [Allelic analysis revealed that in the S-population, out of the total seven clusters co-located for oleic acid and/or linoleic acid, four clusters had higher trait alleles from only one parent i.e. ‘SunOleic 97R’ or ‘NC94022’, while the other three clusters had higher alleles from different parents for different traits which co-localized in a particular cluster. In the cluster CL-S3 on linkage group a09_S  between analysis . Similaranalysis , 33. The2A genes . These c2A genes . In this2A genes . TherefoFAD2 genes has well been analyzed for different fatty acids. It has been observed that if both the mutant alleles together increase the production of oleic acid and gadoleic acid while decreased the production of linoleic acid, palmitic acid, stearic and arachidic acid  with 352 and 248 individuals, respectively, were used in this study [ine F435  while ‘Nle FAD2A , while ‘The complete sets of the S- and T-population along with parental lines were grown in three replications in the field in 2010 and 2011 at the Bellflower Farm, Tifton, Georgia, USA.The harvested pods from all the three replications were dried, packed and sent for the determination of fatty acid profiles to Plant Genetic Resources Conservation Unit, USDA-ARS, Griffin, USA. An Agilent 7890A  gas chromatograph with a flame ionization detector (FID) was used to determine the fatty acid composition. Oil was extracted from ~150 mg of finely grounded peanut seed tissue in 5.0 ml of n-heptane (Fisher Scientific). Then it was trans-esterified to fatty acids methyl esters (FAME) with 500 μl of 0.5 N sodium methoxide (NaOCH3). After 120 minutes, organic layer was separated from the aqueous layer and seed residue by adding 7.0 ml of distilled water. The organic layer contains methyl esters; 1.5 ml of the aliquot was transferred to autosampler for gas chromatography (GC) analysis. Peak separation was done on a DB-225 capillary column (5 m × 0.25 mm id with a 0.25 μm film) from Agilent Technologies, Santa Clara, California, USA. At a split ration of 60:1, 1 μl of the sample was injected onto the column maintained isothermally at 280°C. The temperature for inlet was same as that of column while that of detector was set to 300°C. Helium was used as a carrier gas and was set to a flow rate of ~1.0 ml/min (38 cm/sec). A FAME standard mix RM-3  was used to establish peak retention times. Each sample took 12 mins for running. A total of six fatty acids palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0) along with oleic acid (C18:1) and linoleic acid (C18:2) was measured in each peanut sample.The DNA from each RIL was extracted from fresh leaves along with their parental genotypes as described in Qin et al. . After aAll six fatty acids were used as traits for QTL mapping. Since the data was generated for two years, QTL analysis was performed for individual year  as well as the mean (trait_mean). Windows QTLCartographer version 2.5  was usedS1 TableThis table shows details on location, flanking marker loci, LOD value, phenotypic variance explained and additive effects of 40 M-QTLs detected by QTLCartographer.(XLSX)Click here for additional data file.S2 TableThis table shows details on location, flanking marker loci, LOD value, phenotypic variance explained and additive effects of 97 M-QTLs detected by QTLCartographer.(XLSX)Click here for additional data file.S3 TableThis table shows details on location, flanking marker loci, phenotypic variance explained and additive effects of 6 M-QTLs detected by QTLNetwork.(XLSX)Click here for additional data file.S4 TableThis table shows details on location, flanking marker loci, phenotypic variance explained and additive effects of 21 M-QTLs detected by QTLNetwork.(XLSX)Click here for additional data file.S5 TableThis table shows details on search range, F value, phenotypic variance explained, number of interacting loci and names interacting loci for 27 E-QTLs detected by QTLNetwork.(XLSX)Click here for additional data file.


Clostridium botulinum group III strains producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of fourteen strains of Clostridium botulinum producing type C/D and two strains producing type D/C isolated in France, and one strain producing type D/C that originated from New Caledonia.Animal botulism is mainly associated with  C. botulinum strains that produce type C and D toxins, or a chimeric fusion of C and D termed C/D or D/C toxin , producing 75 to 146 contigs  according to the manufacturer’s instructions for Gram-positive bacteria, with an additional RNase A (Roche) treatment. Libraries were prepared using the Nextera XT kit (Illumina). Whole-genome sequencing was performed using an Illumina MiSeq platform (Illumina) according to the manufacturer’s instructions. Three MiSeq runs were carried out, two with paired-end 150-nucleotide (nt) reads on MiSeq V2 and V2 microchemistry, another with paired-end 300-nt reads on V3 chemistry. The raw reads were trimmed  and assembled in CLC Genomics Workbench version 7.5.1 by  contigs . The medd 68 kbp . The seqThe average size of the genomes in this study is 3.09 Mb, 2.82 Mb being the smallest genome size, with an average G+C content of 28.9% (isolate 58752, Clostridium botulinum group III strains were deposited in DDBJ/ENA/GenBank under the accession numbers listed in The annotated draft whole-genome sequences of these


Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers. Hyperthermostable enzymes are usually isolated from hyperthermophiles grown at extremely high temperatures Compared to their mesophilic homologues, some hyperthermostable proteins have larger oligomeric sizes A. Pyrophilus (ApSOD) Superoxide dismutases (SODs) are evolutionally highly conserved enzymes that catalyze the disproportionation of superoxide radicals Tris, dithiothreitol (DTT), dithiobis(succinimidylpropionate) (DSP), isopropyl-1-thio-β-d-galactopyranoside (IPTG), glutaraldehyde and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Corporation. The vector pET28a was purchased from Novagen. All other chemicals were local products of analytical grade.5′-CGGGATCCATGCCAGTGGATAAGTTA-3′; K12A-forward, 5′-CGGGATCCATGCCAGTGCATAAGTTAGAGCCAAAGAACCATCTTGCGCCTTC-3′; K12A-reverse, 5′-CCAAGCTTTTATTTCACAAAATCCTTGAGGGTTTCATAAGC-3′; E23A-forward, 5′-CATTTCCAACGCGCAGATTGAG-3′; E23A-reverse, 5′-GGCTCAATCTGCGCGTTGGAAAT-3′; E165A-forward, 5′-ACTTACGCACACGCCTAC-3′; E165A-reverse, 5′-AGTAGGCGTGTGTGCGTAAGTGT -3′; E165 V-forward, 5′-ACTTACGTACACGCCTAC-3′ and E165 V-reverse, 5′-AGTAGGCGTG TACGTAAGTGT-3′. The WT and mutated proteins were overexpressed in E. coli BL21 induced by IPTG and purified by Ni-NTA affinity chromatography as described previously The cloning of the genes of wild type (WT) tcSOD and two truncation mutants, M202 (residues 1–201) and M211 (residues 1–210) has been described elsewhere The 12.5% SDS-PAGE and 10% native-PAGE of the proteins were conducted according to the standard methods. The 10% native-PAGE was run using the protocol similar to that of SDS-PAGE except for the absence of SDS in the loading buffer and the gel. SOD activity was assayed according to the standard pyrogallol autoxidation methods Spectroscopic measurements were performed at 25°C using a protein concentration of 0.27 mg/ml in 20 mM Tris-HCl buffer, pH 7.4 as those described elsewhere Cross-linking by glutaraldehyde was performed according to the method described previously with some modifications The separation of the tetramers and high-order oligomers was performed by ion exchange using the 1 ml Resource Q column equipped on an ÄKTA FPLC . The column was equilibrated with 20 mM PBS. About 2 ml protein solutions with a concentration of 1 mg/ml was injected in the column and eluted with a linear gradient of 0–500 mM NaCl in 20 mM PBS.The size-exclusion chromatography (SEC) experiments were carried out on a Superdex 200HR 10/30 column on an ÄKTA FPLC. The column was pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.4), and then 100 µl protein solutions were injected into the column. All samples were run at a flow rate of 0.5 ml/min at 25°C.We evaluated the oligomeric equilibrium of tcSOD by SEC and spectroscopic analysis by varying protein concentrations. The elution volume and spectroscopic characteristics remained unchanged for protein concentration ranging from 0.1 mg/ml to 1.0 mg/ml  and CD signal.(PDF)Click here for additional data file.


Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages  and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following  Infections by parasitic worms are very common, and controlling them is a major medical and veterinary challenge. Very few drugs exist to treat them, and the parasites can develop resistance to these. In order to find new ways to control worm infections, understanding how our immune system responds to them is essential. Many important parasitic worm infections move through the host lung. In this study we show that a major secreted protein in the lung, Surfactant Protein D (SP-D), is essential for immunity to a parasitic worm infection. We found that this protein binds to worm larvae in the lung to help the immune system kill them. Infecting mice that do not express SP-D with worms demonstrates SP-D is important in this immune response. These mice are unable to launch an effective anti-worm immune response and have many more worms in their intestine compared to mice that do express SP-D. We also show that if we increase SP-D levels in the lung the mouse has better immunity to worms. Together this shows for the first time that SP-D is very important for immunity to worm infections. Surfactant Protein (SP)-D is a constitutively expressed C-type lectin, which has a well recognized role in innate pulmonary immunity against viruses, bacteria and fungi, as well as in maintaining pulmonary homeostasis . Direct SP-D is primarily secreted by alveolar epithelial type II (ATII) cells ; ATII ceS. mansoni driven inflammasome activation [Schistosoma mansoni [Helminth infections are likely to have contributed to the evolution of both type 2/TH2 immunity and associated mechanisms that regulate the strength of this response , 13. Hostivation . Only on mansoni  however,Nippostrongylus brasiliensis induced a striking type 2-dependent increase in the levels of host SP-D. This induction of SP-D was associated with an increase in type-2 anti-parasite immune responses. Moreover, we found that immunity to infection required direct interaction of SP-D with both the fourth stage (L4) larvae and host alveolar macrophages, driving the latter to an enhanced AAM phenotype. SP-D therefore represents a previously un-described but pivotal mechanistic contributor to host immunity to helminth infection.In the study presented here we demonstrate that infection with the experimental model nematode N. brasiliensis infection [N. brasiliensis infection increased pulmonary and systemic levels of SP-D. Analysis of BAL  and serum  of N. brasiliensis-infected mice showed a temporal relationship between SP-D levels and progression and resolution of N. brasiliensis infection. The highest levels of SP-D were found at the peak of infection; namely day 7 post primary infection in both BAL and serum, highlighting an association with host protective immunity to N. brasiliensis. SP-D production has been shown to be dependent on IL-4, IL-13 and STAT6 [N. brasiliensis results in enhanced host secretion of IL-4 and IL-13, with IL-13 being essential for resolution of infection [N. brasiliensis infection. WT, IL-4/IL-13-/- mice  and IL-4Rα-/- mice  were infected with N. brasiliensis and, at 5 days post-infection, SP-D levels in BAL fluid and serum  were quantified. Significantly higher SP-D levels were found in WT mice when compared to both IL-4/IL-13-/- and IL-4Rα-/- mice.Type 2 cytokine-associated increases in SP-D levels have previously been shown in bronchoalveolar lavage (BAL) and serum of mice following challenge with a range of antigens and pathogens , but notnfection , 19, we nfection . We inveN. brasiliensis we infected wild type C57/BL-6 and SP-D-deficient (SP-D-/-) mice [. Levels of the cytokine IL-13, essential for resolution of N. brasiliensis infection, were significantly reduced at day 9 PI in the intestine, but not the lung, of SP-D-/- mice when compared to wild type mice . Equivalent levels of the alarmins IL-25, IL-33 and TSLP were detected in the lung and intestine of both WT and SP-D-/- mice at days 9 and 16 PI .To test the association between elevated SP-D levels and immunity to /-) mice  with theN. brasiliensis infection. Numbers and proportions of ILCs, which are required for resolution of N. brasiliensis infection [. Proportions and numbers of alveolar macrophages (AlvM) was significantly increased in SP-D-/- mice when compared to wild type mice . However, expression of a hallmark of alternative activation, resistin-like molecule (RELM) alpha/FIZZ1 (RELM-α), within AlvM was significantly decreased in SP-D-/- mice compared to wild-type by day 9 post-infection . Additionally, analysis of total RELM-α protein levels in the lung revealed a significant reduction of RELM-α levels in SP-D-/- mice when compared to WT, although total levels of YM1 were equivalent in both groups 7-rSP-D,SP-D−/−). Significantly reduced N. brasiliensis numbers in the intestine in doxycycline treated mice (SP-Don) when compared to untreated mice (SP-Doff) demonstrated that SP-D expression in the lung is a major component of host immunity against N. brasiliensis. However, numbers of worms in SP-Don mice were significantly higher than in WT mice indicating possible SP-D mediated inputs to immunity at other sites.We also quantified the numbers and proportions of innate lymphoid cells (ILCs) and alveolar macrophages at day 9 post infection to identify if SP-D was required for the development of these cells, which are essential for optimal resolution of nfection , were siN. brasiliensis by immunofluorescence using third stage larvae (L3), fourth stage larvae (L4), and adult worms. rfhSP-D binding to the surface of parasites was only seen for L4 . Moulting of N. brasiliensis L3 to L4 takes place in the lungs of the host. This would suggest that elevated pulmonary SP-D levels seen in the lung  could induce protective immunity by coating L4 parasites and enhanced immune recognition of these parasites both in the lung and intestine. We tested this by infecting mice intranasally with rfhSP-D-coated L4, prior coating with rfhSP-D did not result in reduced parasite viability . Examination of host alarmin responses revealed no effect on IL-33 or IL-25, but a significant reduction in TSLP . Recipients of rfhSP-D-coated L4 had increased numbers of ILC2 while AlvM populations showed a heightened expression of the markers of alternative activation RELM-α chitinase-3-like protein Chil3 (Ym1) .SP-D control of infection is associated with its ability to opsonize and enhance immune recognition of pathogens. We assessed the ability of a recombinant homotrimeric fragment of human SP-D (rfhSP-D) to directly bind to N. brasiliensis and that rfhSP-D binding of L4 parasites could enhance host immunity to the infection we next tested if heightened levels of pulmonary SP-D prior to N. brasiliensis infection conferred enhanced immunity against the parasite. Naïve mice treated with rfhSP-D for 4 days did not display detectable levels of IL-13 or IL4 in the lung. Numbers and proportions of ILC2s were also equivalent . At day 5 post-infection, intra-nasal administration of rfhSP-D prior to N. brasiliensis infection  significantly reduced intestinal burdens of adult N. brasiliensis in rfhSP-D-treated mice compared to bovine serum albumin (BSA)-treated controls . Protection was associated with increased pulmonary type 2 cytokines IL-4 and IL-13 but not IL-33 at day 5 post infection . Additionally, type 2 innate lymphoid cells (ILC2) required to control N. brasiliesis infection were increased in numbers and proportions in the lung of rfhSP-D treated mice. Moreover, a higher percentage of ILC2s produced IL-13 in rfhSP-D treated mice when compared to untreated mice .Having established in a loss-of-function system that SP-D is required for immunity to -/- mice have a loss of homeostatic regulation of macrophage function [N. brasiliensis recall infection is dependent on IL-4Rα-dependent macrophage polarization to the alternatively activated phenotype in the lung [Heligmosomoides polygyrus [Schistosoma mansoni [N. brasiliensis in the lung, we hypothesized that this may be due, at least in part, to an effect on polarisation of AlvM to AAM.SP-D is known to regulate alveolar macrophage function , 24. SP-function , 26 whicfunction . Host cothe lung . AAMs arolygyrus  and Schi mansoni . As SP-DFig 5A). To test if these SP-D-dependent enhanced AAM response contributed to increased protection against N. brasiliensis, we isolated AlvMs from mice which had been infected with N. brasiliensis and treated with rfhSP-D or not, and transferred these cells intra-nasally into naïve mice. Recipients of rfhSP-D-treated macrophages had reduced intestinal worm burdens at day 5PI when compared to recipients of untreated macrophages .Intranasal administration of rfhSP-D resulted in an increased expression of the AAM markers YM1 and Relm-α in AlvMs when compared to BSA-treated controls at day 5 post-infection . Collectively, these data show that SP-D can enhance AAM-dependent immunity to N. brasiliensis.It is typically considered that polarization to alternative activation by macrophages following an nd IL-13 . We direN. brasiliensis. The ability of the rfhSP-D CRD domain to bind antigen can be blocked with maltose. We used this approach to block the ability of rfhSP-D to bind via the CRD to N. brasilieinsis. Mice treated with maltose-blocked rfhSP-D had higher worm burdens than mice treated with rfhSP-D alone; moreover, mice treated with rfhSP-D blocked with maltose did not demonstrate enhanced ILC2 and AAM responses seen in mice treated with rfhSP-D alone .SP-D typically confers protection against infection by binding of the carbohydrate recognition domain (CRD) to the pathogen and, improving opsonisation and neutralization . We testN. brasiliensis could enhance killing by alveolar macrophages. Macrophages isolated into serum-free medium from the lungs of N. brasiliensis infected mice were added to L4 N. brasiliensis in the presence or absence of rfhSP-D. In the presence of rfhSP-D and alveolar macrophages the ratio of live to dead parasites decreased when compared to parasites cultured with macrophages only .We next tested if rfhSP-D opsonisation of L4 To date it has never been demonstrated if SP-D or any other member of the collectin sub-family of C-type lectins can directly mediate innate protection against a parasitic helminth. In this study we show for the first time that SP-D is an important component of innate immunity to helminth infection.N. brasiliensis. This is in agreement with other reports where SP-D levels increase in response to acute lung stress following pulmonary challenges with, for example, lipopolysaccharide [Aspergillus fumigatus [Actinobacillus pleurpneumoniae or Staphylococcus aureus [We find that levels of SP-D in the lung and serum increase significantly and rapidly, in a manner analogous to an alarmin response, following infection with ccharide , bleomycccharide  and ovalccharide  Similarlumigatus , Actinobs aureus . Our datN. brasiliensis; namely IL-4/IL-13 signaling via IL-4Rα. Previous demonstrations of increased type 2 immunity in SP-D-/- mice [N. brasiliensis infection [N. brasiliensis infections. Our results demonstrated that SP-D deficiency impaired innate type-2 responses associated with immunity to N. brasiliensis infection [N. brasiliensis.Elevated SP-D production can also be driven by the type 2/TH2 cytokines IL-4 and IL-13, and in turn SP-D can impart negative feedback control of type 2/TH2 responses; indeed in the absence of SP-D TH-2 cytokine levels are raised . Our fin-/- mice  may havenfection  would hanfection ; moreoveOpsonisation of pathogens and allergens by SP-D enhances host neutralization of them in the lung, primarily by improved recognition by host cells, such as alveolar macrophages . It is wN. brasiliensis is dependent on the CRD. Furthermore, SP-D binding through the CRD promotes innate type 2 responses including ILC2 induction and alternative activation of alveolar macrophages. We also show that SP-D can directly enhance L4 killing by alveolar macrophages following exposure to N. brasiliensis infection. These data clearly show that SP-D can act as an opsonin of L4 N. brasiliensis to enhance parasite killing by alveolar macrophages.In accordance with studies demonstrating the requirement of the carbohydrate recognition domain (CRD) in mediating pathogen binding and enhanced phagocytosis and clearance by neutrophils and macrophages , our stuN. brasiliensis promotes parasite clearance via induction of innate type 2 responses including alternative activation of macrophages but also enhanced ILC2 expansion. To the best of our knowledge this is the first description of SP-D influencing ILC2 biology and we suggest that the decreased type 2 cytokine levels in the lungs may be a function of a loss of an interaction between ILC2 and SP-D in SP-D-/- mice.Our results therefore clearly demonstrate that binding of SP-D to the L4 lung stage of in vivo studies show that SP-D deficiency increased the proportions of alveolar macrophages in the lung, key cells that maintain lung homeostasis and promote parasite clearance [N. brasiliensis infection. Moreover, we show that in the absence of SP-D total levels of RELM-α in the lung were significantly reduced. Like SP-D, RELM-α regulates type 2 immunity; RELM-α-/- mice develop heightened pathology following experimental S. mansoni infection [Additionally, our learance , 27. Ourlearance . Howevernfection . Therefonfection .Induction of type 2 immunity to helminths is significantly dependent on epithelial cells  secreting cytokines such as IL-33  along wiN. brasiliensis. This enhanced immunity is coincident with an increased induction of cells associated with the resolution of infection; namely ILC2 and alternatively activated macrophages. Thus, helminth induction of SP-D is essential for host resolution of helminth infection.In conclusion, we show for the first time in both gain of function and loss function approaches that SP-D is required for immunity against -/-[-/-[7-rSP-D,SP-D−/− [6-10-week-old BALB/c, C57/BL6, IL-4/13-/-, IL-4Rα--/-[-/-, SP-D-/-,SP-D−/− , 47 miceN. brasiliensis L3 larvae and killed at various times post infection as previously described [N. brasiliensis was carried out using techniques adapted from Harvie et al [N. brasiliensis were isolated 2 days post- N. brasiliensis infection from lung tissues. L4 N. brasiliensis were then incubated with rfhSP-D or bovine serum albumin (BSA) control for 1 hour at 37°C. L4 N. brasiliensis infection of mice was carried out by intranasal administration of 250 viable L4 worms in 50 μl to lightly anaesthetized mice. Adult worm burdens were determined by removing the small intestine and exposing the lumen by dissection. The intestines were incubated at 37°C for 4 hrs in 0.65% sodium chloride (NaCl) to allow the worms to migrate out, after which the numbers of worms were counted under a dissecting microscope.Mice were infected subcutaneously with 500 escribed . Intra-nie et al . Briefly2) for 1 hr at 37°C. Mice were killed on days indicated in results.Mice were lightly anaesthetized and treated with 20 μg of rfhSP-D or BSA control (diluted in PBS) intra-nasally in a volume of 50 μl on day 0, 1, 2 and 3. For blocking of rfhSP-D CRD head region, 20 mM Maltose was incubated with rfhSP-D in the presence of 1 mM calcium chloride  by administration of 1 ml sterile PBS containing 0.25 mM Ethylenediaminetetraacetic acid (EDTA) intra-tracheally. Lungs were lavaged three times. BAL fluid was centrifuged at 1200 rpm for 5 mins and the supernatant was frozen at -80°C.The left lobe of the lung was snap frozen in liquid nitrogen and subsequently stored at -80°C until analysis. To prepare lung homogenates, 400 μl of lysis buffer  was addeApproximately 500 μl of blood was collected by cardiac puncture and the serum isolated by centrifugation (4000 rpm for 20 mins).Whole lungs were removed from individual mice, finely cut and digested in Iscove’s modified Eagle medium (IMDM) (Invitrogen) containing 50 U/ml collagenase type I (Invitrogen) and 13 μg/ml DNase (Roche) at 37°C for 90 mins. Digested lung tissue were pushed through 70 or 100 μm nylon cell strainer  and subjected to red cell lysis.6 single cell suspensions from individual lungs were stained in MACS buffer with anti-Siglec-F PE (E50/2440) and anti-CD11c APC (HL3) antibodies to stain for AlvMs and eosinophils. To stain for ILC2, the antibodies anti-Lineage PE , anti-CD45 AF700, anti-CD90 PacBlu, anti-CD127 PE-Cy7 or APC (SB/199), anti-ICOS Biotin or FITC (7E.17G9) anti- Sca-1 V450 (D7) and anti-T1/ST2 FITC PerCP-Cy5.5 (DJ8) were used. Anti-FcR (2.4G2) was used to block non-specific binding of immunoglobulins to the FCγII/III receptors.1 x 10For intracellular staining, cells were stained with surface markers, fixed in 2% paraformaldehyde before being permeabilized with buffer containing saponin. Cells were subsequently stained with anti-Ym1 biotin (ECF-L) and anti-RELM-α (E19). For intracellular staining of ILC2 cells were fixed and permeabilized with Fix/perm (eBioscience) and stained with IL-13 PE-Cy7. Cells were acquired using a FORTESSA Flow cytometer (BD Biosciences) and data analyzed using FlowJo software . Antibodies were purchased from BD Pharmingen, eBioscience and Biolegend.N. brasiliensis infected mice were analyzed for SP-D content by ELISA. 96-well flat-bottom plates  were coated overnight at 4°C with 50 μl of appropriate coating antibody diluted in 1X PBS. Plates were washed four times in Tris-Buffered Saline containing % Tween (TBST), blocked with 200 μl blocking buffer overnight at 4°C. Following this, appropriate dilutions of samples and standards were prepared, loaded into wells and incubated overnight at 4°C. The plates were further washed and 50 μl of appropriate biotinylated secondary antibodies were added and incubated at 37°C for 3 hrs. 50 μl of Streptavidin-coupled horseradish peroxidase (HRP) was then added after washing and plates were incubated for 1 hr at 37°C. The plates were developed with 3,3’,5,5’-Tetramethylbenzidine (TMB) microwell peroxidase substrate system, and the reaction was stopped with 1 M H3PO4. The plates were read at an absorbance of 450 nm using a VersaMax microplate reader . All antibodies were from BD Pharmingen, San Diego, CA.Whole lung and intestine homogenates were used to quantify cytokines, and BAL fluid or serum from +Siglec F+Autoflourescenthigh using a FACS Aria cell sorter (Becton Dickinson), purity was also confirmed by microscopic analysis . 1 x 105 macrophages were then transferred intra-nasally into naïve BALB/c mice 24 hrs prior to N. brasiliensis infection.Mice were treated with 20 μg of rfhSP-D or BSA at D0, 1, 2, 3, 6 and 7 post-infection. Single-cell suspensions of pooled lungs were prepared at day 8 post-infection and AlvMs were stained with anti-CD11c APC-conjugated and anti-Siglec-F PE conjugated monoclonal antibody (MAb) (BD Pharmingen) before they were isolated (> 95% purity) as CD11c+Siglec-F+AutoFlourescenthigh) were isolated from single cell suspensions of lung tissue by FACS Aria as described above and plated in duplicates at 4x105 cells per well. Cells were stimulated with either recombinant mouse IL-4/IL-13, IL-4/IL-13 + 20 μg/ml of rfhSPD or left untreated. The cultures were incubated for 60 hrs at 37°C. Thereafter, cells were washed and stained for alternative activation markers, YM1 and Relm- α as described above, and acquired with LSRFORTESSA (BD Biosciences).Naïve AlvMs  used at 1/200 and left overnight at 4°C. To detect the SP-D binding, the organisms were subsequently incubated with Streptavidin cy3 (1/500) for 2 hrs at room temperature (RT). All sections were viewed with Axiovert LSM 510 Meta NLO microscope (Zeiss).L3, L4 and adult stage larvae of Data were expressed as mean ± standard deviation and analyzed using Mann-Whitney nonparametric T test or ANOVA with a 95% confidence interval. p values are represented as p< 0.05 (*), p<0.01 (**) and p<0.005 (***).S1 FigN. brasiliensis infection (a). SP-D levels of IL-4/IL-13-/- mice (b) were measured in serum at day 5 PI and compared to wild type controls. Data are representative of one individual experiment. N = 5 mice per group. **P<0.01.Kinetics of SP-D levels was measured by ELISA in serum following (TIFF)Click here for additional data file.S2 Figa). Data are representative of 2 individual experiments. WT, CCSP-rtTA, (tetO)7-rSP-D,SP-D −/− mice–doxycycline (SP-Doff) and CCSP-rtTA, (tetO)7-rSP-D,SP-D −/− mice + doxycycline (SP-Don) were infected with 500 x L3 N. brasiliensis and intestinal worm burdens established at day 5 PI (b). Data are representative of 2 individual experiments. N = 4–6 mice per group.Relative concentration of RELM-α and YM1 in individual lung samples of day 9 PI WT and SP-D-/- mice as detected by Western Blot and quantified by densitometry ((TIFF)Click here for additional data file.S3 FigN. brasiliensis parasites with anti-IgG2b isotype control (a). Untreated N. brasiliensis L4 or N. brasiliensis L4 pre-incubated for 1 hr with either 20 μg/ml BSA or 20 μg/ml SP-D. Worm motility was assessed by time lapse photography (b). Top row shows bright field, bottom row shows standard deviation of overlay of 20 sequence pictures; white indicates movement. Data are representative of two individual experiments.Labeling of (TIFF)Click here for additional data file.S4 Figa). Total numbers and proportions of lung ILC2s (b). Data are representative of 2 individual experiments. N = 4 mice per group.BALB/c mice were treated with 20 μg rfhSP-D for 4 days. IL-4, IL-13 and IL-33 cytokine levels in lung homogenates were detected by ELISA at day 5 PI ((TIFF)Click here for additional data file.S5 FigN. brasiliensis infected and N.brasiliensis + SP-D treated mice (a). Expression levels of YM1 and RELM-α on alveolar macrophages isolated from N. brasiliensis infected and N. brasiliensis infected + SP-D treated mice (b). Data are representative of 2 individual experiments.Light microscope analysis of macrophage morphology in cells isolated from (TIFF)Click here for additional data file.


Dynamic changes in protein S-palmitoylation are critical for regulating protein localization and signaling. Only two enzymes - the acyl-protein thioesterases APT1 and APT2 – are known to catalyze palmitate removal from cytosolic cysteine residues. It is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. Using a dual pulse-chase strategy comparing palmitate and protein half-lives, we found knockdown or inhibition of APT1 and APT2 blocked depalmitoylation of Huntingtin, but did not affect palmitate turnover on postsynaptic density protein 95 (PSD95) or N-Ras. We used activity profiling to identify novel serine hydrolase targets of the APT1/2 inhibitor Palmostatin B, and discovered that a family of uncharacterized ABHD17 proteins can accelerate palmitate turnover on PSD95 and N-Ras. ABHD17 catalytic activity is required for N-Ras depalmitoylation and re-localization to internal cellular membranes. Our findings indicate that the family of depalmitoylation enzymes may be substantially broader than previously believed.DOI:http://dx.doi.org/10.7554/eLife.11306.001 Proteins play important roles in many processes in cells. Some of these proteins can be modified by the addition of a molecule called palmitate. This process, termed “palmitoylation”, helps direct these proteins to the compartments within the cell where they are needed to carry out their roles. One target of palmitoylation is N-Ras, which is a protein that can promote the development of cancer.We understand quite a lot about how palmitate is added to proteins, but much less about how it is removed. So far, researchers have only identified two enzymes – known as APT1 and APT2 – that can remove palmitate from proteins, but it is possible that there are others. Identifying other “depalmitoylase” enzymes could help us find ways to block the removal of palmitate from N-Ras, which could lead to new treatments for some cancers.Lin and Conibear used several biochemical techniques to search for depalmitoylase enzymes in human cells. The experiments reveal that although APT1 and APT2 are important for removing palmitate from some proteins, they are not needed to remove palmitate from N-Ras. Instead, Lin and Conibear found that an enzyme called ABHD17 removes palmitate from N-Ras. The next step following on from this work will be to find out what other proteins ABHD17 acts on in cells. A longer-term challenge will be to develop specific chemicals that inhibit ABHD17 activity and test if they are able to reduce the growth of cancer cells.DOI:http://dx.doi.org/10.7554/eLife.11306.002 Protein S-palmitoylation involves the post-translational attachment of the 16-carbon fatty acid palmitate to cysteine residues . While aPalmitoylation is mediated by a family of DHHC (Asp-His-His-Cys) proteins , whereasHere, we show that APT1 and APT2 inhibition or knockdown reduces palmitate turnover on some substrates but has no effect on N-Ras and PSD95. We identified members of the ABHD17 family as novel PalmB targets that depalmitoylate N-Ras and promote its relocalization to internal membranes. This demonstrates the enzymes responsible for protein depalmitoylation are more diverse than previously believed, which has important implications for understanding the selectivity and regulation of dynamic palmitate turnover.APT1 and APT2 were proposed to act universally and constitutively to remove mislocalized proteins from intracellular membranes and allow their re-palmitoylation at the Golgi . ReporteAPT1 and APT2 are reported to have differential substrate specificity . We founPrevious studies suggested that APT1, APT2, and PPT1 were the sole mSHs targeted by PalmB , whereasThe set of candidates inhibited by both PalmB and HDFP  includesSelective inhibitors that target the remaining four candidates have not been identified. Therefore, we used pulse-chase click chemistry to test if increased expression of these enzymes enhances palmitate turnover. High levels of ABHD16A, ABHD6, or APT1/2 had little effect on N-Ras  or PSD95We focused on ABHD17A, which showed the strongest effect in promoting palmitate turnover on N-Ras and PSD95. The ABHD17 proteins are targeted to membranes by a palmitoylated N-terminal cysteine cluster . We founWe next examined the cellular consequences of ABHD17A expression. Disrupting N-Ras palmitoylation by mutating the palmitoylated residue (C181S) or treating cells with the inhibitor 2-bromopalmitate (2-BP) relocalized N-Ras from the plasma membrane to internal organelles, as previously described  Figure . OverexpTo determine if the endogenous ABHD17 proteins regulate palmitate cycling in vivo, we investigated the effect of ABHD17 knockdown on N-Ras depalmitoylation in HEK293T cells. RT-qPCR (Reverse transcription quantitative polymerase chain reaction) showed efficient silencing of ABHD17A alone, or ABHD17A, ABHD17B, and ABHD17C in concert, after 72 hr with siRNA treatment . ABHD17ACaenorhabditis elegans ABHD17 homologue AHO-3 regulates starvation-induced thermotactic plasticity . Plasmids expressing Myc-hAPT1, GOLIM4-GFP, FLAG-SPRED2, and GAD65-GFP were generous gifts from Dr. Takashi Izumi (Gunma University), Dr. Adam Linstedt (Carnegie Mellon University), Dr. Akihiko Yoshimura (Keio University), and the late Dr. Alaa El-Husseini (University of British Columbia), respectively. Venus-tagged Rab5, Rab7, and Rab11 plasmids were gifts from Dr. Nevin Lambert (Georgia Regents University). EGFP-ITM2B was cloned by polymerase chain reaction (PCR) amplification of the ITM2B ORF (open reading frame) from MGC Fully Sequenced Human BRI3 cDNA, clone ID 3163436 , using the forward primer 5’-ATTTAACCCGGGATGGTGAAGATTAGCTTCCAGCC-3’ and the reverse primer 5’-ATTTAAGGTACCTCACACCACCCCGCAGAT-3’, followed by restriction digest and ligation with BspEI/KpnI-digested pEGFP-C3 vector from Clontech . EGFP-N-Ras-C181S was generated by Quikchange mutagenesis  using the forward primer 5’-CAACAGCAGTGATGATGGTACCCAGGGTAGTATGGGATTGCCATGTGTGG-3’ and the reverse primer 5’-CCACACATGGCAATCCCATACTACCCTGGG TACCATCATCACTGCTGTTG-3’ with EGFP-N-Ras as the template.For cloning of mSHs for activity-profiling studies, plasmids containing corresponding human ORFs were purchased from DNASU  and OpenBiosystems, or obtained as clones from the hORFeome v8.1 Collection . Genes oThe ABHD17A-FLAG construct was used as the template to generate ABHD17A mutant and mCherry-tagged plasmids. S211A-FLAG in pCINeo was generated by Quikchange mutagenesis, and ABHD17A ∆N-FLAG was amplified by PCR then subcloned into pCINeo. ABHD17A-mCherry wild type and mutant plasmids were generated by pairing each forward oligo with the reverse ABHD17A-mCherry-Linker oligo as listed in plus SMARTpool siRNAs targeting APT2, ABHD17A, ABHD17B, or ABHD17C, as well as Non-Targeting control siRNA, were purchased from Dharmacon .The pSUPER vector and the shRNA pSUPER-APT1 plasmid used in knockdown studies was a generous gift from Dr. Gerhard Schratt (University of Marburg), and ON-TARGETH-1,2,3-triazol-4-yl)methyl]amine (TBTA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Triton-X 100 (TX-100), sodium deoxycholate, CuSO4, palmitic acid, and 2-bromopalmitate were obtained from Sigma-Aldrich . 17-ODYA, C75, WWL70, and RHC-80267 were purchased from Cayman Chemical . HDFP, C83, and C115 were gifts from Dr. Brent Martin (University of Michigan), and FP-rhodamine was generously provided by Dr. Benjamin Cravatt (Scripps Institute).Lipofectamine 2000, Lipofectamine RNAiMax, sodium dedocyl sulfate (SDS) solution, L-azidohomoalanine (L-AHA), Alexa Fluor 488-azide (AF488-az), Alexa Fluor 647-alkyne , TRIzol reagent, and Prolong Gold Antifade Mountant with DAPI were purchased from Life Technologies . X-tremeGENE 9 was purchased from Roche . Palmostatin B was purchased from Merck Scientific . Tris-palmitate labeling of H-Ras (p<0.005). In contrast, we specifically measured depalmitoylation rates using a pulse-chase protocol that detects rapid palmitate turnover. Our results demonstrate that ABHD17 isoforms play a more important role than APT1/2 in regulating dynamic palmitate cycling on specific substrates including N-Ras, but do not preclude a function for APT1/2 in regulating steady-state palmitoylation of H-Ras.Minor points:1) Although not essential, it would be of some value to assess the depalmitoylation of Ras palmitoylated peptides by ABHD17A as well as APT1 and APT2 to see if the cellular results can be observed in a purified system.in vitro palmitoylation assay would be valuable. However, this is not a trivial experiment because we have shown ABHD17A must be membrane-anchored to display full activity (We agree an activity . A more 2) I am not really sure what is meant by the dashed lines and the cropping of lanes in several of the blots. Given that some of the effects are modest (ca. less than 2-fold), it would be better to just repeat experiments and represent on one complete gel.The images in the original figures represented single gels that had been cropped to remove extraneous lanes. All quantitation was performed on the uncropped gels, thus we believe cropping had no effect on the results. Nevertheless, we repeated the experiments in 3) Some binding to ABHD4 is possible, as our analysis showed PalmB reduced the activity of ABHD4 by approximately 25%, consistent with our observation that PalmB binds additional mSHs. However, to find the most significant PalmB targets, we chose the set of enzymes showing >50% inhibition for more detailed study.4) In We clearly see no effect on the rate of Ras or PSD95 de-palmitoylation in the presence of these mutants, yet baseline palmitoylation does appear to be elevated in an activity-independent manner. This would be worth future investigation, but is beyond the scope of the current study.


Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant. Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in poultry world-wide and raise concerns about a novel pandemic due to repeated zoonotic transmissions to humans2346789912131412131213234R65) leads to 100% morbidity and 30% lethality in chickenTo reveal HPAIV gene constellations sufficient for high virulence, we performed selection experiments with the genes of HPAIV A/Swan/Germany/R65/2006 (H5N1) (R65) versus those of LPAIV A/Teal/Germany/Wv632/2005 (H5N1) (TG05). To this end, we co-transfected plasmids encoding the R65 and TG05 genes, used the resulting supernatants for infection of chickens, took oral swabs and screened them for reassortant genotypes. Since we already could demonstrate that the introduction of the R65 HA into the genetic background of TG05 , we co-transfected the TG05 polymerase gene plasmids  and the R65 HA plasmid together with mixtures of the NA, M, and NS plasmids originating both from TG05 and R65. Sanger sequencing of the post-transfection supernatant indicated the simultaneous presence of the NA and M genes from both TG05 and R65 plus the presence of the TG05 NS gene . Oculonaall R65 genes, we then co-transfected the TG05 plasmids except the HA plasmid together with all eight R65 plasmids. Sanger sequencing of the post-transfection supernatant revealed mixtures of the PB2, PB1, NP, NA, and M genes, whereas PA and NS appeared to originate from TG05 predominantly , whereas reassortant TG05-HA/NA/M/NSR65 carries the HA plus the NA, M, and NS genes from R65 but the polymerase complex genes of TG05. Oculonasal inoculation of ten chickens with 105 pfu of TG05-HA/PB2/PB1/PA/NPR65 resulted in a lethality of 30% like TG05-HAR65R65 exhibited 100% lethality resembling R65. Furthermore, the 2.9 days (d) mean death time (MDT) of TG05-HA/NA/M/NSR65 is close to that of R65 with 3.8 d   , Fig. 3,R65, TG05-HA/NA/NSR65, TG05-HA/M/NSR65, or the double reassortant TG05-HAR65NAR65. TG05-HA/NA/MR65 and TG05-HA/NA/NSR65 displayed 100% lethality with increased MDT of 3.6 d and 5.2 d, respectively, suggesting a decrease in virulence compared to the quadruple reassortant TG05-HA/NA/M/NSR65 (MDT 2.9 d)  displays 100% lethality, although at a prolonged MDT of 5.9 d (range 5–7 d), proving that introduction of the R65 NA gene into TG05-HAR65 resulted in a highly pathogenic phenotype  is responsible for high virulence, we infected chickens with the three triple reassortants TG05-HA/NA/MT 2.9 d) . In conthenotype . Since MR65 or TG05-HAR65NAR65 and added four contact animals after one day. In both groups, all primarily infected animals died on days 4 to 6. All contact animals succumbed to death on days 7 to 8 with TG05-HA/NA/MR65 and days 8 to 9 with TG05-HAR65NAR65   , displaythality) . TherefoHPAIV evolve from low-pathogenic precursors by acquisition of a polybasic HACS1213141516234First, to reveal minimal gene constellations conferring high virulence, we selected random reassortants by co-transfection of plasmids from both the HPAIV R65 (H5N1) and LPAIV TG05 (H5N1) followed by infection of chickens. In contrast to the classical technique to obtain reassortants by double-infection21R65 resulted in prolonged mean death times indicating limited contribution of the R65 polymerase, M, and NS genes to virulence .Secondly, we investigated specific reassortants from R65 and TG05 in chicken. Stepwise reduction of the R65 genes beginning with reassortant TG05-HA/NA/M/NSirulence . RemarkaR65NAshort-TG05 increased lethality and transmission to 100% compared with its parental virus TG05-HAR65 displaying 30% lethality242526We then investigated whether the NA stalk deletion in R65 is crucial for high virulence and transmission to contact animals. Repairing the stalk deletion led to reduced virulence and prevented transmission, whereas introduction of the NA stalk deletion into the TG05 NA as in the mutant TG05-HAmax at a nearly unaffected Km has been demonstrated in case of a small substrate28Current understanding on functional consequences of the NA stalk deletion is still limited. An elevated Vmajor but non-essential plus the polybasic HACS as the prime and essential virulence determinant. However, without the NA stalk deletion, a highly pathogenic phenotype is displayed by the reassortant R65-NATG05 which in addition to the R65 HA carries the R65 polymerase and NP genes together with the R65 M and NS genes. Thus, the aa exchanges of those genes can be considered an alternative set of minor virulence determinants among which a single exchange contributes to the virulence to only a minute extent. Therefore, HPAIV like R65 may contain at least two different sets of virulence determinants. Taken together, HPAIV require an adapted HA with polybasic HACS as essential plus either several adaptive aa changes in polymerase, NP, M, and NA genes as minor virulence determinants or an NA stalk deletion as major virulence determinant.In general, the pairwise comparison of an HPAIV versus its putative low-pathogenic precursor may be limited to only a partial subset of a concealed set of virulence determinants, all together conferring high virulence. Such a complete set may be formed by an NA stalk deletion as a DQ464354-DQ464361) and A/Teal/Germany/Wv632/2005 (H5N1) (TG05) (Genbank accession numbers CY061882-9) have been described129Madin-Darby canine kidney (MDCK) cells were cultivated in minimal essential medium containing 10% fetal bovine serum. Plasmids encoding the gene segments of strains A/Swan/Germany/R65/2006 (H5N1) (R65) (Genbank accession numbers CY061887) like that in the R65 NA (Genbank accession number DQ464355), we removed nucleotides 165–224 corresponding to aa 49–68. To elongate the stalk region of R65 NA, we inserted nucleotides 165–224 from the TG05 NA gene between nucleotides 164 and 165 of the R65 NA. Those modifications were introduced by site-directed QuikChange mutagenesis (primer sequences available on request).To generate a stalk deletion in the TG05 NA  and gained governmental approval (registration number LALLF M-V/TSD/7221.3-1.1-018/07). Four- to eight-weeks-old White Leghorn specific-pathogen-free chickens  were infected oculonasally with 10How to cite this article: Stech, O. et al. The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken. Sci. Rep.5, 13493; doi: 10.1038/srep13493 (2015).