Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values . Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN. Diabetes mellitus is a chronic disease with potentially devastating complications. For example, diabetes mellitus is associated with macrovascular complications, such as cardiovascular and cerebrovascular diseases, and microvascular complications, including diabetic nephropathy (DN) and retinopathy . DN is aDN occurs in 15% to 25% of type 1 diabetic patients and 30% to 40% of type 2 diabetic patients . The use of microalbuminuria to predict incipient DN, particularly in type 2 diabetic patients, is limited for several reasons : the mic Recently, to compare DN patients with non-DN patients, proteomic technologies have been developed to identify urinary marker candidates that are associated with the development of DN. Various proteomic approaches have been used for this purpose, including 2-DE, 2-DE DIGE, and SELDI-TOF , 11, 12.E. coli, mammalian cells, yeast, plant cells, and human biological fluids  phosphine) were added to each sample and incubated for 1 hr at 60°C. Each sample was allowed to cool at room temperature, and 1 μL of cysteine blocking reagent (200 mM methyl methanethiosulfonate (MMTS) in isopropanol) was added and incubated for 20 min at room temperature. The tubes were digested with trypsin  at a protein-to-enzyme ratio of 10 : 1 at 37°C overnight, and the contents of one vial of iTRAQ reagent, dissolved in 70 μL of ethanol, were added to each peptide mixture and incubated for 1 hr at room temperature. Aliquots of 100 In this study, 3 iTRAQ experiments were performed. The detailed iTRAQ labeling strategy is summarized for the specified NA/MA urine samples in μL of buffer A (25% v/v acetonitrile (ACN) and 5 mM ammonium formate, adjusted to pH 2.7 with formic acid) and loaded onto a PolySULFOETHYL A column  in a HP1100 series HPLC . The column was equilibrated for 5 min in buffer A, and the peptides were eluted using a gradient of 0% to 30% buffer B (25% v/v ACN and 1 M ammonium formate [pH 3] with formic acid) over 80 min and 30% to 90% buffer B for 40 min at a flow rate of 0.7 mL/min. Absorbance was monitored at 280 nm, and the fractions were collected every 2 min after injection.iTRAQ-labeled samples were subjected to LC-MS/MS at the National Instrumentation Center for Environmental Management, Seoul National University, and fractionated using strong cation exchange (SCX) chromatography, as follows. Dried samples were reconstituted in 500 μm fused silica emitter tip . Fractions were reconstituted in solvent A and injected into an LC-ESI-MS/MS system. LC-MS/MS was performed using an integrated system, which consisted of an autosampler switching pump and a micropump  with a hybrid quadrupole-TOF LC-MS/MS spectrometer  that was equipped with a nanoelectrospray ionization source (Applied Biosystems) and fitted with a 10 μm id × 5 mm, 5 μm, 100 Å; Agilent Technologies), washed for 10 min with 98% solvent A (water/ACN [98 : 2 v/v] and 0.1% formic acid) and 2% solvent B (water/ACN [2 : 98 v/v] and 0.1% formic acid) at a flow rate of 10 μL/min, and separated on a Zorbax 300SB-C18 capillary column  at a flow rate of 300 nL/min. The LC gradient was run at 2% to 35% solvent B over 120 min and from 35% to 90% over 10 min, followed by 90% solvent B for 15 min, and finally 5% solvent B for 35 min. The resulting peptides were electrosprayed through a coated silica tip (New Objective) at an ion spray voltage of 2300 eV.Peptides were first trapped on a Zorbax 300SB-C18 trap column (300 m/z for a 1 sec TOF-MS survey scan to detect precursor ions. The 5 most abundant peptides (count >20) with charge states of +2 to +4 were selected to perform the information-dependent acquisition (IDA) of MS/MS data. Once selected, the precursor ions were dynamically excluded for 60 sec at a mass tolerance of 100 ppm.For data acquisition, the mass spectrometer was set in the positive ion mode at a selected mass range of 400–1600  Data file processing, protein identification, and relative abundance quantification were performed using ProteinPilot v.2.0.1  and the Paragon algorithm . DatabasThe confidence threshold for protein identification was an unused ProtScore >1.3 . ProteinPilot computes a percentage confidence that reflects the probability that a hit is a false positive; thus, at the 95% confidence level, the false positive identification rate is approximately 5% , 25. Althttp://www.pantherdb.org/), provided in the ProteinPilot output when a Celera human database is used. To construct a graphical representation of differentially excreted proteins, MultiExperiment Viewer (Version 4.3) was used, allowing us to generate a “heatmap” of differentially excreted proteomes (http://www.tm4.org/mev/).The “biological process” and “molecular function” classifications were analyzed using PANTHER ID numbers , vitamin D-binding protein , α1-acid glycoprotein , and haptoglobin .Twenty-four urine samples that were matched for gender and age  were selected from the urine sample groups  and subjected to Western blot validation of the 6 representative candidates from the iTRAQ experiments Figures . The prim/z for beta-galactosidase peptide) [In addition to Western blot, multiple reaction monitoring (MRM) was performed to verify the candidate biomarkers using 9 NA and 14 MA urine samples from the urine sample groups  . In our peptide) . In the For the iTRAQ experiments, 3 biological replicates , replicate 2 was labeled (d), and replicate 3 was labeled (e)); 2 technical replicates  and (c), replicate 2 was labeled (b) and (d)) were generated from normoalbuminuric and microalbuminuric urine . The 3 bSeven hundred ten proteins were identified from 21,610 peptides of the 3 combined biological replicates at a minimum confidence level of 95% (unused ProtScore > 1.3). Of the proteins that were identified by iTRAQ, 27% comprised 1-peptide proteins; 14% was 2-peptide proteins; 8% was 3-peptide proteins; 5% was 4-peptide proteins; 46% comprised proteins that had 5 or more peptides. In our iTRAQ experiment, 83 proteins (unused ProtScore > 1.3) were common to all 3 biological replicates at a minimum confidence level of 95%, using 3 different pooled urine samples.P value <0.05 for the 115/114 and 117/116 reporter ions. The 70 proteins were used to monitor technical variations and confirm the threshold for meaningful differences. To generate the quantitative proteome using iTRAQ labeling, we first determined the labeling efficiency, which exceeded 98% (data not shown). Next, the cutoff for significant fold-change was determined based on the 2 technical replicates ((a) and (c) of iTRAQ experiment 1, (b); (d) of iTRAQ experiment 2) . In the 2r = 0.9527 and 2r = 0.9178, respectively . Further, biomarker candidates were confirmed and validated by 2-DE, Western blot, and MRM.P value < 0.05, EF < 2, more than 2 unique peptides with >95% confidence level, and protein expression >1.25 or <0.80 for all reporter ions; 99 and 97 proteins were upregulated and downregulated, respectively , ceruloplasmin precursor (CP), mannose-binding lectin-associated serine protease-2 precursor (MASP2), alpha-1-antitrypsin (A1AT), haptoglobin (HP), and basement membrane-specific heparin sulfate proteoglycan core protein (HSPG) were associated with glomerular dysfunction; except for MASP2 and HSPG, all were upregulated in microalbuminuric versus normoalbuminuric urinary proteomes  and alpha-1-acid glycoprotein 1 precursor (AGP1) were selected for further validation. VDBP and AGP1 were upregulated in microalbuminuria versus normoalbuminuria  and PSCA (prostate stem cell antigen) correlate with other types of disease, and PSCA was selected for further validation. In this iTRAQ experiment, FABP was downregulated, whereas PSCA expression increased in the microalbuminuric versus normoalbuminuric urinary proteome  were upregulated, and 5 proteins  were downregulated in microalbuminuric urine , FABP (spot 5: 0.27 ± 0.037 and iTRAQ: 0.29), and MASP2 (spot 6: 0.10 ± 0.005 and iTRAQ: 0.29) were significantly downregulated in both techniques (AMBP (spot 7) was downregulated by 2-DE (0.20 ± 0.002) but upregulated in the iTRAQ experiment (1.44). Two proteins were identified by 2-DE alone—regulator of telomere elongation helicase 1 (spot 8: 3.0 ± 0.5) was upregulated and fibulin-5 precursor (spot 9: 0.12 ± 0.007) was downregulated  that were associated with pathogenic status were subjected to Western blot. The Western blot results were consistent with the iTRAQ findings (TF (4.66 ± 1.41 and P < 0.0005), CP (11.16 ± 0.38 and P < 0.01), A1AT (3.36 ± 0.03 and P < 0.005), VDBP (2.88 ± 0.11 and P < 0.05), AGP1 (1.82 ± 0.08 and P < 0.05), and HP (7.28 ± 5.52 and P < 0.05) were upregulated in microalbumiuric versus normoalbuminuric urine.To validate the differentially excreted proteins from the iTRAQ results, 6 proteins , MRM was performed using 9 individual normoalbuminuric and 14 microalbuminuric samples  values , as did CP and VDBP, to a lesser extent   . Particu, resp.) .TF, CP, A1AT, VDBP, AGP1, and PSCA were upregulated in microalbuminuric versus normoalbuminuric urine, whereas HP was down-regulated. TF, CP, A1AT, VDBP, AGP1, and PSCA had the same excretion patterns by iTRAQ and western blot; conversely, HP had the opposite excretion pattern.In the interactive plots, To identify and quantify proteins that were associated with diabetic nephropathy in microalbuminuric and normoalbuminuric urine, we used relative quantitative proteomic techniques, such as iTRAQ, 2-DE, Western blot, and MRM. In our iTRAQ experiment, 710 urinary proteins were identified at a >95% confidence level, of which 196 were differentially excreted by >1.25 or <0.80—99 and 97 proteins were up- and down-regulated, respectively .Recently, the Urinary Protein Biomarker (UPB) database was constructed and published, in which 205 publications were curated manually . Using tThe “molecular function” subcategories accounted for 196 differentially excreted proteins, of which “immunity” represented 25 of the quantitated proteins—the largest component . This prHSPG, FABP, and MASP2) that were identified by 2-DE had the same pattern of differential excretion in the 2-DE and iTRAQ experiments  were validated by Western blot, for which the patterns of excretion were consistent with the iTRAQ results  is an acute phase protein that binds to free hemoglobin (Hb) with the highest affinity. Formation of the Hb-HP complex prevents the loss of renal iron and oxidative damage that are driven by free Hb. Specifically, HP mediates complement-dependent podocyte damage [HP, which was downregulated and is associated with diabetic nephropathy and type 2 diabetes mellitus, appeared in the UPB database. However, in our iTRAQ and Western blot, HP was commonly upregulated in the comparison of microalbuminuric and normoalbuminuric urinary proteome , IL-6, and tumor necrosis factor alpha (TNF-α), which are associated with type 2 diabetes [AGP1 is an indicator of tubular disorder in multiple myeloma [AGP1 levels are elevated in type 2 diabetic patients with kidney disorders [AGP1 was listed in the UPB database, showing upregulation, and is associated with diabetic nephropathy, diabetic kidney disorder, preeclampsia, and acute appendicitis. In our iTRAQ and Western blot, AGP1 was also commonly upregulated in the comparison of microalbuminuric and normoalbuminuric urinary proteome  correlated with other types of diseases [FABP was not listed in the UPB database, and in this iTRAQ and 2-DE, FABP was commonly downregulated in the comparison of microalbuminuric and normoalbuminuric urinary proteome  were confirmed in 9 normoalbuminuric and 14 microalbuminuric urine samples by MRM  and β-chain (aminoacid sequence: 162–406), which are connected by disulfide bridges. In the MRM experiment, the transition (amino acid sequence: 203–215) in the β-chains was used for the relative quantitation of HP. In contrast, portions of both the α-chain and β-chain were used in the iTRAQ quantitation (sequence coverage: 31.0%), and an antibody that targeted a sequence in the α-chain was used for the Western blot analysis. It is conceivable that these disparate targets resulted in contradictory patterns between iTRAQ, Western blot, and MRM. Regardless of the methods or targets, reproducible patterns must be obtained with each method.In the interactive plots, Furthermore, we performed a multiplex assay to improve AUC values with 3 biomarker candidates , obtaining a merged AUC value of 0.921, which is greater than those of the individual proteins  .A1AT, AGP1, and PSCA are excellent biomarker candidates, with AUC values > 0.8; combining the candidates improved the AUC value of 0.921. Accordingly, the other differentially expressed proteins from our iTRAQ experiment in Although our results require further validation in a larger collection of urine samples that contains various control samples, it appears that Microalbuminuria is used as a noninvasive index for the detection of diabetic renal disease. Yet, more specific and accurate biomarkers for DN are required, particularly in type 2 diabetic patients, due to several reasons, including nonspecific detection in nondiabetic renal disease, cardiovascular disease, inflammation, and hypertension. In our iTRAQ experiments, 710 urinary proteins were identified at a >95% confidence level, of which 196 were differentially excreted by >1.25 or <0.80—99 and 97 proteins were up- and down-regulated, respectively .A1AT, AGP1, and PSCA, which had AUC values > 0.8, are good biomarker candidates, and we improved the AUC value to 0.921 on combining the 3 proteins.We prioritized 196 proteins to select preliminary biomarker candidates by characterizing them with regard to “biological process” and “molecular function” and associating them with pathogenesis. Consequently, 10 proteins were selected. To confirm and validate these candidates, 2-DE, Western blot, and MRM were performed. Based on the MRM results, Further validation studies on other differentially excreted proteins might contribute to a greater understanding of the mechanism of renal dysfunction and its association with the pathogenesis of DN, facilitating the development of better biomarkers for DN.


Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster  thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as “Candidatus Nitrosotenuis uzonensis”, is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, “Candidatus N. uzonensis” also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in “Ca. N. uzonensis”, which potentially include genetic determinants of ecological niche differentiation.The discovery of ammonia-oxidizing archaea (AOA) of the phylum  Thaumarchaeota [The discovery of ammonia-oxidizing archaea revealed a new microbial group involved in nitrogen cycling -3. All Archaeota ,5, whoserchaeota . While urchaeota ,8, it harchaeota –12, ocearchaeota –15 and srchaeota ,17.amoA)-like genes in high temperature habitats up to 97°C has been demonstrated. So far, such amoA-like gene sequences have been found in subsurface thermal springs [in situ nitrification [amoA gene transcription [In contrast to AOB, AOA also play a crucial role for nitrification in high temperature habitats -20. In h springs ,24, many springs ,20,25-27 springs ,29. Meascription ,30 in secription ,32 and tcription ,33, in tcription -37.Despite the global distribution and ecological significance of AOA, knowledge of the physiology and genomic make-up of these organisms are still relatively sparse. This knowledge gap can partly be attributed to the difficulties of cultivating the slow-growing AOA under laboratory conditions. Thus, only few enrichment or pure cultures of AOA are currently available ,22,38-44Candidatus Nitrosotenuis uzonensis”, represents a lineage of AOA that is globally distributed in terrestrial high temperature environments, but was not brought into culture previously. Furthermore, a near-complete genome sequence of “Candidatus Nitrosotenuis uzonensis” was obtained and we demonstrate the presence of (i) genes typically found in chemolithoautotrophic thaumarchaeotes, (ii) provide evidence for flagellation and chemotaxis, and (iii) confirm the status of “Candidatus Nitrosotenuis uzonensis” as a new species and member of a new genus by average nucleotide identity analyses.In this study, we inoculated mineral medium with samples from a hot spring located in the Uzon caldera on the Kamchatka peninsula (Russia). Stable ammonia-oxidizing enrichment cultures were established and grown at 46°C. We present evidence that in one of these cultures a novel, moderately thermophilic AOA strain is solely responsible for the observed oxidation of ammonia. We demonstrate that this thaumarchaeote, which we designate as “Nitrospira [An ammonia-oxidizing enrichment culture seeded with material from a geothermal spring located in the Uzon caldera on the Kamchatka peninsula (Russia) was established and maintained in the laboratory for seven years. During the initial enrichment phase, different incubation temperatures were tested and the enrichment was observed to oxidize ammonia within a range of 28-52°C. For all further incubations 46°C was chosen, because it resulted in the highest ammonia removal rates. In later stages of the enrichment nitrite accumulated  and no ntrospira .-1) and streptomycin (50 mg L-1) followed by inoculation into fresh medium (10 vol% of the culture). This procedure was repeated three times and subsequently the culture was further purified by repeated subculturing from diluted suspensions. At the time of manuscript submission, only one morphotype was visible in the enrichment culture by phase microscopy, but PCR screening with three different primer sets (Table S10 in -1) and rifampicin (20 mg L-1). While most archaea are resistant to rifampicin, many rifampicin-sensitive halobacterial and methanogenic species have been reported [-1) of organic substrates, namely formate, acetate, succinate, and pyruvate (shown to have a growth-stimulating effect on N. viennensis EN76) [-1 salinity with an optimum at 0.5 g salinity (data not shown). In contrast to the primary enrichment, culture N4 ceased to oxidize ammonia in the CaCl2-based medium as soon as the pH dropped below 6 (for comparison the pH at the sampling site was 6.5). Re-adjusting the pH did not restore ammonia-oxidizing activity. Consequently, culture N4 was till then maintained exclusively in CaCO3-based medium.A highly enriched AOA culture, in the following referred to as “culture N4”, was obtained by consecutive application of vancomycin   did not amoA genes from this culture were unsuccessful even when up to 40 PCR cycles were used. Similarly, no AOB were detected by fluorescence in situ hybridization (FISH) using established rRNA-targeted probes that are specific for betaproteobacterial AOB  [Nitrosopumilus maritimus SCM1  . The nexamoA and amoB (ammonia monooxygenase subunits A and B) genes, and 39 and 12 clones, respectively, were randomly picked and sequenced. All obtained amoA and amoB gene sequences were highly similar (≥99.1% nucleotide identity for each gene) and clearly differed from those of other identified AOA . Intriguingly, the amoA sequence of the novel thaumarchaeote is closely related to sequences that have been obtained from various geographically distant high temperature terrestrial environments  FISH using probes Arch915 and Cren512  to the sequences retrieved from the aforementioned clone libraries. No sequence indicative of the presence of ammonia or nitrite oxidizing bacteria or other archaea was found in the metagenome.The genome of the novel AOA is represented by a single scaffold containing 14 contigs with a total length of 1,649,125 bp, mean sequence coverage of 39.4 and an average GC-content of 42.2%. Other key features of the genome are listed in N. maritimus SCM1  are present. The amo genes are clustered with an arrangement identical to the order in the genomes of N. maritimus SCM1, “Ca. N. limnia” SFB1 and “Ca. N. koreensis” MY1  [The genome of the enriched thaumarchaeote encodes all proteins that have been hypothesized to be involved in the oxidation of ammonia and subsequent electron transport in C, amoB) ,51,52,55N. maritimus SCM1, “Ca. Nitrosopumilus Salaria” and “Ca. N. limnia” sp. [Ca. Cenarchaeum symbiosum” A was reported to harbour all essential genes for ureolysis [Nitrososphaera cluster [Ca. N. gargensis” [While two AmtB-type ammonia transporters were identified  ,51,56. Tlture N4 , which clture N4 . This fi N4 . As in other AOA, canonical PHA depolymerases are absent from the strain N4 genome. In contrast to “Ca. N. gargensis”, Raman micro-spectroscopic analyses of strain N4 cells did not reveal signature bands of polyhydroxyalkanoates (data not shown), but this does not exclude the production of this storage polymer under different growth conditions.As all other genome-sequenced AOA, strain N4 encodes a polyhydroxyalkanoate synthase, the key enzyme for the production of polyhydroxyalkanoate (PHA), which most probably starts from the 3HP/4HB-cycle intermediate 3-hydroxybutyryl-CoA . Similarrgensis” , the enzN. maritimus SCM1 and “Ca. C. symbiosum” A, but were identified in “Ca. N. limnia” sp. as well as in the moderate thermophile “Ca. N. gargensis”, and are typically clustered in a single region  and euryarchaeotal/bacterial (ftsZ) cell division machinery components  that mainly contain genes specific for the novel thaumarchaeote. This analysis revealed numerous RGP with a total length of ~503 kb  of the novel thaumarchaeote and 1,205 CDS (67.1%) of N. maritimus SCM1 are organized in 106 syntenic regions (syntons) with an average length of 11 CDS. A genome-wide dot plot  of the novel thaumarchaeote and 1,125 CDS (55.5%) of “Ca. C. symbiosum” A. However, these syntons were shorter with an average length of 7 CDS and much more scattered, with genome-scale synteny being very low  and 64.8% (compared to "Ca. C. symbiosum" A) are far below the threshold of 95-96%, which corresponds to DNA-DNA hybridization values of 60-70% and has been suggested as the ANI boundary for circumscribing archaeal and bacterial species [N. maritimus SCM1) and 0.55 (compared to "Ca. C. symbiosum" A). In general, much higher correlation coefficients (>0.99) correspond to ANI values above 96% [The phylogenetic analyses of four marker genes  indicate species . Tetranubove 96% . Thus, mNitrosopumilus cluster that is responsible for the observed oxidation of ammonia. We propose the following Candidatus status for this archaeon: “Nitrosotenuis uzonensis” fam et. gen. et sp. nov. In this study we demonstrate that strain N4, which we obtained in our moderately thermophilic enrichment culture, is a thaumarchaeote of the Nitrosus (Latin masculine adjective), nitrous; tenuis (Latin masculine adjective), small/slender; uzonensis (Latin neutrum genitive), from Uzon.A terrestrial thermal spring located in the Uzon caldera on the Kamchatka peninsula, Russia.Nitrosopumilus cluster as defined by [Thaumarchaeota within the domain Archaea.A chemolithoauotrophic ammonia oxidizer affiliated with the fined by  of the pCa. N. yellowstonii” [Nitrosocaldus cluster and “Ca. N. gargensis” [Nitrososphaera cluster, this archaeon is the third identified species of AOA growing at elevated temperatures. “Ca. Nitrosotenius uzonensis” is the first cultured representative of Nitrosopumilus-cluster AOA with worldwide distribution in terrestrial geothermal habitats. Furthermore, as evidenced by the phylogenetic clustering of our AOA with the recently cultivated strains “AOA-AC5” and “AOA-DW” [In addition to “wstonii”  of the Nrgensis”  of the N“AOA-DW” , this ph2PO4, 74.4 mg KCl, 49.3 mg MgSO4 ×7H2O, 584 mg NaCl, 33.8 µg MnSO4, 49.4 µg H3BO3×7H2O, 43.1 µg ZnSO4×7H2O, 37.1 µg (NH4)6Mo7O24×4H2O, 97.3 µg FeSO4×5H2O, and 25.0 µg CuSO4×2H2O. Furthermore, either 147 mg CaCl2 or 4.0 g CaCO3 L-1  was added. At late stages of the enrichment, only CaCO3-based medium was used. Typically, fresh medium contained 1 mM NH4Cl. In the case of CaCl2-medium, the pH was adjusted to 7.4-7.8 by adding 1% (w/v) NaHCO3 solution. To further enrich for AOA, consecutive application of vancomycin (100 mg L-1) and streptomycin (50 mg L-1) was performed, which was followed by inoculation into fresh medium (10 vol%). Sodium salts of either formate, acetate, pyruvate, or succinate were added (20 mg L-1) to test whether strain N4 used these organic substrates. The enrichment culture was regularly transferred by inoculation of flasks containing fresh medium with 10 vol% of the culture. The concentrations of nitrite and nitrate were regularly measured by using Merckoquant test stripes . For recording substrate turnover, ammonium, nitrite, and nitrate concentrations were precisely measured as described recently [An ammonia-oxidizing enrichment culture, inoculated with water and sediment from a terrestrial thermal spring located in the Uzon caldera on the Kamchatka peninsula (Russia), was established in 2006 and maintained for seven years. A temperature of 45°C and a pH of 6.5 were measured at the sampling site. The concentration of ammonium was 34.4 µM, whereas nitrite and nitrate were not detectable at the time of sampling [recently ,74.2 and 1 U of Taq-polymerase  in combination with 50 pmol of the respective oligonucleotide primers. In all PCR 30 cycles were used to amplify the target DNA and a final elongation step of 10 min at 72 °C was performed. A list of all primers with the applied annealing temperatures is provided in Table S1 in amoA genes was attempted by using primers amoA1F and amoA2R [amoA gene of Nitrosomonas sp. as template was used as positive control. Cloning and sequencing were performed as described elsewhere [hcd genes were screened, prior to sequencing, by RFLP analysis using the restriction endonucleases MspI and AluI (16S rRNA) or RsaI (hcd) , whereas amoA and amoB clones were randomly chosen for sequencing without RFLP analysis. For each unique RFLP pattern at least one representative clone was sequenced. Phylogenetic analyses were conducted using the programs ARB [DNA was extracted from enrichment culture N4 either by phenol/chloroform-extraction including a bead-beating step  or by ud amoA2R , and a plsewhere . The clorams ARB  and Phylrams ARB  with comrams ARB  without in situ detection and visualization of bacterial and archaeal cells in enrichment culture N4, FISH and CARD-FISH were performed as described elsewhere [-1) and were microscopically analyzed using a Zeiss LSM 510 confocal laser scanning microscope and the included software. The absence of unspecific fluorescence after CARD-FISH was confirmed by using probe NON-EUB .For obtaining biomass for metagenome sequencing, culture N4 was grown aerobically in 300 ml Erlenmeyer flaks as described above. Biomass was harvested by centrifugation  and frozen (-20 °C) until phenol/chloroform-based DNA extraction (see above) was performed. The genomic DNA was submitted to mechanical fragmentation through nebulization (hydroshear) with a 3 kb mean fragment size. After recircularization using an adapter, the DNA was fragmented using an E210 ultrasound DNA-shearer , and 600 bp fragments containing the adapter were selected for sequencing. Three quarters of a 454 Titanium mate-paired run were performed, generating ~250 Mb of sequence in 796,942 reads. In addition, Illumina (GA-IIx) single read sequencing was performed, generating 34,689,904 reads of 36 bp each. The 454-reads were subjected to assembly by using the Newbler software package, generating 739 contigs on 41 scaffolds with a cumulative size of ~5.5 Mb. The largest of these scaffolds, containing 14 contigs with a total length of 1,649,125 bp and a mean contig coverage of 39.4, represents the genome of strain N4. The Illumina reads were used to correct 454 sequencing errors as described by Aury et al. . CorrectCa. Nitrosotenuis uzonensis” has been deposited at the European Nucleotide Archive with the master reference number CBTY000000000.The genome sequence of “Figure S1Ca. Nitrosotenuis uzonensis”.Phylogenetic analysis of a selection of AmoA sequences obtained from geothermal sites that are closely related to the sequence of “ The tree shows sequences affiliated with the Nitrosopumilus cluster, while sequences belonging to the Nitrososphaera, Nitrosotalea and Nitrosocaldus clusters have been used as outgroup. If available, temperature, pH and sample type are indicated for clones obtained from geothermal systems (in red font). Circles on tree nodes indicate parsimony bootstrap support ≥90%. Numbers in parentheses indicate the number of sequences within a group. The scale bar equals 10% estimated sequence divergence.(TIF)Click here for additional data file.Figure S2Ca. N. uzonensis” chromosome.Circular representation of the “ Contigs are separated by white space in all rings. Predicted coding sequences (rings 1+2), RNA genes (ring 3), regions of genomic plasticity compared to N. maritimus SCM1 (ring 4) or “Ca. Cenarchaeum symbiosum” (ring 5), and local nucleotide composition measures (rings 6+7) are shown. Very short features were enlarged to enhance visibility.(TIF)Click here for additional data file.Figure S3Ca. Nitrosotenuis uzonensis” to the genomes ofDot plots representing genome-wide alignments of “ (A) Nitrosopumilus maritimus SCM1, (B) “Ca. Nitrosoarchaeum limnia” SFB1, and (C) “Ca. Cenarchaeum symbiosum” A. Forward matches are shown in red, while reverse matches are shown in blue.(TIF)Click here for additional data file.File S1Combined file containing tables S1-S10.(XLSX)Click here for additional data file.


Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-β-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan. One potential source for discovering novel molecular mechanisms that protect against malignancy is a longevity model, because a major cause of premature death in mice can be attributed to this disease process. One such model, which we discovered and described, is the knockout of adenylyl cyclase type 5 (AC5). These mice live a third longer than wild-type (WT) littermates and are protected from the cardiomyopathy of aging  is a ubiquitous enzyme that catalyzes the synthesis of cAMP, activating PKA and the cAMP response element-binding protein (CREB)  approved for antiviral therapy in patients, Vidarabine, also is a specific inhibitor of AC5  and a lower mammary tumor incidence . The results were striking: by day 521 all WT MMTV-Her-2 neu mice developed mammary carcinomas and died, but there were no deaths in AC5KO-MMTV-Her-2 neu at that time  and we confirmed that AC5KO × MMTV-Her-2 neu mice have significantly lower body weight (P < 0.01).It is well known that obesity and diabetes are established risk factors for the development of postmenopausal breast cancer and are associated with the increased risk of other cancers, for example, endometrium, kidney, colon, and esophagus . These results were striking: at day 27, tumor volume in WT was 6-fold greater than in AC5KO , while average wet tumor weight was less than half in AC5KO (P < 0.01). These data suggest that the effects of AC5KO might be not only at the level of cellular transformation, but also at the level of tumor growth and progression.The third goal was to examine the extent to which AC5KO mice are protected against syngeneic tumor growth for which we used the standard B16F10 melanoma model. After B16F10 melanoma cells of C57BL/6 origin were inoculated s.c., AC5KO demonstrated decreased tumor growth compared with AC5WT , P < 0.01.We next assessed the mechanisms responsible for reduced B16F10 melanoma growth. AC5KO mice were protected through several mechanisms that regulate tumor growth, for example, angiogenesis, apoptosis, and tumor cell proliferation , demonstrating its ability to block endogenous AC in these tumor cells. Inhibiting cAMP reduces expression of PKA-CREB-dependent genes involved in angiogenesis, inflammation, and tumor growth  compared with vehicle-treated mice (P < 0.05 and P < 0.05). We measured the intratumor cell proliferation by nuclear staining of Ki-67 antigen and observed that tumors from AC5 inhibitor-treated mice exhibited a significantly reduced cell proliferative index (P < 0.01). In addition, the AC5 inhibitor doubled the apoptotic index in B16F10 tumors . We also found that the percentage of cells with apoptosis was increased in LP07 cells treated with the AC5 inhibitor . To elucidate the effects of the AC5 inhibitor on endothelial cells, we used HUVEC cells that have a population doubling time of approximately 24 h and found that the viability of HUVEC was reduced with the AC5 inhibitor (50 μm) after 24 h .The AC5 inhibitor also protected against tumor angiogenesis. Staining of tumors with CD31 after 22 days of treatment showed that intratumor microvessel density was reduced by 50%  and the forskolin-induced secretion (P < 0.01). Angiogenesis was also evaluated by staining the tumors after 30 days of treatment with the AC5 inhibitor or vehicle with CD31 and found reduced microvascular density. Lastly, in vivo angiogenesis was also evaluated. The density and length of vessels induced by the LP07 tumors were reduced in AC5 inhibitor-treated mice  for in vitro experiments.LP07 lung adenocarcinoma cell line was obtained ad libitum. Studies were approved by the Animal Care and Use Committees of Clemson University, Instituto de Oncologia ‘Angel H. Roffo’ and New Jersey Medical School.AC5KO mice on C57BL/6 or mixed 129SVJ/C57BL/6 background were used. FVB/N Tg (MMTVneu) 202Mul/J mice, expressing the WT rat neu transgene under the control of the mouse mammary tumor promoter (MMTV-neu), were obtained from Jackson Laboratories . AC5  mixed 129SVJ/C57BL/6 background, and AC5  × MMTV-neu female mice were obtained and monitored for tumor incidence along their life. BALB/c and C57BL6, 2–4 month-old mice were used for the short-term mouse tumor experiments. They were weight-matched and kept in our Animal Care Division. Mice were housed in a pathogen-free facility under a 12:12 h light–dark cycle with access to water and food 4 B16F10 cells (C57BL/6 origin) suspended in 0.2 mL medium were injected s.c into the right flank of mice (n = 4–9/group). (B) AC5 inhibitor on B16F10 melanoma growth: miniosmotic pumps  containing vehicle or AC5 inhibitor (50 mg kg−1 day−1) were implanted s.c in C57BL6 mice 3 days after being treated; 8 × 104 B16F10 were injected into the right flank of mice (n = 14/group) and treatments continued till the end of the experiment. (C) AC5 inhibitor on LP07 lung cancer growth: LP07 cells (3 × 105) were inoculated s.c. in the right flank of mice BALB/c (n = 9). When tumors became palpable, animals were treated daily with Vidarabine (50 mg kg−1 day−1) or vehicle intraperitoneally during 30 days. Vehicle was a 0.25% sodium carboxymethylcellulose solution that was stored at −20 °C, thawed overnight, and completed with povidone stock solution  to a 0.085% final concentration. Vidarabine (50 mg kg−1 day−1) or vehicle was prepared weekly, and a magnetic stir bar was added to the bottle to keep the suspension phase throughout the dosing. Tumor growth was assessed by measurement of tumor diameter in the longest dimension (L) and at right angles to that axis  using a Vernier caliper. Tumor volumes were estimated using the formula, L × W × W × π/6.(A) AC5  experiments: 8 × 10et al.  with 8 × 104 B16F10 cells in 250 μL DMEM plus trypan blue  cells in BALB/c mice (n = 5/group) in 0.1 mL MEM plus trypan blue to localize the site of inoculation. After 7 days, mice receiving Vidarabine (50 mg kg−1 day−1) or vehicle were euthanized and the inner skin was exposed, and the vascular response was observed with a dissecting microscope using 6.5× magnification. Inoculated sites were photographed using a high-resolution camera. A microscope micrometer was used to calibrate length in mm. The total length of vessels was calculated based on the calibration by the Image Pro Plus software. In vivo angiogenesis was represented by the total length of vessels around the tumors and expressed as percentage of inhibition compared with AC5WT or vehicle-treated mice.Ten-week-old female AC5WT, AC5HET, AC5KO mice were injected intradermally in the dorsal midscapular part of mice  and CD31  proteins, respectively. Proliferation was assessed by counting the number of tumor cells with positive nuclei (for Ki-67) at 400× magnification. Ten fields were counted per sample and results were expressed as the proliferation index: proportion of the positively staining cells over the total number of cells. Microvessel density was assessed by counting the number of CD31 positive vessels at 400× magnification in the fields with the highest vascularization -mediated dUTP nick-end labeling (TUNEL)  as described previously  following manufacturer’s recommendations.et al., m Tris–HCl (pH 7.6), 150 mm NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS with phosphatase inhibitor cocktails 1 and 2 (Sigma), protease inhibitor cocktail (Thermo Scientific), and 1 mm NaF. Equal amounts of protein (20–90 μg) were subjected to SDS-PAGE. After electrophoresis, samples were transferred to millipore immobilon-P membrane and immunoblotted with antibodies against β-actin , Bcl-2, total caspase-3 (Santa Cruz Biotechnologies), and cleaved caspase-3 .The expression of AC5 in tumor cell lines was quantified using a monoclonal antibody as described previously (Hu 3), flat-bottomed plates in 10% FBS MEM or DMEM media or 2% FBS EGM-2 and allowed to adhere overnight. Then, fresh medium containing vehicle or Vidarabine (0–50 μm) in serum-free or 2% FBS medium was added. At 24–72 h, the culture medium was replaced with 3--2.5-diphenyltetrazolium bromide . The reaction was stopped 4 h later by removing the medium and then adding 100 μL DMSO. Absorbance was measured after 30 min at 560 nm in a 96-well plate reader. IC50 values were calculated using GraphPad Prism software .LP07, B16F10, LLC1 (ATCC), and HUVEC were seeded in 96-well . 2.5 × 105 LP07, LLC1, or B16F10 cells were seeded in 96-well plates in basal medium containing vehicle or AC5 inhibitor (0–5 μm) and incubated for 90 min. Then, the medium was discarded; cells were fixed, and stained with 100 μL of methanol/crystal violet solution. After 3 h of incubation, wells were washed with PBS and then cells were lysed with 250 μL SDS. Absorbance was measured with spectrophotometer at 595 nm.10m) and 105 LLC1 or B16F10 cells were seeded on top of the membrane of each transwell insert in basal DMEM with vehicle or AC5 inhibitor (0–5 μm), and migration was evaluated as described previously  LP07 subconfluent monolayers were pretreated overnight with 2% FBS MEM containing vehicle or Vidarabine  overnight. Using the Imagine Pro Plus software, the total capillary tube length was calculated.HUVEC capillary network m) for 24 and 48 h. Then, cells were trypsinized, washed, suspended in ice-cold PBS (pH 7.4), counted, and fixed overnight in 50% ethanol at 4 °C. Then, (106 mL−1) cells were resuspended in 1.12% sodium citrate with RNase A (500 units mL−1) for 30 min at 37 °C and then propidium iodide (50 μg mL−1) was added. Red fluorescence of single events was recorded using an argon ion laser at 488-nm excitation wavelength and 610-nm emission wavelength to measure DNA index on a Coulter Epics XL flow cytometer. The percentage of cells present in each phase of the cell cycle was determined using ModFitLT V2.0 software from Verity Software House . The percentage of apoptotic cells as well as cells in G0/G1, S, and G2/M phases was calculated.LP07 cells were treated with vehicle or Vidarabine (0–5 μm). Thirty-six hours later, cover slips were stained with DAPI and mounted on slides with PBS: Glycerol (1:1). Cells were observed for nuclear condensation/fragmentation as an indicator of apoptosis with a fluorescent microscope. Results were expressed as the percentage of total apoptotic cells vs. total number of cells.LP07 cells were treated with vehicle or Vidarabine .spss  and GraphPad InStat  softwares. Log-rank, Chi-square test, Student’s t-test, and Fisher exact test were used, and one-way analysis of variance (ANOVA) was used when comparing more than two groups. After ANOVA, Tukey–Kramer multiple comparisons test was used. The level of statistical significance for all tests was P < 0.05.Statistical comparisons were performed by


Carnobacterium gilichinskyi strain WN1359, previously isolated from Siberian permafrost and capable of growth under cold (0°C), anoxic, CO2-dominated, low-pressure (0.7-kPa) conditions in a simulation of the Mars atmosphere.We report the complete genome sequence of  Carnobacterium sp. strain WN1359, isolated from Siberian permafrost, was capable of growth under a combination of low-temperature (0°C), low-pressure (0.7-kPa), and CO2-enriched anoxic conditions intended to simulate the atmosphere of Mars . As part of an effort to further investigate the molecular basis of the response of this organism to simulated Martian conditions, we report here the complete genome sequence of the strain.Recently it was reported that C. gilichinskyi strain WN1359 is from the corresponding author’s strain collection and is deposited as strain DSM 27470T in the German Collection of Microorganisms and Cell Cultures (DSMZ) (http://www.dsmz.de). Its genome was sequenced at the University of Florida Interdisciplinary Center for Biotechnology Research (UF-ICBR) using the PacBio SMRT system . A total of 78,692 reads were obtained, with a mean read length of 3,850 bp. The initial PacBio reads were error corrected using the PacBio RS_PreAssembler.1 module with minimum subread length of 500 bp, minimum read quality of 0.80, and minimum seed read length of 6,000 bp. The error correction process yielded 10,736 reads with an average length of 6,421 bp. A single scaffold was assembled directly from the error-corrected reads using Celera assembler (CA) version 7.0 software. The initial genome assembly was further refined using the PacBio RS_Resequencing.1 module with Quiver consensus calling. This process removes sequencing errors remaining in the initial CA assembly and produces the final consensus genome sequence. The chromosome has 2,347,813 bp and an overall GC content of 35.26%. We detected the presence of five plasmids in the genome, consisting of 9,615 bp (designated pWNCR9), 12,656 bp (pWNCR12), 15,476 bp (pWNCR15), 47,068 bp (pWNCR47), and 64,492 bp (pWNCR64). Open reading frame (ORF) prediction and annotation were performed through the Rapid Annotations using Subsystems Technology (RAST) pipeline (CP006812 (chromosome) and CP006813, CP006814, CP006815, CP006816, and CP006817 (plasmids).The results of this whole-genome shotgun project have been deposited with GenBank under accession numbers


According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK.Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF- Panax ginseng C.A. Meyer) is a representative herbal which is ethnopharmacologically well known in East Asian countries, including Korea, China, and Japan for about 2,000 years. This plant is indeed widely used as a supplementary herbal medicine for treating numerous diseases such as cancer, diabetes, and atherosclerosis [Korean ginseng  . Thus, ised mice , 7–9. RGsed mice , 11. Fursed mice .Although previous papers indicated its immunostimulatory roles in various immune cells, the exact molecular mechanism of RGAP in macrophages has not been fully elucidated. In this study, therefore, we aimed to explore how RGAP can stimulate functional activation of macrophages by measuring molecular events and characterizing surface receptors and also understand how the immunostimulatory activity by RGAP occurs. Panax ginseng C.A. Meyer) as described previously [E. coli 0111:B4) were purchased from Sigma Chemical Co. . Piceatannol, SP600125, U0126, PP2, and pam3CSK were obtained from Calbiochem . β-glucan was purified from Lentinus edodes [κBα,γ-tubulin, β-tubulin, and β-actin were obtained from Cell Signaling . RGAP isolated from Korean red ginseng was performed by steaming and drying fresh ginseng root  were plated in 100 mm tissue culture dishes for 4 h at 37°C in a 5% CO2 humidified atmosphere.Peritoneal exudates were obtained from wild-type or RGS-2 knockout C57BL/6 male mice  by lavaging 4 days after intraperitoneal injection of 1 mL of sterile 4% thioglycollate broth  as reported previously . After w2. For each experiment, cells were detached with a cell scraper. Under our experimental cell density (2 × 106 cells/mL), the proportion of dead cells was less than 1%, according to Trypan blue dye exclusion tests. Peritoneal macrophages and RAW264.7 cells were cultured with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum , glutamine, and antibiotics (penicillin and streptomycin) at 37°C under 5% CO6 cells/mL) for 18 h, cells were treated with RGAP (0 to 4 mg/mL) or LPS (1 μg/mL) for 24 h. The inductive effect of RGAP on NO production was determined by analyzing NO levels with the Griess reagent, as described previously [After preincubation of RAW264.7 cells , according to the manufacturer's instructions. The total RNA was stored at −70°C until use. Semiquantitative RT reactions were conducted as reported previously , 22. TheκBα, IKKβ, Akt, p85/PI3K, PDK1, γ-tubulin, and nonreceptor tyrosine kinases (Src and Syk) was done according to previously published methods [Preparation of total lysates and nuclear extracts from LPS-treated RAW264.7 cells pretreated with RGAP or LPS was done using a method previously published , 24. Imm methods .P < 0.05 was considered a statistically significant difference. All statistical tests were carried out using the computer program SPSS .Data Figures , and 6, Lentinus edodes is composed of the basic structure of a β-1,3-glucan with β-1,6-glucopyranosidic branches and has also showed immunostimulating properties [Polysaccharides isolated from basidiomycetes have been reported to act as immunostimulators. The fungal polysaccharides  originating from operties –28. In coperties , but theγ-glutamic acid, lactic acid bacterium-derived peptidoglycans, and β-glucans [μg/mL) was distinct from that of RGAP (2 mg/mL). For example, the morphological change of macrophages observed by LPS exposure was not induced by RGAP and IL-4 , AP-1 (c-Jun and c-Fos), CREB, ATF-2, and STAT-1 required for iNOS promoter activity [κB activation in macrophages have been reported previously [To confirm whether the production of NO by RGAP is managed by cellular transcription factor activation, levels of iNOS expression and transcription factors required for iNOS gene expression were explored. As activity  was alsoeviously . Howeverβ-glucans and lactic acid bacterium-derived peptidoglycans [κB, AP-1, CREB, and ATF-2 in RGAP-induced macrophage activation was also demonstrated by measuring the phosphorylation levels of their upstream signaling enzymes [κB signaling, RGAP enhanced the phosphorylation of Akt and IκBα within 5 to 15 min, while LPS only strongly enhanced IκBα at 5 min (κBα at 5 min is critically regulated by Syk activity [κB stimulated by RGAP and LPS. For MAPK activation, ERK, JNK, and p38 seemed to be activated at 5 min. In contrast, LPS-induced MAPK signaling events were distinctly seen at 15 to 30 min (To compare the difference in transcriptional activation levels of macrophages between LPS and RGAP, intracellular signaling events were also investigated. As shown in oglycans . These d enzymes . For NF-at 5 min . Accordiactivity , Syk seeo 30 min . Therefoβ-glucan and Zymosan [β-glucan (a dectin-1 ligand; Finding the molecular target of RGAP is important in understanding the molecular mechanism of action for RGAP-induced immune responses. Considering that RGAP is not able to penetrate into cell membranes like other glucose moiety-containing immunogens , it is e Zymosan . Indeed,β-glucans and polysaccharides from mushrooms [Finally, even though ginseng has been known to boost the body's immune responses, the stimulatory effect of ginseng polysaccharide fraction was marginal, as assessed by NO production . Compareushrooms . TherefoκB, AP-1, and CREB. Evaluation with specific enzyme inhibitors, phosphorylation levels of intracellular signaling proteins, and inhibitory patterns with antagonistic antibodies suggested that ERK and JNK were the most important signaling enzymes for RGAP and TLR2 may be a surface receptor for RGAP. Lastly, macrophages from RGS2 knockout mice and wortmannin exposure in RAW264.7 cells demonstrated marked upregulation of RGAP-treated NO production. Therefore, our results strongly suggest that RGAP can be used as an immunostimulatory remedy via TLR2-mediated functional activation of macrophages, which can be boosted by wortmannin-targeted enzymes. In summary, we found that RGAP treatment induced NO production in RAW264.7 cells without altering morphological changes, unlike LPS. RGAP-induced NO production was associated with enhanced levels of iNOS and nuclear transcription factors such as NF-


Histochemistry and ex vivo glycerol releasing assay showed that combined treatment accelerated lipid mobilization in adipose tissue. Combined treatment also increased the transcription of glut4, hsl, atgl and adiponectin, and decreased that of plin1, cd11c, ifnγ and leptin. Combined treatment markedly elevated the transcription of fgf21 in liver but not in adipose tissue. These results suggest that concurrent activation of LXR and PPARα as a strategy to control glucose and lipid metabolism in obesity is beneficial but could lead to elevation of lipid accumulation in the liver.Liver X receptor (LXR) activation improves glucose homeostasis in obesity. This improvement, however, is associated with several side effects including hyperlipidemia and hepatic steatosis. Activation of peroxisome proliferator-activated receptor alpha (PPARα), on the other hand, increases fatty acid oxidation, leading to a reduction of hyperlipidemia. The objective of this study was to investigate whether concurrent activation of LXR/PPARα can produce synergistic benefits in treating obesity-associated metabolic disorders. Treatment of high fat diet-induced obese mice with T0901317, an LXR activator, or fenofibrate, the PPARα agonist, or in combination alleviated insulin resistance and improved glucose tolerance. The combined treatment dramatically exacerbated hepatic steatosis. Gene expression analysis in the liver showed that combined treatment increased the expression of genes involved in lipogenesis and fatty acid transport, including  Liver X receptors (LXR) are transcription factors belonging to the nuclear receptor superfamily. Since their initial discovery in 1995, LXRs have emerged as powerful metabolic regulators in different tissues and cell types. LXRs have been shown to regulate cholesterol, bile acid, triglyceride and glucose homeostasis as well as inflammation and intestinal lipid absorption et al. and Laffitte et al. show that LXR activation improves glucose tolerance in diabetic animal models LXR activation produces a variety of beneficial effects in managing metabolic disorders. For example, previous studies by Cao On the other hand, activation of PPARα accelerates lipid absorption and increases fatty acid oxidation, leading to an improvement in lipid metabolism and a reduction of hyperlipidemia The focus of the current study is to assess the effects of concurrent activation of LXR and PPARα on systemic metabolism and hepatic fat accumulation under the status of obesity in which the metabolism of glucose and lipids are dysregulated. We demonstrate that combined treatment by T0901317, a potent activator of LXR, and fenofibrate, an agonist of PPARα, alleviated insulin resistance and improved glucose tolerance. Surprisingly, this combined treatment dramatically exacerbated hepatic steatosis in obese mice. Mechanistic studies suggest the exacerbation effect is caused by increased lipogenesis in the liver and accelerated lipid mobilization in the adipose tissue.The use of animals in this study was in compliance with relevant federal guidelines and institutional policies and the animal protocol was approved by the IACUC of the University of Georgia.i.p.), fenofibrate  or a combination of T0901317 and fenofibrate , respectively. The injections were performed daily for 5 days while keeping animals on high fat diet. After the last injection, mice were fasted overnight and sacrificed using carbon dioxide.Male C57BL/6 mice  were fed a high fat diet  for 12 weeks and became obese. These mice were then divided into four groups (5 each), including the control group, a T0901317-treated group, a fenofibrate-treated group and a group with a combined treatment of T0901317 and fenofibrate . Mice in the control group were injected with carrier solution (dimethyl sulfoxide), and mice in treated groups were injected with T0901317  at 1.5 g/kg, and the time-point was set as 0 min. Blood glucose levels at 0, 30, 60 and 120 min were measured using glucose meters and glucose test strips. Mice utilized in ITT were fasted for 4 h before the injection of insulin  from Eli Lilly , and blood glucose levels were measured at 0, 30, 60 and 120 min after insulin injection.IPGTT and ITT were performed using 4 groups of obese mice (5 each) treated with the same regimen. Mice employed in IPGTT were fasted for 6 h before the test. Glucose solubilized in phosphate buffered saline was injected (For haematoxylin and eosin (H&E) staining, tissues of interest were dissected and fixed overnight in 10% neutral buffered formalin. The samples were dehydrated using gradient ethanol and embedded in paraffin. Tissue sections were cut at 6 µm in thickness and dried at 37°C for 2 h. H&E staining were performed using a commercial kit . For the frozen section and Oil-red O staining, liver samples were frozen in liquid nitrogen and sectioned at 8 µm in thickness using a Cryostat. These sections were placed on slides and washed with 60% isopropanol before being stained with Oil-red O (Electron Microscopy Sciences) for 30 min and counterstained using haematoxylin. The tissue slides were examined using Nikon ECLIPSE-Ti optical microscope and pictures were taken and analyzed using Nikon NIS-Elements AR software.Liver triglyceride levels were determined following a previously reported method Blood samples were collected from animals which were fasted overnight and centrifuged at 3,000 rpm for 10 min to isolate plasma. Plasma concentrations of glucose (Thermo-Scientific), insulin (Mercodia), triglyceride (Thermo-Scientific), cholesterol (Genzyme Diagnostics) and free fatty acids (BioAssay Systems) were determined using commercial kits. Based on fasting glucose and fasting insulin levels, we also calculated the value of Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR) using the following formula: HOMA-IR = [fasting insulin (ng/mL) × fasting plasma glucose (mg/dL)/405] Liver and white adipose tissue samples were freshly collected and immediately frozen at −80°C until use. Total RNA was purified using TRIZOL reagent from Invitrogen . The purified RNA samples were dissolved in RNase-free water and kept at −80°C and quantitative real-time PCR (qPCR) was performed using SYBR Green as detection reagent. The primers for qPCR were synthesized at Sigma  and their sequences are listed in the ex vivo glycerol releasing assay was conducted following a previously reported method 4 and 0.33 mM CaCl2) with 3.5% fatty acid-free BSA and 0.1% glucose. Supernatants were collected at pre-determined time-points and their glycerol levels were measured using a commercial kit purchased from Cayman Chemical .The p value below 0.05 (p<0.05) was considered significantly different.Statistical analysis was performed using one-way analysis of variance. The results were expressed as the mean ± SD. A p<0.01) and T0901317-treated group (p<0.05), suggesting that combined treatment had a synergistic effect on hepatic steatosis in high fat diet-induced obese mice. We also measured liver weight . Less impact was seen in animals treated with fenofibrate (∼7.5 mM). The most significant impact was seen in animals who received the combined treatment reaching blood concentration at 4.9±0.7 mM (p<0.05 vs T0901317-treated group). Similarly, the combined treatment has the highest impact on insulin level with fasting insulin concentration of 1.9±0.5 ng/ml, comparing to 7.0±2.0 ng/ml in control animals, and 3.2±0.9 ng/ml and 4.7±1.0 ng/ml in animals treated with T0901317 or fenofibrate, respectively . Similarly, fenofibrate treatment markedly elevated mRNA levels of cyp4a10 and cyp4a14, 2 target genes of PPARα, by ∼29.8-fold and ∼32.1-fold, respectively . To explore the possible mechanism, we measured the expression of genes involved in lipid and glucose metabolism in the liver. The srebp-1c and chrebp are two master genes for lipogenesis in the liver. Combined treatment significantly increased the expression of srebp-1c and chrebp by 2.2±0.2-fold and 1.6±0.4-fold, respectively . Additionally, we determined the transcription of fgf21 whose expression is regulated by both PPARα and LXR. As expected, activation of either PPARα or LXR markedly increased the transcription of fgf21 .Combined treatment reduced the size of epididymal white adipose tissue (WAT) , indicatex-vivo was ∼15.8, ∼33.8, ∼26.6, and ∼31.8 (µg/100 mg tissue/hour) for control, T0901317-treated, fenofibrate-treated animals, and animals with combined treatment, respectively. Similarly, the value of area under curve (AUC) also shows combined treatment increased glycerol releasing by ∼2 folds  mice acc1, fas and scd1 , finofibrate (FF) or in combination (T09+ FF) on the mRNA level of selected genes in the liver. (A) – (C) The mRNA levels of LXR target genes including cyp7a1, abcg5 and abcg8. (D) – (E) The mRNA level of PPARα target genes including cyp4a10 and cyp4a14. (F) – (C) The mRNA level of a set of nuclear receptor genes including lxrα, pparα, pparγ and pparδ. Values represent average ± SD (n = 4); **p<0.01 vs control.(TIF)Click here for additional data file.Table S1Primer sequences for gene expression analysis.(DOC)Click here for additional data file.


To the Editor: Although most human enterovirus (EV)  infections are asymptomatic, they can cause upper respiratory illness, febrile rash, aseptic meningitis, pleurodynia, encephalitis, acute flaccid paralysis, and neonatal sepsislike disease . The 5´ terminal domain was obtained by reverse transcriptase–PCR with specific primers EV75_sense: 5´-GAAAGCTTYTTCCAAGGGGA-3´ and EV75_anti: 5´-GAGAAGTGKGACCAWCCATC-3´. Phylogenetic analysis of the Spanish isolates and representatives of all other species B EVs showed that the Spanish isolates clustered (bootstrap value 100, To our knowledge, this is the first isolation of EV75 in Spain. Indeed, isolation of EV75 has not been reported in Europe. Given that the European EV75 isolate grows easily in a variety of cell lines, is detected by common EV genus-specific antibodies, and that EV surveillance and typing were performed in Spain since 1988 (The European strains of EV75 appear to represent a different evolutionary lineage than those previously described in the United States, Asia, and Africa (


Epidemics of HFMD are elevated every year globally, especially in mainland China. The disease now presents as an increasing threat to public health worldwide.Five hundred and seventy-one EV71-infected HFMD patients in Beijing You'an Hospital were grouped by disease severity: Mild (no severe complication) (n = 221), and Severe group , and/or pulmonary edema (PE) (n = 350)). Clinical and laboratory findings and levels of 7 serum cytokines were analyzed.9/L (p = 0.040) were associated with severe cases. Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)). In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. “GLU>8.3 and CL<98 and RR>26” (confidence ration (CR) = 100%) is the top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%).Univariate analysis showed that (RR)>26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10Serum levels of IL-2, IL-4, IL-10, IFNγ, GM-CSF, and TNFα were higher in severe cases than in mild cases. A new evaluation scoring system by scoring each risk factor 1 and independent risk factor 2 was developed for early identification of severe HFMD cases.Five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention. Enterovirus genus in the Picornavirus family. Although HFMD could be seen globally, Mainland China is one of the main regions where EV71 outbreaks could be seen, in almost every year recentlyHand, foot and mouth disease (HFMD) is a common disorder in young children. It is considered as a potentially life-threatening diseaseAccumulating evidence from global reports on HFMD epidemics supports the fact that the ratio of severe cases is elevating gradually, along with mortality rateThe objective of this study was to analyze the clinical and laboratory data of a group of pediatric HFMD patients admitted to Beijing You'an Hospital, Capital Medical University, and evaluate the correlations between early clinical-laboratory findings and disease severity. This study aims to identify early indicators of disease severity so that prophylactic measures can be taken to reduce mortality. In addition to evaluate individual markers, we tried to use different combinations of markers as indicators of severity.The case definition is described elsewhereInformation of the study population consisted of 571 children who met the case definition described above was collected retrospectively. The patients were consecutively admitted to Beijing You'an Hospital, Capital Medical University  between Mar and Oct, 2012. All these cases were tested for serum cytokine levels.Patients were divided into two categories based on severity: (1) Mild: patients with HFMD without BE or PE; (2) Severe: HFMD patients complicated by BE or/and PE.All of the parameters included in the investigation were collected by reviewing the patient medical records that were preserved in the medical record library and the medical computerized database at Beijing You'an Hospital, Capital Medical University. The patient records were retrospectively examined for primary set of data, which included demographic characteristics (age and sex), clinical parameters (signs and symptoms), laboratory values , radiologic data, outcome at discharge (recovered or died), admission and discharge dates, and length of hospital stay. Clinical findings and laboratory results analyzed in this study were from the examination done right upon admission of all involved patients.You'an Hospital Ethics Committee has approved this study. It covered the retrospective analysis of the 571 records. Parents or caretakers of all 571 participants have given a written informed consent on behalf of the children participants for their information to be stored and used for research. Human experimentation guidelines of PR.China were followed in the conduct of this clinical research.The blood samples for cytokine determination were collected immediately upon patient admission. The plasma was harvested within 30 min at 37°C of venipuncture from EDTA-anticoagulated blood samples and stored at −70°C until analyzed. The Bio-Plex Human 8-plex kit  was used to detect IL-2, IL-4, IL-6, IL-10, IFNγ, GM-CSF and TNFα levels on Luminex200 xMAP analyzer system , according to the manufacturer's instructions.2 or Fisher's exact test. Continuous data were analyzed by using Spearman's rho correlation analysis. Mann-Whitney U test was used for data that did not have a normal distribution. Correlation among cytokine levels was analyzed by hierarchical cluster analysis. All analyses were performed by SPSS software . P value <0.05 was considered to be statistically significant.Average of data was presented as The demographic and clinical data of 571 HFMD patients are collected and summarized in 9/L (p = 0.040). Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)) >26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10 0.045)) Table 2.Using single parameter to predict disease severity is not a reliable and accurate way to assist diagnosis and initiate early intervention. Therefore, we analyzed various combinations of different important parameters we found above. We tested the positive rates of different combinations of the seven markers and calculated Popularity, Confidence ratio (CR) and Support Table 3.The most reliable combination is “GLU>8.3 and CL<98 and RR>26”, which offered 100% CR, which means all patients with this combination belonged to severe cases. The second most reliable combination is “ALT>40 and LYM>40% and RR>26 and Age<4 yo”, whose CR was 92.9%. Among the top 10 combinations, there are two very simple ones, “GLU>8.3 and CL<98” and “GLU>8.3 and RR>26”, whose CR were 87.5% and 87.1%, respectively. The combination “RR>26 and Age<4 yo” gave the best Popularity (59.3%), which came with the highest Support (42.3%) and a CR of 71.4%.To further analyze the links between risk factors and severe cases, and correlations among risk factors, we created a correlation web Figure 2Cytokines are among the key mediators of immune responses to invasion of virus and other pathogens. Therefore, the levels of IL-2, IL-4, IL-6, IL-10, IFNγ, GM-CSF, and TNFα in all enrolled subjects were tested upon admission, and results are presented in Based on our analysis, we tried to create a practical evaluation system for severe HFMD cases, since most combinations with high CR came with a very low popularity, indicating that using only CR as an indicator for severe cases was not practicable Table 3.9/L were found to be associated with severe cases. And results of multivariate analysis indicated five independent risk factors  >26/min, blood glucose (GLU)>8.3 mmol/L, age<4 yo, percentage of lymphocytes (LYM)>40%, blood chlorine (CL)<98 mmol/L, and white blood cell counts (WBC)>1.2×10>40 U/L) Table 2.In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. Based on 3 parameters , combinations of risk factors were evaluated. Combinations with the best confidence ratio should be the best indicator for severe cases by definition. Therefore, “GLU>8.3 and CL<98 and RR>26” (CR = 100%) is our top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%). All combinations with a CR> = 80% came with a case number<32 and a support<4.9%, except “CL<98 and LYM>40% and RR>26 and Age<4 yo”, whose case number was 73 and support was 10.4%. On the contrary, combinations with a lower CR (70.0% to 79.9%) had a much higher case number and support in average, if compared with those with a CR>80.0%. Among combinations with a lower CR, 37.9% (11/29) had a case number>100 , and 48.3% (14/29) had a support>10%. This implies that these combinations are more commonly seen in clinical practice and should not be ignored. Although combinations with high CR may help identifying severe cases, their popularity is very low Table 3.Prior studies have demonstrated the correlation between certain cytokines and HFMD. The study on ratio of cytokine levels in CSF over in plasma had suggested the possible role of IL-8 in response to EV71 infection complicated with PEIn conclusion, five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention.


Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission.Spores are the major transmissive form of the nosocomial pathogen  Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission. Clostridium difficile is the leading cause of nosocomial diarrhea worldwide C. difficile-associated disease (CDAD) range from mild diarrhea to pseudomembranous colitis and even death. Although CDAD is primarily a toxin-mediated disease C. difficile infections largely arises from its ability to form endospores C. difficile to resist antibiotic treatment and persist in healthcare-associated settings. Thus, spores are the primary vectors for transmission The Gram-positive, spore-forming obligate anaerobe C. difficile spores ingested from the environment must germinate into toxin-producing vegetative cells in the intestinal tract C. difficile spores germinate specifically in response to small molecules known as germinants C. difficile, these germinants are bile salts, which are abundant in the small intestine In order to initiate an infection, sleC in both C. difficileC. perfringensC. perfringens, SleC undergoes several processing events. During sporulation, the N-terminal prepeptide is removed to produce pro-SleC, which consists of an N-terminal propeptide attached to the hydrolase domain In the Clostridia, the primary cortex hydrolase appears to be SleC, since disruption of C. perfringens germination exudates have shown that a fraction containing three serine proteases  can proteolytically activate SleC hydrolase activity in vitrocspB gene, and disruption of this gene abrogates SleC cleavage and spore germination C. difficile, three csp homologs are present in a bicistronic operon  and the subtilase protease family C. difficile. By analyzing CspBA in sporulating C. difficile and purified spores, we demonstrate that CspBA is processed to CspB during spore assembly and that CspB undergoes autoprocessing. We also present the first crystal structure of the conserved Csp family of proteases at 1.6 Å resolution and define its key structural domains. These biochemical and mutational analyses reveal that, in contrast to previously characterized prokaryotic subtilases, wildtype CspB forms a stable complex with its prodomain. Similar to other subtilases C. difficile spore germination in the environment or prevent spore germination during disease transmission and recurrence.In this study, we investigated the protease activity of CspBA in cspBA fusion gene is encoded in the genomes of only five clostridial species (C. difficile is unique among these species in that the CspA portion of CspBA (CD2247) lacks an intact catalytic triad (cspBA-cspC (cd2247-cd2246) operon  −sleC mutants spores were consistently ∼3-fold lower than in wildtype spores (C. difficile spore assembly.The  species . C. diffic triad  and S2. ) operon . In spor ∼55 kDa . The fore spores . NeverthcspBAC locus was previously identified by transposon mutagenesis as being essential for C. difficile spore germination cspBAC in C. difficile prevented SleC cleavage during germination  and CspB perfringens, along with mutants with the catalytic serine inactivated, and compared their apparent MW by SDS-PAGE. Whereas mutation of the catalytic serine caused both CspB difficile and CspB perfringens to run at their expected MWs of ∼60 kDa, the wildtype CspB proteins migrated with MWs of ∼55 kDa  to Gln66 (data not shown). Alignment of this autocleavage site with previously mapped processing sites for CspA, CspB, and CspC of C. perfringensperfringens P1 serine to a bulky, charged Arg did not affect autoprocessing  in trans restored folding to the subtilase domain  or LYTS (P4-P1) permitted labeling of the CspB active site, whereas the full-length prodomain expressed in trans prevented labeling  cspBAC complementation strain and degradation products were apparent  or jellyroll domain (Δjelly) of CspBA. Taken together, these results indicate that CspB catalytic activity and its jellyroll domain are required for efficient C. difficile spore germination.To determine the role of CspBA autoprocessing in cleavage , while lcleavage . NeverthThe observation that ∼5% of pro-SleC undergoes cleavage during germination of S461A mutant spores  raised tIf CspB activity is regulated similarly to PCSK9, CspB protease activity should be dispensable once autoprocessing has occurred. To test this hypothesis, we co-expressed the CspBA prodomain with a CspBA variant lacking its prodomain such that the CspBA produced is identical to wildtype CspBA after autoprocessing Q66, . The proClostridium sp. pathogens such as C. perfringens and C. difficile to initiate infection in vitro and in vivo, the structure reveals that Csps are subtilisin-like proteases with two distinctive functional features: a central jellyroll domain and a retained prodomain.Spore germination is essential for in vitro . Media for C. difficile were supplemented with 10 µg thiamphenicol (Thi) mL−1, 50 µg kanamycin (Kan) mL−1, 8 µg mL−1 cefoxitin (TKC); 10 µg thiamphenicol mL−1; or 5 µg erythromycin mL−1 (Erm) as needed. C. difficile strains were maintained at 37°C in an anaerobic chamber (Coy Laboratory Products) with an atmosphere of 10% H2, 5% CO2, and 85% N2. E. coli strains were grown at 37°C in Luria-Bertrani (LB) broth. Antibiotics were used at 100 µg mL−1 carbenicillin for pET22b, 30 µg mL−1 kanamycin for pET28a, and 20 µg mL−1 chloramphenicol for pMTL83151 and pMTL84151 vectors in DH5α E. coli strains, and 100 µg mL−1 and 20 µg mL−1 in HB101 E. coli strains.Bacterial strains and plasmids used in this study are listed in Table S2 in For details, see C. difficile strains were inoculated from frozen stocks onto BHIS plates containing 0.1% taurocholate. After 24 hr growth, a heavy streak of the strain was transferred to a 70∶30 plate and spread uniformly across the plate. Whereas <0.1% of cells are sporulating on BHIS plates et al.C. difficile samples, cell pellets harvested from the sporulation assay were subject to three cycles of freeze-thaw. On the final thaw, 100 µL of EBB buffer  was added, and the sample was boiled with occasional vortexing for 20 min. The lysate was pelleted for 5 min at 13,000×g and then resuspended; 7 µL of 4× loading buffer  was added. The sample was boiled again for a minimum of 5 min, pelleted at 13,000×g, and 15 µL was resolved on a 7.5%  or an 11 or 12% SDS-polyacrylamide gel .For sporulating 6 spores were pelleted at 15,000×g for 5 min. The spore pellet was resuspended in 50 µL EBB buffer, boiled for 20 min with periodic vortexing, pelleted at 13,000×g for 5 min, and resuspended to further solubilize proteins. Five µL of 4× loading buffer was added, and the sample was boiled for 5 min. After pelleting at 13,000×g, 10–15 µL of the sample was resolved on an 11% or 12% SDS-polyacrylamide gel.For analyses of purified spores, ∼5×106-tagged CspB(1–548 aa), full-length His6-tagged CspB perfringens, His6-tagged CspC, His6-tagged SleC and His6-tagged CD1433. Samples resolved by SDS-PAGE were transferred to Immobilon-FL PVDF membranes (Millipore). The membranes were blocked in 50∶50 PBS∶LiCOR blocking buffer (LiCOR) for 30 min, after which Tween 20 was added to 0.1% v/v, and polyclonal antisera was added at 1∶1,000 for all antibodies with the exception of the anti-SleC antibody, which was used at a 1∶5,000 dilution. After a minimum of 1 hr incubation with shaking, the membranes were washed a minimum of 3 times in PBS+0.01% v/v Tween. Anti-rabbit secondary antibodies conjugated to IR800 dye (LiCOR) were added at 1∶30,000 dilution in 50∶50 PBS∶LiCOR blocking buffer containing 0.1% v/v Tween and 0.1% v/v SDS then incubated with shaking for 1 hr. The membranes were washed a minimum of 3 times in PBS+0.1% v/v Tween before imaging on an Odyssey Clx scanner (LiCOR). Western blot quantitation was performed using the indicated loading controls and LiCOR ImageStudio software.All antibodies used in this study were raised in rabbits by CoCalico Biologicals, with the exception of the SleC antibody, which was raised in rabbits by Pacific Immunology. The antigens used were HisSporulation was induced for 3–4 days on five 70∶30 plates. Spores and cell debris were scraped off the plate into 1 mL ice-cold sterile water and purified as previously described 7 spores were resuspended in a total volume of 100 µL sterile H2O. The spores were heat activated at 60°C for 30 min, cooled for 2 min on ice, then 100 µL of 2× BHIS was added. 100 µL of the spores were removed to a tube containing 2 µL of 10% sodium taurocholate to induce germination. Both samples were incubated at 37°C for 20 min after which spores were serially diluted 10-fold into PBS. 10 µL of the dilutions was spotted onto either BHIS or BHIS+0.1% taurocholate agar plates in triplicate and incubated anaerobically at 37°C for ∼24 hr before assessing spore viability. Equivalent numbers of viable spores were recovered on untreated spores plated on BHIS+0.1% taurocholate plates and taurocholate-treated spores plated on BHIS or BHIS+0.1% taurocholate plates. Because spore clumping increased the variability in counting spores, CD1433 Purified spores were enumerated using disposable semen test counting chambers (InCyto C-Chip). Approximately 5×10For details see For details see 3 in 27–30% (v/v) ethylene glycol buffered to pH 5 with 50 mM sodium acetate. Crystals grew in space group P212121, with unit cell dimensions a = 73.87, b = 138.17, and c = 140.08 Å and two molecules in the asymmetric unit for an estimated 57% solvent content Appropriate protein concentrations for crystallization were determined using Pre-Crystallization Test . Hanging drop crystallization experiments were conducted with CspB (11 mg/mL) in 150 mM NaCl, 10 mM Tris-HCl pH 7.5 and Crystal Screen 2 (Hampton Research). Crystal trays were incubated at 12°C and initial crystal hits in 25% (v/v) ethylene glycol (Condition 3) were discovered within 24 hours. After refinement of crystallization conditions, crystals grew reproducibly to about 100*250*60 µmA complete 1.6 Å single-wavelength data set of a representative selenomethionyl-CspB crystal was collected at the selenium edge (0.9794 Å) at 100 K at the General Medical Sciences and Cancer Institutes Structural Biology Facility (GM/CA @ APS) beamline 23ID-B at the Advanced Photon Source, Argonne National Laboratory . Two putative Na+ and three Cl− atoms (confirmed by sodium iodide soaks) were placed in the model, in addition to ethylene glycol, a crystallization reagent.Although CspB is a dimer in the asymmetric unit, gel filtration chromatography experiments see  indicatetion see  was seenWildtype CspB and its mutant variants were diluted to 15 µM in 10 mM Tris pH 7.5 buffer in a total volume of 150 µL. Twenty-four microliters of the mixture were transferred into 8 well strip tubes. One microliter of chymotrypsin  was added, and the mixture was mixed then incubated for 60 min at 37°C. Chymotrypsin activity was quenched by the addition of 8 µL of 4× loading buffer. The samples were boiled for 3 min at 95°C and then 7 µL was resolved on a 15% SDS-PAGE gel and visualized by Coomassie staining.E. coli cultures were grown as described for protein purification. One hour after IPTG induction, a 1 mL sample was removed, the OD600 measured, and the sample pelleted at 13,000×g for 2 min. Cells were lysed in 1× loading buffer (10 OD600/mL). To obtain cleared lysate samples, 30 µL of the supernatant produced upon high-speed centrifugation of sonicated lysates was added to 10 µL of 4× loading buffer. For eluate samples, 30 µL of the eluate was added to 10 µL of 4× loading buffer. All samples were boiled at 95°C for 5 min, pelleted at 13,000×g for 5 min, then 2.5 µL of induced and cleared lysate samples or 5 µL of eluate samples were resolved on a 12% SDS-PAGE gel and analyzed by Western blotting.Wildtype CspB and its mutant variants were diluted to 10 µM in 10 mM Tris-HCl pH 7.5 buffer in a total volume of 155 µL. Twenty-five microliters were aliquoted in triplicate into strip tubes. 0.25 µL of 100 µM FP-Rh (fluorophosphanate-rhodamine probe) was added to CspB and incubated at RT for 10 min. Labeling was quenched by adding 8 µL 4× loading buffer to the sample and boiling at 95°C for 3 min. Six microliters of the labeling reaction was resolved on a 15% SDS-PAGE gel, and fluorescence was imaged using a Biorad PharosFX scanner.www.rcsb.org) under the accession number 4I0W.Coordinates and structure factors have been deposited in the Protein Data Bank  Schematic of sleC and csp genes in C. perfringens ATCC 13124 (gas gangrene isolate) C. perfringens SM101 (food poisoning isolate) C. difficile 630. (b) Western blot analyses of sporulating cells and (c) germinating spores for −cspBAC complementation strains. Sodium taurocholate was used to stimulate germination for 20 min at 37°C. The number of viable spores obtained upon plating on BHIS plates containing 0.2% w/v taurocholate is given as colony forming units (cfus).(TIF)Click here for additional data file.Figure S2ClustalW sequence alignment of CspBA proteins. Completely conserved identical residues are blocked in blue, conserved identical residues in green, and conserved similar residues in yellow. A red triangle indicates catalytic triad residues . Note that the catalytic His of CspB difficile did not align with the other CspBA homologs, despite being conserved in position in alignments with isolated CspB proteins . Because of this discrepancy, the ClustalW alignment was altered to reflect the conservation of the catalytic His. CspBAs from C. butyricum (ZP_045298777), C. botulinum E3 (ZP_04529497), C. acetobutylicum (AE007820_5), Candidatus arhtomitus (EGX28514), and C. difficile 630 (YP_001088762.1). We also note that C. tetani E88 encodes an N-terminally truncated CspBA homolog lacking the first Asp in the catalytic triad (AAO36820), but this was not included in the alignment.(TIF)Click here for additional data file.Figure S3ClustalW sequence alignment of major classes of bacterial subtilisin-like proteases. Completely conserved identical residues (blue), conserved identical residues (green), and conserved similar residues (yellow). A red triangle indicates catalytic triad residues (boxed in red). The central insertion corresponds to the jellyroll domain. Major intracellular serine proteases (ISP): B. subtilis str. 168 (NP_389202.1) and C. difficile (YP_001088508.1); extracellular serine proteases: AprE from B. amyloliquefaciens (YP_003919715.1) and subtilisin E from B. subtilis str. 168 (NP_388911.2); CspB: C. perfringens ATCC 13124 (YP_697251.1) and C. difficile 630, 1–548 aa (YP_001088762.1).(TIF)Click here for additional data file.Figure S4ClustalW sequence alignment of diverse Csp proteases. Completely conserved identical residues are blocked in blue, conserved identical residues in green, and conserved similar residues in yellow. A red triangle indicates catalytic triad residues . C. perfringens Csp prodomain cleavage sites are boxed in pink C. difficile CspB (1–548 aa) autoprocessing site is boxed in orange. α-helices and β-sheets in the CspB perfringens structure are indicated as a helix or a bracket above the sequence alignment, respectively. CspA Roseburia intestinalis (CBL08898), CspB Blautia hansenii (ZP_05853381), CspA Ruminococcus gnavus (ZP_02042115), CspA Coprococcus catus (CBK81001), CspC C. perfringens ATCC 13124 (YP_697250), CspA C. perfringens ATCC 13124 (YP_697252), CspB C. perfringens ATCC 13124 (YP_697251), and CspB C. difficile 630, 1–548 aa (YP_001088762.1).(TIF)Click here for additional data file.Figure S5Interaction interfaces within CspB perfringens. The prodomain is shown in teal, the subtilase domain in purple, and the jellyroll domain in green. Each residue involved in a predicted hydrogen bond is shown as a stick model. Bonds predicted by PDBePISA (TIF)Click here for additional data file.Figure S6Csp prodomain transcomplementation. (a) Schematic of transcomplementation constructs. The source of the prodomain is indicated. (b) Western blot and Coomassie stain showing the purification of CspB transcomplementation mutants. Cultures expressing cspB variants were induced with IPTG, and aliquots were removed 30 minutes later (“induced” sample). During the purification process, a sample of the soluble fraction was removed (“cleared lysate” sample). These samples were resolved by SDS-PAGE and analyzed by Western blotting using an anti-CspB perfringens antibody. Following affinity purification of the His6-tagged CspB variants, equivalent amounts of the “eluate” were loaded and analyzed by SDS-PAGE and Coomassie staining.(TIF)Click here for additional data file.Figure S7Electron density maps of CspB perfringens. (a) Stereo view of bias-free, density-modified experimental map produced from SHELX/C/D/E (TIF)Click here for additional data file.Text S1The file includes supplemental , Tables S1, S2, S3, S4, and associated references.(DOC)Click here for additional data file.