Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821T, a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.Functional variation of Rpf, a growth factor found exclusively in  Micrococcus luteus (Mycobacterium (Myc.) tuberculosis  with lysozyme-like activity that is approximately 70 amino acids long (The resuscitation promoting factor (Rpf) is a protein that has been shown to promote the growth of active cells and to resuscitate non-dividing cells . Thus fas luteus , Mycobacrculosis , and Corutamicum , have bereported . Putativids long . The exaids long , 16. Rpfids long , 19, 29.Actinobacteria Rpf have been proposed and two other families with distantly related proteins in Firmicutes have been putatively identified. The functions of eight Rpf proteins have been studied: Rpf from M. luteus from the LysM subfamily; five Rpfs from Myc. tuberculosis that are in Rpf subfamilies A, B, C, D, and E; C. glutamicum Rpf2 from the RpfB subfamily, and C. glutamicum Rpf1 from the Corynebacterium subfamily. Now with the availability of over 2,100 completed bacterial genome sequences , a BLASTp search using the Rpf domain sequence indicated that it is exclusively found within the phylum Actinobacteria , a novel member of the suborder Corynebacterineae isolated from a permafrost ice wedge in Fox Tunnel, Alaska  was aerobically grown at 20°C, the optimum growth temperature for this strain (2HPO4 and 37.4 mM NH4Cl and using 27.7 mM fructose as the carbon source. All experiments were started with cultures that had been grown to the mid-logarithmic growth phase and stored in glycerol solution (20% v/v) at −80°C. Glycerol stock culture (2% v/v) was first grown in TSBF medium until the mid-log phase and then transferred (1% v/v) to an appropriate growth medium. Escherichia coli TOP 10  used for cloning and expression was grown in Luria-Bertani (LB) medium supplemented with 50 μg mL−1 ampicillin (LB-amp)  or kanamycin  where appropriate.s strain , with shs strain  that wasT. biformata was extracted and purified following a previously described method (rpf gene fragment from T. biformata was obtained by PCR of purified DNA (50 ng) using degenerate primers 2RpfF (5′-AYCAACACSGGHAACGG-3′) and 2RpfR (5′-CCABGCKCCCCARCCCTG-3′) conserved in the Rpf domain of M. luteus Rpf protein (GenBank accession no. Z96935 positions 181–333). Touchdown PCR amplification was used , and 1 min at 72°C; followed by 20 cycles of 1 min at 95°C, 1 min at 51°C, and 1 min at 72°C; and finally 10 min at 72°C). The amplicons were purified using the SUPREC-PCR kit , inserted into the pGEM-T Easy vector system , transformed, and sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit  following the manufacturers’ protocols. The sequence of the amplicon was analyzed using BLASTx (Hind III-digested T. biformata genomic DNA. An approximately 1.9-kbp Hind III fragment hybridized with the probe was self-ligated to obtain a circular DNA product that was used as the template (10–50 ng) for inverse PCR using KOD Plus Ver. 2 . Two primers InvJ2F1 (5′-GCAGATCGAGAT TGCCGAGAAG-3′) and InvJ2R1 (5′-GGTGCTGGGGGAGAA CTGCA-3′), which were constructed within the probe sequence, were used to obtain the entire ORF of the putative rpf gene. The thermal cycler conditions used for inverse PCR were 5 min at 95°C; then 10 cycles of 10 s at 98°C, 30 s at 63°C (decrease 0.5°C per cycle), and 2 min at 68°C; followed by 20 cycles of 10 sec at 98°C, 30 sec at 58°C, and 2 min at 68°C; and finally 5 min at 68°C. The purified products  were inserted into the pCR-Blunt II-TOPO cloning vector  and sequenced. Sequence assembly and analysis were carried out using Genetyx . Potential signal peptide and conserved domains within the putative rpf gene were determined using the Signal P 3.0 server  and stored at −80°C until RNA was extracted. RNA was extracted using the RNeasy Mini Kit (Qiagen) after cells had been digested using lysozyme (Wako) and Proteinase K (Qiagen) and mechanically disrupted in Lysing Matrix B  using a Multi-beads Shocker . Reverse transcription of template RNA (20 ng) was performed using T. biformata rpf gene specific primers  and the OneStep RT-PCR Kit (Qiagen) following the manufacturer’s protocol. Absence of DNA contamination was confirmed by omitting the reverse transcription step according to the manufacturer’s protocol.Cultures of M. luteus  and pBADgIIIb XbaIR2 (5′-GC TCTAGAACGCGGAGGCCAAGCTTGCTG-3′) (restriction sites are in bold) using the following thermal cycler program: 5 cycles of 10 s at 98°C, 30 s at 70°C (decrease 0.5°C per cycle), and 72 s at 69°C; followed by 25 cycles of 10 s at 98°C, 30 s at 67°C, and 72 s at 69°C; and finally 7 min at 68°C. The restriction enzymes, Xho I and Xba I, were used to digest the ends of the PCR product for insertion into the corresponding sites in the pBADgIIIB vector. After the sequence was verified, the pBAD/gIIIB plasmid carrying the rpf homolog with a hexa-histidine tag was transformed into E. coli TOP 10.Recombinant Rpf (rRpf) was produced using the pBAD/gIIIB expression vector (Invitrogen) and purified as previously described for . luteus , except E. coli clone was grown in LB-amp medium and expression of the rpf gene was induced by addition of 0.01% (w/v) L-arabinose . Recombinant E. coli cells without L-arabinose induction were processed in parallel to obtain a recombinant protein-negative fraction for activity analysis. Cells were lysed following the manufacturer’s protocol. Supernatants were collected and His-tagged recombinant Rpf protein was recovered by nickel affinity chromatography  and separated on an AKTA Explorer 100 system (GE) using a 20 to 400 mM imidazole gradient at 4°C according to the manufacturer’s protocol.The T. biformata .The expressed proteins were detected by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot hybridization using the anti-His antibody. Protein fragments from the SDS-PAGE gel transferred by electroblotting to a PVDF membrane , following the manufacturer’s instructions, was used for N-terminal sequence determination. Amino acid sequence analysis was performed on a Procise 492 HT (Applied Biosystems) in the Instrumental Analysis Division, Equipment Management Center, Hokkaido University, Japan. Freshly prepared protein solutions (used on the same day or stored at 4°C for use the next day) were desalted and separated from low molecular weight molecules by centrifugation through an Amicon Ultra-4 filter unit , diluted and sterilized by passage through a 0.22-μm PVDF membrane (Millipore). Protein concentration was determined using the Quick Start Bradford Reagent kit  with spectrophotometric detection  following the manufacturer’s protocol. The approximate protein concentration used in experiments was estimated based on the deduced molecular weight of the putative Rpf from Non-dividing cells were induced by incubation under oxygen-limited conditions. Inoculum prepared from a glycerol stock pre-culture was transferred into 20-mL mMMF in 30-mL tubes (in triplicate) that were tightly capped with butyl rubber stoppers  and crimped with a 20-mm aluminum seal cap  then incubated at 20°C with shaking (140 rpm) for 10 d followed by 50 d without shaking. Numbers of culturable cells were determined by plating cultures onto TSBF agar in triplicate and incubating at 20°C for 7 d. Live and membrane-damaged cells from the liquid cultures were discriminated by staining with the Live/Dead Baclight Bacterial Viability kit (Invitrogen) following the manufacturer’s instructions modified by using a SYTO 9:PI proportion of 1:2. Stained cells were counted in a Neubauer counting chamber  using an epifluorescence microscope . Fluorescent signal from at least 10 fields was captured by CCD camera  to count more than 1,000 total cells. The estimated percentage of non-dividing cells was calculated using the formula:Differences in morphological characteristics of cells were determined by TEM analysis of cultures from (i) non-dividing cells (60 d of oxygen limitation in mMMF), (ii) early logarithmic growth (TSBF for 42 h), and (iii) stationary growth (TSBF for 72 h) phases. Cultures were harvested and prefixed in glutaraldehyde  at 4°C overnight, followed by fixation in 1% (w/v) osmium tetroxide at 4°C for 60 min. The fixed cells were suspended in 0.5% (w/v) aqueous uranyl acetate for 2 h at room temperature and embedded in 1.5% (w/v) agarose prior to dehydration using an ethanol gradient series. The dehydrated blocks were embedded in Epon 812 resin. Ultrathin sections were cut using an ultramicrotome , mounted on copper grids, and stained with uranyl acetate and lead citrate. Sections were imaged using a transmission electron microscope .T. biformata. Cells in the logarithmic phase were grown in 10-mL TSBF at 20°C, harvested, washed twice with mMMF, and resuspended in the same volume of mMMF medium. Serially diluted cell suspensions (100 μL) were transferred to 10-mL mMMF containing rRpf fractions at final concentrations of 0.25 or 5 μg mL−1. For non-dividing cell assays, bacteria from oxygen-limited cultures were harvested, washed, and serially diluted to eliminate the remaining dividing cells from suspensions used for resuscitation experiments. Diluted cell suspensions were transferred to 5-mL mMMF medium containing the rRpf fraction at a final concentration of 2 or 20 μg mL−1. Triplicate cultures were grown with shaking (140 rpm) in test tubes fitted with silicon stoppers. Cell growth was monitored daily by OD600 measurements using a spectrophotometer . Sterilized distilled water (SDW) and eluted non-induced cell lysate were used as negative controls.The biological activity of rRpf protein was determined using low inoculum concentrations of cells in the logarithmic phase and non-dividing cells of T. biformata putative rpf gene sequence and all known and putative Rpf proteins from all other species with completed publically available genome sequences obtained from the Integrated Microbial Genome (IMG)   databaseme (IMG) . Amino ame (IMG)  and the me (IMG)  based onrpf gene from T. biformata was deposited in GenBank under accession number AB723682.The nucleotide sequence for the entire T. biformata using degenerate primers 2RpfF and 2RpfR had a deduced amino acid sequence with 74% similarity to Rpf protein in M. luteus. Using the PCR fragment as a probe, it hybridized to an ~1.9 kb Hind III digested fragment of T. biformata genomic DNA. The nucleotide sequence of this 1,865-bp fragment included a putative 1,119 bp ORF. BLASTn analysis indicated that the highest sequence match was to Rhodococcus opacus B4 , in which Rpf has not been studied. No other sequences provided a match over the full length of the gene. A short region  matched the transglycosylase domain of some other Actinobacteria. BLASTp analysis indicated that the highest amino acid sequence match, 58% identity and 73% similarity, was to RpfB from Rhodococcus erythropolis strain SK121 (GenBank accession ZP_04388698) and strain PR4 (GenBank accession YP_002767809). Messenger RNA corresponding to the rpf gene homolog was detected by RT-PCR throughout the exponential growth phase of T. biformata cells, indicating that this gene was being expressed in growing cultures.A 162-bp fragment initially amplified from genomic DNA of rpf ORF (372 aa) included a 30 amino acid signal peptide, two copies of the DUF348 (domain of unknown function) domain in the N-terminal region, a G5 domain with putative cell-wall adhesive function  and Rpf2 from C. glutamicum . The tree also indicated that RpfB sequences from the same genera tended to branch together. Outside of the conserved amino acids in the Rpf domain the sequences varied among different Rpf sequences  from Myc. tuberculosis is active in resuscitating non-dividing cells of Myc. bovis compared to RpfA (1.6 pM)  .E. coli clone resulted in substantial lysis of the cells, previously described to be related to Rpf accumulation  in mMMF medium. The apparent lag phase was shortened from 12 d to 10 and 5 d in the culture amended with rRpf of 0.25 and 5 μg mL−1, respectively , increases in OD600 were observed at 15 and 19 d with the addition of 20 and 2 μg mL−1 rRpf protein, respectively (T. biformata required to promote growth and resuscitation was much greater than the pM amounts of rRpf from M. luteus and Myc. tuberculosis reported in previous studies (T. biformata Rpf. This hypothesis warrants more in-depth genetic analysis in the future.The rRpf fraction resuscitated cultures . Two indectively . In the ectively ; however studies , 19. Thirpf gene homolog was identified in T. biformata, a novel member of the Actinobacteria isolated from a unique environment. Phylo-genetic analysis of the deduced protein product indicated that T. biformata Rpf formed a monophyletic clade in the RpfB subfamily of Rpf and was distant from other functionally tested Rpfs. The whole length of the rpf gene from T. biformata had nucleotide sequence similarity to only one other bacterium, Rhodococcus opacus B4, in which Rpf has not been studied. The gene was successfully cloned and expressed to demonstrate that despite differences in sequences from previously studied Rpf, the protein was functional and could promote the growth of dividing cells in low-inoculum cultures and resuscitate non-dividing cells. This study provides additional genetic and functional information on the divergence of Rpf produced by different genera in Actinobacteria. It adds to the current body of knowledge of functional Rpf and will aid in future studies to understand other significant roles played by Rpf, such as intercellular communication among Actinobacteria.As we initially hypothesized, a new


PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression  Accompanying changes in life style and dietary choices there is a growing global pandemic of obesity and related metabolic disorders, which include non-alcoholic steatohepatitis or NASH3Col1a1/Col1a2) and alpha smooth muscle actin (Acta2). Several sequence-specific transcription factors contribute to the reprogramming of HSC transcriptome, among which SMAD3 is the most extensively investigated.Hepatic stellate cells (HSCs) are believed to play an essential role in liver fibrosisSMAD3 has long been documented as a mediator of the signaling pathway downstream of transforming growth factor (TGF-β), the preeminent pro-fibrogenic humoral factor in the liver781114db/db mice were fed on a methionine-and-choline deficient (MCD) diet for 4 weeksCol1a1), there was a concomitant increase in PIAS4 expression and a decrease in SIRT1 expression in the livers of MCD-fed mice. Meanwhile, other PIAS family members including PIAS1, PIAS2, and PIAS3 did not show appreciable changes in the livers of MCD-fed mice compared to the control mice. Chromatin immunoprecipitation (ChIP) assay indicated that there was enhanced occupancy of PIAS4, but not that of PIAS1, PIAS2, or PIAS3, on the SIRT1 promoter in the livers of MCD-fed mice compared to the control mice  are a major source of liver fibrogenesisCol1a1, Col1a2) and alpha smooth muscle actin (Acta2) were markedly decreased in MCD-fed mice receiving shPias4 lentivirus than those receiving SCR lentivirus , lentivirus-mediated delivery of short hairpin RNA targeting PIAS4 (shPias4) alleviated steatotic injury as demonstrated by ALT levels  and H&E ntivirus . TherefoSMAD3 is a sequence-specific transcription factor essential for HSC activation and liver fibrogenesisFinally, we tackled the mechanism by which PIAS4 regulates fibrogenesis in hepatic stellate cells. High glucose stimulated the activity of SMAD3 as measured by a reporter gene under the control of tandem repeats of SMAD response element (SRE) in HSC-T6 cells while over-expression of PIAS4 further enhanced the activation by glucose; pre-treatment of SRT1720, a specific SIRT1 agonistTranscriptional regulation highlights the progress of liver fibrosis during NASH pathogenesis212324We show here that in mice fed on a pro-fibrogenic MCD diet, PIAS4 directly binds to the SIRT1 promoter and represses SIRT1 transcription. Furthermore, PIAS4 also mediates the trans-repression of SIRT1 in cultured HSCs exposed to high concentrations of glucose. It is noteworthy that we have used an over-simplified cell model (glucose stimulation) to extrapolate the animal experiments. As such these data must be interpreted with extreme precaution as they only reflect a small part of a very complicated pathophysiological process. In addition to glucose, several other nutrients including free fatty acids and fructose when present in excess have been shown to promote fibrosis in the context NASH2628db/db mouse model. Although fibrosis occurs relatively fast in this model (4~6 weeks as opposed to 10~12 weeks in the high fat diet model), these mice lose significant weight, which is contrary to typical human NASH pathogenesis. Therefore, similar data based on alternative  NASH animal models should be sought before the relevance of the current finding to human pathology can be convincingly established.The current dataset suggests that in response to glucose stimulation, PIAS4-mediated SIRT1 repression leads to deacetylation of SMAD3, which in turn switches on the pro-fibrogenic transcriptional program. Several caveats exist for this proposal. First, since SIRT1 has been shown to suppress epithelial-to-mesenchymal transition (EMT), a key process contributing to fibrosis14153132We show here that lentivirus mediated PIAS4 knockdown in MCD-fed mice alleviates liver fibrosis, which could be explained by PIAS4 mediated SIRT1 trans-repression in HSCs. Alternatively, pro-inflammatory cells and circulating mediators are known to promote HSC activation and liver fibrosis36In conclusion, we present evidence that PIAS-mediated transcriptional repression of SIRT1 contributes to HSC activation and liver fibrosis in the context of NASH pathogenesis. More investigations are warranted to further clarify the role of PIAS4 in this process before the effort of targeting PIAS4 for therapeutic purposes can be moved forward.Immortalized rat hepatic stellate cells  were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). Primary murine hepatic stellate cells were isolated as described previouslyPIAS expression constructsdb/db mice were fed on an MCD diet  for 4 consecutive weeks. In certain experiments, these mice were injected via tail vein purified lentiviral particles (1 × 109 MOI) that carry short hairpin RNA (shRNA) targeting PIAS4 (GTGCTGTACGGGAAGTACTT) or scrambled shRNA (SCR) every 10 days for the duration of the experiments.All animal protocols were approved by the NJMU Intramural Ethics Committee on Animal Studies and adhere to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”. To induce steatosis, 6~8 week-old male Whole cell lysates were obtained as previously describedRNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed as previously described using a SuperScript First-strand Synthesis System (Invitrogen)ChIP assays were performed essentially as described before43Histological analyses were performed essentially as described before14Supernatants containing pro-inflammatory mediators were collected from cultured hepatocyte or liver homogenates and ELISA was performed to measure IL-1, IL-6, and MCP-1 using commercially available kits (R&D).p values smaller than 0.05 were considered statistically significant (*).One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package. Unless otherwise specified, How to cite this article: Sun, L. et al. Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis. Sci. Rep.6, 28432; doi: 10.1038/srep28432 (2016).


Emerging data suggest that innate immunity may play a role in asthma, particularly the toll-like receptors (TLRs). Some studies pointed to an involvement of TLRs 2 and 4 in the pathogenesis of allergic asthma, and other studies related TLRs to IgE. However, there are not any studies that have comprehensively evaluated the expression of TLRs 2 and 4 in inflammatory cells, in peripheral blood and induced sputum specimens from asthmatic patients, according to their total serum IgE.We studied 44 asthmatic patients . On a single visit, all patients underwent: induced sputum, pulmonary function tests, determination of exhaled nitric oxide fraction, venipuncture for blood analysis and skin prick allergy tests. The induced sputum cellularity was analyzed by flow cytometry, where expression of TLRs 2 and 4 was studied using fluorochrome-conjugated monoclonal antibodies.P = 0.048). Furthermore, we observed a correlation (but weak) between the percentage of macrophages expressing TLR4 in induced sputum and the total serum IgE level .Asthmatic patients with high total serum IgE showed, a higher percentage of macrophages expressing TLR4 (42.99 % ± 22.49) versus asthmatic patients with normal total serum IgE (28.84 % ± 15.16) (Asthmatic subjects with high total serum IgE show increased macrophage expression of TLR4 in induced sputum. This outcome may result from a link between innate immunity and IgE-mediated, adaptive immune responses in asthma, and point to TLR4 as a potential therapeutic target. IgE plays a prominent role in the pathophysiology of allergic asthma. IgE binds to receptors on the surface of different types of immune effector cells causing them to release a variety of mediators that promote airway hyperresponsiveness, mucus secretion and increased vascular permeability . SeveralTLRs are a family of cell surface proteins involved in the recognition of pathogen-associated molecular patterns (PAMPs). Upon stimulation, TLRs can modulate subsequent adaptive immune responses. TLR2 and 4 are expressed on the cellular surface and migrate to phagosomes after activation on recognising the ligand. Just because these receptors are expressed on the cellular surface makes them easy to measure. Our laboratory has expertise in measuring them .TLRs 2 and 4 function as signal receivers for gram-positive and negative bacteria (endotoxin recognition) –8. In soPrevious studies addressing the role of TLRs 2 and 4 in asthmatic patients had several limitations. No studies approached a differential analysis of TLRs 2 and 4 in blood versus induced sputum. Most of the studies were limited to blood cells (frequently monocytes) , 19, in The study was approved by the Institutional Review Board and registered at ClinicalTrials.gov with identifier NCT02028637.The study was conducted in accordance with the Declaration of Helsinki principles  and was approved by the Clinical Research Ethics Committee  of our institution. The participants signed their informed consent to participate in this study and personal identification data were anonymized.This was an observational, cross-sectional study performed at a hospital outpatients clinic. Forty-four subjects on maintenance treatment for asthma, aged 18 to 75 years, were included. All subjects were non-smokers on study inclusion and had asthma diagnosed as per the Global INitiative for Asthma (GINA) criteria . FurtherSociedad Española de Neumología y Cirugía Torácica (SEPAR) [All study procedures were performed at a single clinic visit. After signing the informed consent, the participating patients fulfilled an Asthma Control Test (ACT) questionnaire  and unde (SEPAR) . FeNO wa (SEPAR)  and refe (SEPAR) . Total s (SEPAR) . Induced (SEPAR)  and proc6 cells/g were not included in the analysis. Cell viability was determined by trypan blue dye exclusion and was required to be greater than 40 % as quality criterion. Differential leukocyte counts were done on a minimum of 400 cells and were expressed as cell percentage of lymphocytes, neutrophils, eosinophils and macrophages. Cell count reference values were previously established [Induced sputum specimens were processed according to a consensus standard procedure . Mucus pablished . The spuablished  cells/tuPeripheral venous blood was treated with trisodium citrate as anticoagulant, then aliquoted into 100 μl/tube samples, and erythrolysed with TQ Prep Sample Preparation and COULTER PrepPlus 2 , and immunostaining for flow cytometry followed.Sputum and blood cell suspensions were blocked with mouse serum and immunostained with saturating concentrations of the following fluorochrome-conjugated monoclonal antibodies, for 15 min at room temperature in the dark: phycoerythrin (PE) anti-TLR4 (clone HTA125), PE anti-CD66b (G10f5) and PE/Cyanin-7 anti-CD14 (clone M5E2) from Biolegend ; Alexa Fluor 488 anti-TLR2 (clone 11G7) and PE anti-CD125 (A14) from BD Biosciences ; PE anti-CD16 (3G8), PE anti-CD45 (MEM28), fluorescein isothiocyanate (FITC) anti-CD16 (3G8), FITC anti-CD66b (B13.9), and PE/Dy-647 anti CD45 (MEM28) from Immunotools  . PE mousFlow cytometry data acquisition was performed with a FC500 equipment . Ten thousand events were analyzed for all sample runs. Gating of sputum leukocytes was based on side light scatter versus CD45 expression, which allowed for the discrimination of lymphocyte, macrophage and granulocyte populations. The mean fluorescence intensity (MFI) of the cells stained with control antibody was subtracted from the MFI of the cells stained with receptor antibodies to provide a measure of receptor-specific fluorescence.Asthma inflammatory phenotypes were classified according to sputum cellularity. Patients were classed as neutrophilic asthma if the neutrophil count was >61 %, eosinophilic asthma if eosinophils >3 %, as and those with <61 % neutrophils and <3 % eosinophils were considered paucigranulocytic asthma .Values are presented as percentages and frequencies for qualitative data and mean ± standard deviation for quantitative data. Comparisons between asthma with high versus normal total serum IgE were analyzed with Student’s t-test. Categorical variables were contrasted through contingency tables and tested with chi-square, or Fisher’s test where appropriate. Pearson’s coefficient was employed for correlation analysis. The level of statistical significance was set as α = 0.05. Analysis was done with SPSS software version 18.0 for Windows .Out of the 44 subjects with asthma studied, 15 had high total serum IgE and 29 had normal total serum IgE. Demographics, clinical and functional data are shown in Table TLR expression data are shown in Table The percentage of macrophages expressing TLR4 in induced sputum was significantly higher in asthma with high total serum IgE versus asthma with normal total serum IgE Fig. . In both+ macrophages was not influenced by the patient’s GINA therapeutic step  in GINA-3, n = 11; 28.99 % (12.83 %) in GINA-4, n = 9; and 38.66 % (23.93) in GINA-5, n = 16; P = 0.501).The percentage of TLR4We observed a correlation (but weak) between total serum IgE and the percentage of macrophages expressing TLR4 in induced sputum . A correlation was also found between the percentage of macrophages and neutrophils expressing TLR4 in induced sputum .The main outcome of the present study was that the percentage of macrophages expressing TLR4 in induced sputum was higher in asthmatic patients with a high total serum IgE than in asthmatics with normal total serum IgE. Furthermore, we observed a correlation (but weak) between total serum IgE and the percentage of macrophages expressing TLR4 in induced sputum. This result is consistent with a possible link between innate immunity and the adaptive immune response in the pathogenesis of IgE-mediated asthma. The role of TLRs in the inflammatory process has been the subject of recent investigations. However, most of the studies have evaluated cellular responses in vitro or systemic reactions, and few studies focused on the role of TLRs in lung inflammation. In the present study, we analyzed the cellular expression of TLRs 2 and 4 on concurrent induced sputum and peripheral blood samples in adult asthmatics, and the data were stratified according to the type of asthma in terms of high versus normal level of total serum IgE. The study was limited to an observational assessment of TLR expression, yet the work was performed on a large sample of clinically well characterized patients, where all data were collected together at a single time point.TLRs are a family of proteins responsible for the recognition of PAMPs, which include repetitive microbial molecular domains such as lipopolysaccharides, flagellin, mannose and nucleic acids from viruses and bacteria. TLRs 2 and 4 are expressed on the cell surface and translocate to phagosomes after activation, upon binding their ligands. TLR4 activation leads to a series of events including bronchoconstriction, expression of adhesion molecules by vascular endothelial cells and the release of cytokines. Such actions result in neutrophil recruitment and activation of pulmonary dendritic cells and macrophages , as well+ macrophages and the total serum IgE level, are consistent with the idea of a relationship between respiratory exposure to PAMPs and asthma with increased IgE, and suggest an involvement of the macrophage in such relationship.There are findings showing that bacterial and viral infections can modify the course of allergic diseases by affecting high-affinity IgE receptor (FcεRI)-dependent mast cell activation. Such studies reported that the ligation of TLRs by bacterial or viral antigens can affect IgE-dependent mast cell degranulation and preformed mediator release, as well as eicosanoid production . FurtherMacrophages are key cells in the pulmonary innate immune responses, since they are the most abundant leucocyte in the air spaces and one of the first cells to encounter inhaled proteins that may act as allergens. Depending on the signals received, macrophages can be pro- or anti-inflammatory. Macrophage stimulation can result from a variety of stimuli including TLR engagement in the presence of IL-10 or other cytokines . Our anaThe role of TLR2 in airway allergic inflammation is not fully clear. In our work we did not find any significant relationship between TLR2 expression and total serum IgE. However, TLR2 stimulation by its ligands was reported to potentiate Th2 responses and exacerbate airway hyperresponsiveness , 44. TheOur study has some limitations: (i) the effect of inhaled corticosteroids on expression of TLRs in induced sputum is unknown; (ii) we only analyze receptors that are expressed on the cell surface (TLR4 and 2); (iii) it is a descriptive study. Our results were significant but, should be studied with a higher population size.In summary, this work is the first evaluation of the expression of TLRs by blood and induced sputum leukocytes in subjects with asthma. The main finding was that asthmatic patients with a high total serum IgE have a higher percentage of macrophages expressing TLR4 in induced sputum, compared with patients with normal total serum IgE values. This outcome supports the possibility of therapeutic approaches for some forms of immune-mediated lung disease through stimulating or blocking TLRs with agonists or antagonists , and may


X-ray crystal structures of a variant of human carbonic anhydrase II in complex with four imidazole derivatives  have been determined in order to identify the binding sites for such compounds, and a mechanism to explain the effects on catalytic activity is proposed.Human carbonic anhydrases are zinc metalloenzymes that catalyze the hydration and dehydration of CO 2 and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O) in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives  have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the ‘in’ and ‘out’ rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO The first step of catalysis, in the hydration direction, is a nucleophilic attack on an incoming CO2 by the zinc-bound OH− (ZnOH−) to produce HCO3−. The binding of HCO3− at the metal is weak and is accordingly displaced by a water molecule (1)−. This is thought to occur through a network of well ordered hydrogen-bonded waters in the active site, and to be facilitated by the protonation/deprotonation of the imidazole side chain of His64 (2)The catalysis relies on a zinc hydroxide/water  or Eigen (H9O4+) cations may be involved  in CA II, it causes a reduction in the enzymatic proton shuttling and thus decreases the rate of reaction by ∼20-fold  cells. The culture was grown at 37°C in the presence of ampicillin (100 µg ml−1) until it reached an OD600 of 0.6, and was thereafter induced with IPTG (100 µg ml−1) for protein expression. The lysate was then purified by affinity chromatography using a p-aminomethylbenzenesulfonamide column. Nonspecifically bound proteins were washed off the column using buffers (200 mM sodium sulfate) at pH 7.0 and pH 9.0, finally eluting the protein with elution buffer (400 mM sodium azide). The enzyme was thereafter buffer-exchanged to remove sodium azide and concentrated to 15 mg ml−1 (450 µM) using a 10 kDa filter. The detailed vector preparation and protein purification has been published previously  were equilibrated against the precipitant solution  at room temperature (298 K) for all of the small imidazoles. Crystals were observed after 5 d and, based on visual selection, a crystal of each of the CA complexes was cryoprotected by quick immersion into 20% sucrose precipitant solution and flash-cooled by exposure to a gaseous stream of nitrogen at 100 K. The X-ray diffraction data for the I, 1MI and 4MI complexes were collected on the F1 beamline at the national X-ray facility at Cornell High Energy Synchrotron Source (CHESS). The crystal-to-detector distance was maintained at 100 mm, with 1° oscillation steps and 1 s exposure per image. Diffraction data for the 2MI complex were collected in-house with an R-AXIS IV++ image-plate system on a Rigaku RU-H3R Cu rotating-anode generator operating at 50 kV and 22 mA using Osmic VariMax HR optics. Setting the crystal-to-detector distance to 80 mm and using oscillation steps of 1°, a 5 min exposure time was used per image.Co-crystals of each of the H64A CA II–compound complexes  were obtained using the hanging-drop vapor-diffusion method. Drops of 10 µl , 4hez (1MI), 4hew (2MI) and 4hey (4MI). All four structures had a main-chain r.m.s.d. of <0.2 Å when compared with wild-type CA II  based on their location within the active site. Sites 1 and 2 were defined as the overlapping regions of the inward and outward conformations, respectively, of His64 in wild-type CA II. The binding of imidazoles in sites 1 and 2 provides a possible proton-donor/acceptor group within these locations, thereby restoring the reduced proton-shuttle capability of the enzyme. Site 3 overlaps with the CO2-binding site ; being this far from the zinc, it is improbable that they affect catalysis.Each of the four imidazole compounds I, 1MI, 2MI and 4MI were co-crystallized with H64A CA II, the structures were determined to a resolution of at least 1.7 Å Table 1 and the ed Fig. 2. Of thesed Fig. 2a based 3.1.B factor of 24.5 Å2, which is comparable to the B factors of surrounding solvent molecules and residues (Table 2a). The absence of a methyl group (compared with the other imidazoles in this study) imparts low hydrophobicity to I , thereby reducing the possibility of it being involved in hydrophobic interactions. This could explain why I was not observed at sites 1 and 2. At concentrations as high as 100 mM in the crystallization conditions, I was also bound to the active-site Zn2+  displacing four water molecules including the ZnOH−, indicating inhibition. Kinetics data showing this inhibition are provided in the Supporting Information .A total of nine molecules of I were bound to H64A CA II, with an average s Table 2a. The a3.2.B factor of 26.0 Å2). Although 1MI and I have similar pKa values (pKa ≃ 7.1), the presence of a methyl group makes 1MI more hydrophobic in nature and possibly leads to its ‘capture’ at sites 1 and 2 (Table 2b). There were various other sites (inside the protein and out on the surface) where 1MI molecules were located, but it is improbable that they affect the catalysis of H64A CA II. 1MI binds to the active-site zinc with its unmethylated N atom, and the other methylated N atom lacks the ability to form hydrogen bonds to Thr200, which could explain its absence at site 3.1MI was bound at seven different sites (average 2 Table 2b. There3.3.Ka = 8.2) bound to H64A CA II , both with an average B factor of 19.2 Å2 and in close proximity to each other, interacting with the active-site residues Val121, Phe131, Leu198, Thr199 and Pro202. The computed ΔG values were higher, most likely because of a coordination geometry between imidazole and Zn2+ at site 3 which makes this site the most stable. Occupying both sites 3 and 4, 2MI buried a total surface area of 190.3 Å2 . Inhibition data on 2MI are shown in Supplementary Fig. S2(a).There were only two molecules of 2MI  and has two N atoms that can participate in hydrogen bonds which help 4MI bind at a maximum number of sites. It occupied sites 1 and 2, which readily offers a structural explanation of how 4MI is able to enhance the activity of H64A CA II by acting as the external proton donor/acceptor for either or both of the ‘in’ and ‘out’ conformations of His64 (Table 2d). Another molecule of 4MI, similar to I and 2MI, was observed to be directly bound to the active-site Zn2+, displacing four water molecules including ZnOH− . This displacement of ZnOH− by 4MI clearly suggests the mechanism of inhibition of CA activity by high concentrations of the compound .With an average 4 Table 2d. AnothH− Fig. 3a. This nd Fig. 3b.4.et al., 2001et al., 200218O-exchange method and are included in the Supporting Information. Additionally, the effects of imidazole on wild-type CA II and H64A CA II have also been studied previously, in which the inhibition profiles appeared to be identical for both of them , 1-methylimidazole (1MI), 2-methylimidazole (2MI) and 2-methylimidazole (4MI), with H64A CA II to identify the binding sites for such compounds and propose a mechanism to explain the effects on catalytic activity. This is the first X-ray crystal structure that reports simultaneously (i) an inhibitory binding site for imidazoles (directly bound to the active-site zinc) and (ii) sites occupied by both the ‘in’ (not previously observed) and ‘out’ conformations of His64 in wild-type CA II. Previously published enzyme-kinetics studies . The mechanism behind this enhancement of activity and inhibition has long been discussed. Site 2 is the only site that has previously been found to be occupied by 4MI ; however, the presence of a methyl group on imidazole  does not seem to affect the hydrophobic interactions enough to cause changes in binding orientations or affinities as suggested by MD simulations (data not shown). It can be suggested that molecules of 1MI and 4MI bound at sites 1 and 2 mimic the inward and outward conformations of His64 (in wild-type CA II) and thus enhance the activity of the enzyme by aiding in proton shuttling. Similarly, molecules of I, 1MI, 2MI and 4MI bound to the active-site Zna and 2d). Similarly, bound on the surface near Ile91 at site 5, both 1MI and 4MI exhibit a dual conformation. Although this site is outside on the surface and it is unlikely that it can affect the catalytic efficiency of the enzyme, the presence of both compounds in almost the same orientation with a dual conformer is interesting (Tables 2b and 2d). Most binding sites (on and around the surface of the protein) could be considered unproductive and inconclusive, but the three sites (sites 1–3) within the active site strongly indicate activity enhancement and inhibition. In contrast, a different perspective could also be drawn. The data show that there are so many binding sites for imidazoles that every one of them makes a small contribution, which is why Duda and coworkers did not observe significant effects on activity enhancement when a single binding site (site 2 in this study) was destroyed by mutagenesis  with 4MI even though these sites do not appear to offer substantial binding interactions (Tables 2et al., 2013bet al., 2013aet al., 2013bet al., 2013bet al., 2013In addition to the inhibition of CA by these imidazoles, the presence of such a large number of binding sites also provides insights and preliminary data required for the fragment-addition approach of drug design, as we have previously suggested (Aggarwal, Kondeti H64A CA II, complex with imidazole, 4hf3PDB reference: complex with 1-methylimidazole, 4hezPDB reference: complex with 2-methylimidazole, 4hewPDB reference: complex with 4-methylimidazole, 4heyPDB reference: 10.1107/S2052252514004096/dc5001sup1.pdfSupporting information. DOI:


Orconectes hylas (Faxon)) has been introduced into the St. Francis River watershed in southeast Missouri and has displaced populations of native crayfish. The effects of O. hylas on macroinvertebrate community composition were investigated in a fourth-order Ozark stream at two locations, one with the presence of O. hylas and one without. Significant differences between sites and across four sampling periods and two habitats were found in five categories of benthic macroinvertebrate metrics: species richness, percent/composition, dominance/diversity, functional feeding groups, and biotic indices. In most seasons and habitat combinations, the invaded site had significantly higher relative abundance of riffle beetles (Coleoptera: Elmidae), and significantly lower Missouri biotic index values, total taxa richness, and both richness and relative abundance of midges (Diptera: Chironomidae). Overall study results indicate that some macroinvertebrate community differences due to the O. hylas invasion were not consistent between seasons and habitats, suggesting that further research on spatial and temporal habitat use and feeding ecology of Ozark crayfish species is needed to improve our understanding of the effects of these invasions on aquatic communities.Introductions of alien species into aquatic ecosystems have been well documented, including invasions of crayfish species; however, little is known about the effects of these introductions on macroinvertebrate communities. The woodland crayfish ( Aquatic macroinvertebrates continue to be widely studied because of their unique diversity and ubiquity in streams and rivers worldwide –3. AmongProcambarus clarkii (Girard) into lentic waters of Chozas in León (Northwest Spain) resulted in increased turbidity by decreasing plant coverage by 99%, thus indirectly reducing macroinvertebrate populations by 71%, duck species by 75%, and amphibians by 83%. Houghton et al. [Orconectes rusticus (Girard) into Prairie River, Wisconsin.The effects of alien crayfish on the native crayfish fauna have been documented ; howevern et al.  showed aWhile crayfish have been moved all across the world by various vectors , it shouOrconectes hylas (Faxon)). This species is endemic to the Black River watershed and headwaters of the Meramec and Big Rivers in Missouri and was introduced to the neighboring St. Francis River watershed over 30 years ago [O. hylas, and this invasive alien crayfish can reach relative abundances up to 25% greater than native crayfish in the invaded areas [Orconectes peruncus (Creaser)) and St. Francis River crayfish (Orconectes quadruncus (Creaser)) are endemic to the upper St. Francis River watershed, upstream of Lake Wappapello [O. hylas has invaded [O. peruncus and O. quadruncus are listed as imperiled in Missouri (S2) and globally (G2) [A well documented example of a localized crayfish introduction is the movement of the woodland crayfish  , and as lly (G2) .O. hylas within the St. Francis River watershed in the Ozarks provided an opportunity to study the effects of an invasion on a stream system where the upstream movement of this species was ongoing. While it has been demonstrated that O. hylas is altering the native Missouri crayfish fauna, no research had been conducted to investigate what effects this invasion may have on aquatic macroinvertebrate communities. The objectives of this study were to document the differences in the benthic macroinvertebrate community composition in the presence or absence of the invading O. hylas in an Ozark stream, and to contribute spatial and temporal data of these effects to guide management and regulatory efforts of nuisance and invasive species in Missouri.The range expansion of The study plan conforms to relevant national and international guidelines regarding ethical treatment, use, and preservation of animals. All field collections of macroinvertebrates were conducted using established sampling protocols, and access to study sites was granted by private landowners. At the time of the sample collections, the primary author and one of the co-authors were employed by the Missouri Department of Conservation, and therefore no permits for collection of macroinvertebrates were necessary.Orconectes hylas fits the term of “invasive alien” species due to its range expansion and subsequent range contraction of the native crayfish species.There are many terms for organisms that have been moved outside their native ranges. We will follow the naming protocol outlined in Occhipinti-Ambrogi and Galil . Orconec2) and is primarily forested (87.7%) with low development (2.27%) and cropland (0%) [Crane Pond Creek is a fourth-order Ozark stream with perennial flow located in Iron County, Missouri , 47. It and (0%) .O. peruncus and invading O. hylas, while also exhibiting similar habitat characteristics throughout its drainage. Since no other streams in the region had the same O. hylas invasion status, our study was conducted entirely within Crane Pond Creek. Preliminary sampling indicated that O. hylas had invaded upstream to Highway F in Crane Pond Creek, but had not yet invaded upstream to Highway C , summer (24 June), late summer (29 July), and fall (30 September). Stream discharge was conducted using a Marsh-McBirney Flo-Mate 2000 flow meter at each site during each sampling season. Discharge was calculated as cubic feet per second (cfs) by following the methods listed in the Missouri Department of Natural Resources (MDNR) Flow Measurement in Open Channels Standard Operating Procedure . At eachHabitat quality was evaluated once in September 2011. Stream habitat quality was assessed with the Stream Habitat Assessment Project Procedure (SHAPP) . The SHAAquatic macroinvertebrates were sampled at both sites using the Semi-Quantitative Macroinvertebrate Stream Bioassessment Project Procedure (SMSBPP) , which i2 areas disturbed were composited each for the CS and NF habitats). This resulted in six total samples (three CS and three NF) at each site and for each sampling season . Each composite sample was preserved with 80% buffered ethyl alcohol in 1-L Nalgene bottles, and labeled by habitat type, site, date, and replicate.For each sampling period and habitat, three random replicates (triplicates) were taken with a D-frame rectangular aquatic kicknet . Each sample replicate consisted of a composite of net samples taken at multiple stream locations (three separate 1-mtol) followed the Taxonomic Levels for Macroinvertebrate Identifications document developed by the MDNR [Laboratory processing of macroinvertebrate samples followed the SMSBPP protocol , and incthe MDNR . VoucherTo provide community-level comparisons between sites, a total of 18 macroinvertebrate indicator metrics were calculated from the data . These ip < 0.05) were performed for each metric and included a three-way ANOVA with data for the two stream habitats analyzed as separate samples , and a two-way ANOVA with data for the two habitats pooled at each site and sample replicate within a sampling event .For each site, and within each sample period, means and standard errors were determined from CS and NF habitats. To test for significant differences between sites, a nested, non-parametric analysis of variance (ANOVA) was performed on the means of each macroinvertebrate metric  using version 9.3 of the Statistical Analysis System  and ProcValues for water quality parameters across sampling seasons were all within water quality standards for Missouri  with no Based on the Missouri habitat assessment protocol , both stO. hylas = 95% with O. peruncus absent), and 85 crayfish collected at Site 2 . It should be noted, the sampling protocol employed does not specifically target crayfish, but rather benthic macroinvertebrates in general and therefore only presence/absence can be determined using the crayfish numbers above.All raw benthic macroinvertebrate data collected in Crane Pond Creek in 2011 are found in Stenelmis lateralis:Coleoptera) by habitat (NF) represented a mean relative abundance of up to 52.4% of macroinvertebrates at Site 1; however, most taxa were present in low abundances and represented less than 2% of the macroinvertebrates across all habitats and sampling periods. During all four sampling seasons, riffle beetles in the genus Stenelmis (Elmidae) were the most dominant taxon at Site 1 in both habitats, and this was the only taxon that was among the five most dominant organisms in both habitats and at both sites. Other taxa such as the mayflies Caenis sp. and Stenonema femoratum (Say) were also dominant in the NF habitat at Site 2 during the late summer and fall sampling seasons. The midges Tanytarsus sp., Cladotanytarsus sp. and Polypedilum aviceps (Townes), as well as the caddisfly Cheumatopsyche sp. (Hydropsychidae) were also among the most dominant taxa at Site 2. Overall, there were 13 macroinvertebrate taxa present at Site 2 that were not collected at Site 1. These include two crayfish (O. peruncus and Cambarus hubbsi (Creaser)) and insects belonging to the orders Diptera , Ephemeroptera (1 taxon), Plecoptera (3 taxa), Trichoptera (2 taxa), Odonata (2 taxa), and Hemiptera (1 taxon). In contrast, there were 19 taxa present at Site 1 that were not collected at Site 2. These included mollusks (3 taxa), crayfish (O. hylas), and insects belonging to the orders Diptera (3 midge taxa), Ephemeroptera (3 taxa), Plecoptera (2 taxa), Trichoptera (3 taxa), Odonata (3 taxa), and Coleoptera (2 taxa). However, none of these listed taxa found at only one of the study sites were among the five dominant taxa in any habitat or season, and in most cases made up less than 5% of sample relative abundances. Individual values and ranges across seasons for richness, relative abundance, and dominance of macroinvertebrate taxa, as well as summary statistics for the individual metric values determined from the samples, are given in Freeland-Riggert [Mean taxonomic richness in data pooled by habitat (CS+NF) was similar at both sites across all seasons and ranged between 46‒63 taxa at Site 1 and 57‒70 taxa at Site 2. Individual taxa (-Riggert .rich), mean EPT (EPTrich), and Chironomidae taxa richness (Chirrich) were higher at Site 2 during one or more seasons and habitats  and fall samples (CS only); however, mean EPTrich in NF was significantly higher in fall at Site 1  was significantly higher at Site 1 , Site 2 had significantly higher mean values than Site 1 in both NF and pooled habitat types during late summer and fall samples  and fall , all of the significant interactions observed from the two-way analysis , P x H 12 metrics), and H x S (five metrics). Among these, Tric metrics,n Tables . Only on metrics  and amon metrics . Results metrics . Among tl Tables . Except 2 Tables .O. hylas) which was in the process of displacing the native O. peruncus while the study was being conducted. Identification and enumeration of crayfish specimens found in macroinvertebrate samples confirmed that no O. hylas were present at the upstream site (Site 2) during the entire duration of the study. The ecology of Ozark crayfish species and their feeding and habitat preferences are relatively poorly known, but some aspects can provide further interpretation of our study results. Early research on crayfish has suggested they gain most of their nutritional needs from detritus and plant material, but this belief has been overemphasized [We examined the macroinvertebrate community in Crane Pond Creek, Iron County, Missouri during the upstream expansion of an alien crayfish . Among the parameters that showed a greater difference between sites, flow status scored lower at the downstream site based on a greater relative percentage of exposed gravel in the stream channel. However, this parameter may be highly variable because large exposed gravel bars are a common feature of Ozark streams , and theO. peruncus [O. hylas [O. hylas, the minor differences in habitat between our sites did not provide any evidence that habitat was a factor affecting our observed differences in crayfish abundance. This follows previous Missouri studies in watersheds where introduced crayfish have invaded or native species have been displaced. For example, studies conducted with both O. hylas (introduced into the St. Francis watershed) and one subspecies of the ringed crayfish (Orconectes neglectus chaenodactyleus (Williams) introduced into the Spring River watershed) have shown that overall stream habitat is not limiting [As previously mentioned, it has been documented peruncus  and O. hO. hylas  utilize limiting , 41, 79.O. hylas was present compared to where it was absent. In addition to the 13 macroinvertebrate taxa that were present at Site 2 and absent at the invaded site, at least one of the taxa richness metrics was significantly higher at the non-invaded site during each season and habitat type. Only EPT richness during fall in the NF habitat showed the opposite pattern where richness was significantly higher at the invaded site. Significant site differences were more frequently observed in abundance/composition metrics than in the richness metrics in Crane Pond Creek. During one or more seasons and in one or more habitat types, significantly lower relative abundances of several taxa were observed at the invaded site, including midges (Chircp) and three metrics related to EPT organisms . Similarly, an increase in relative abundance of riffle beetles in the family Elmidae (Elmcp) was also observed at the invaded site. This overall result is similar to McCarthy et al. [O. rusticus crayfish had the greatest negative effects on abundance and composition of benthic macroinvertebrates.Several macroinvertebrate indicators showed significant differences between sites where y et al. , who useO. hylas may have caused a decline in community evenness. In at least one season and habitat, the invaded site had significantly higher values for one or both of the dominant taxa metrics  and significantly lower Shannon Diversity Index values (SDIdd). These results might be expected considering the high abundances of Stenelmis lateralis (Family Elmidae), which was the most dominant taxon at the invaded site during all seasons and in each habitat type [Our data also suggest that the invasion of tat type .cp) and mayflies (Ephcp) were significantly lower in NF and pooled habitats in late summer and fall. Previous literature suggests that effects of invasive alien crayfish on mayfly abundance may be attributed to their behavior. For example, Nyström [Tricorythodes sp.) may be more vulnerable to predation [cp) was significantly higher in NF habitat at the invaded site during fall. Some species in this order are known to migrate to certain habitats during various parts of their life cycle [Pacifastacus leniusculus. Trichoptera abundance (Trichcp) was lower in CS and pooled habitats during summer, late summer and fall. This is similar to McCarthy et al. [O. rusticus reduced numbers of Trichoptera. However, Whiteledge and Rabeni [The results of this study demonstrate the value of including multiple indicator metrics associated with each of the EPT orders of macroinvertebrates when studying the effects of introduced crayfish species. It is known that macroinvertebrates belonging to these insect orders are commonly ingested by crayfish –25, 29,  Nyström , 81. Conredation . This mafe cycle , which mfe cycle  found a rich) and relative percent abundance (Chircp) of this group of insects were significantly lower at the invaded site, with abundance lower in CS and pooled habitats during spring and early summer, and richness lower in one or more habitat types in spring, late summer, and fall. Lower abundance and richness of Chironomidae has been previously documented following the introductions of two other invasive alien crayfish into U.S. waters: the signal crayfish (Pacifastacus leniusculus (Dana)) and the red swamp crayfish (Procambarus clarkii (Girard)) [O. hylas and the native and closely related O. peruncus may provide some insight to reduced abundances of midges at the downstream site during the spring and early summer. As previously discussed, O. hylas has been shown to have reproductive advantages over O. peruncus, as well as O. hylas young exhibiting higher densities in riffle habitat during the spring [O. hylas may feed on these soft-bodied invertebrates at a higher rate as compared to the native crayfish species present in the stream, and that their competitive advantage may result in reduced abundances of Chironomidae during some seasons, as we observed in our study.As previously mentioned, midges (Chironomidae) are a key food item for crayfish. In our study, both richness (ChirGirard)) , 72, 83.Girard)) , 84–86. e spring . This suP. leniusculus in Finland [O. hylas within both habitats during all four sampling seasons. The riffle beetle Stenelmis spp. was the most dominant member of the family Elmidae, and overall, among the most dominant taxa at the invaded site for every season and habitat except NF in the spring. Few studies have examined Elmidae as a prey item for crayfish, however this family is known to exploit multiple habitats in streams because both larvae and adults are aquatic while occupying more than one trophic feeding strategy  [O. hylas. Studies conducted on North American adult Elmidae have confirmed that crayfish, turtles and some fish find them unpalatable as a food item [Significant declines in this family of beetles have been associated with the presence of introduced  Finland . Even thlectors) . A majorlectors) , possiblood item . Adult aood item , 89, andood item . In addiood item , 90, 91.O. hylas invasion (i.e. declines in richness or changes in abundance) would most likely be the result of macroinvertebrate taxa or functional group being utilized as a food source, rather than effects from competition between invasive alien crayfish and other macroinvertebrates.The Crane Pond Creek data were analyzed as a nested study design because all factors were intentionally left as independent variables, and comparable habitats were sampled at both sites independently during the same time period to isolate effects that may result from the crayfish invasion. We did not expect nor anticipate interactions between these factors; therefore, we presented and interpreted our study results for each season and habitat independently. In contrast, a factorial study design would have been more appropriate if our factors were intentionally treated as co-dependent on one another, because interactions are an expected and planned result  and interich (1<2), Elmcp (1>2), and Scfh (1>2). Significant interactions between habitat and site are expected if a habitat type has different availability to crayfish at one site or another, or if foraging habitat or diel feeding preferences of invasive alien crayfish are different than the native crayfish at one site or another. For both nested ANOVA results (2-way and 3-way), this interaction was not significant in five metrics with significant site differences, including Chirrich (1<2), Elmcp (1>2), EPTcp (1<2), SDIdd (1<2), and Scfh (1>2). Significant interactions between period and site are expected if invasive alien crayfish are utilizing both sites differently depending on the season, or if they are feeding uniformly at each site but differently across seasons. For both nested ANOVA results (2-way and 3-way), this interaction was not significant in five metrics with significant site differences, including TTrich (1<2), EPTrich (1<2), Elmcp (1>2), EPTcp,(1<2) and Scfh (1>2). We also might expect significant three-way interactions (Period x Habitat x Site) if invasive alien crayfish are affecting the macroinvertebrate community at the two study sites differently, depending on all combinations of season (4 periods of collection), and habitat type . For both nested ANOVA results (2-way and 3-way), this three-way interaction was not significant for only three metrics, including EPTcp (1<2), SDIdd (1<2), and Scfh (1>2). From the 3-way ANOVA results (CS and NF habitats not pooled), other metrics with significant site differences included Chircp , Pleccp , and the two dominant taxa metrics as indicators of community evenness DT1dd  and DT2 dd . Significant site differences in these indicator metrics were not consistent across habitat types, seasons or interactions, therefore these differences could be caused by the presence of either invasive alien crayfish, other unknown factors that were not measured during the study (i.e. diel crayfish movement or changes in habitat use), or both. However, most site differences we observed in the indicator metrics were consistent across seasons and habitats (1>2 or 1<2), indicating that these macroinvertebrate community attributes are the ones most likely being affected by the presence of O. hylas crayfish at the invaded site.A significant interaction between period and habitat is expected if a certain habitat is being utilized more or less by invasive alien crayfish across different seasons, or if invasive alien crayfish are utilizing a different food source across different habitats. For both nested ANOVA results (2-way and 3-way), this interaction was not significant in three metrics with significant site differences, including ChirO. hylas at the invaded site but not utilized by native crayfish (i.e. O. peruncus) at the upstream, non-invaded site, this might explain why significant interactions were not present for some indicator metrics. These interactions can more easily be explained in relation to the crayfish invasion, and appear to indicate that both sampling season and habitat are affecting the observed differences in macroinvertebrate metrics between sites. Considering our overall results and interpretation of interactions, the presence of O. hylas at the invaded site (Site 1) resulted in decreased richness of total, EPT and Chironomidae, decreases in Chironomidae abundances, changes in functional groups (scrapers) and overall community evenness, increases in Elmidae, and declines in overall diversity. However, our data also suggests that other factors are influencing macroinvertebrate community composition at the study sites at specific habitats or during some seasons, and the relative degree to which these other factors are affecting the macroinvertebrate communities at both sites cannot be determined.Since the habitat assessment we conducted during this study did not include measurements of seasonal habitat availability or spatial/temporal changes in habitat use by crayfish or other macroinvertebrates, the causes of these interactions cannot be determined with any certainty. Literature suggests that some crayfish species forage in different habitats during different seasons and/or times of the day –95. HoweO. hylas crayfish have replaced the native O. peruncus. The invaded site had significantly different total, midge (Chironomidae) and EPT taxa richness, abundances of riffle beetles (Elmidae) and EPT organisms, and community evenness compared to the non-invaded site during more than one sampling season and in more than one habitat. We also observed significant site differences even in cases where interactions between variables were not significant. Because we observed changes in the same macroinvertebrates utilized as prey items by crayfish, it is possible that the invasion has the potential to cause ecological harm to resident macroinvertebrate communities. Overall, literature suggests that the competitive advantages of alien O. hylas over the native O. peruncus  may reduce macroinvertebrate abundances and richness or cause shifts in community dominance.Results from our study highlight differences in macroinvertebrate community composition between an invaded and non-invaded site in Crane Pond Creek, where O. hylas obtain their highest densities in riffle habitat during spring and early summer [O. hylas, and very few of these studies have been conducted on Ozark crayfish species in general [O. hylas may cause changes in the trophic interactions and ecological functions that are beyond the scope of this study.The effects we observed have potential implications for streams in the Ozark region of Missouri. In particular, newly hatched y summer , the samy summer , 12, 73. general . Our resrich) and EPT (EPTrich) richness, Shannon Diversity Index (SDIdd), and the Missouri Biotic Index (Bitol) are the four core metrics used to evaluate wadeable stream quality. In particular, we did not expect a significant difference between our study sites in the BItol, which is a pollution-based indicator that is sensitive to nutrient enrichment [We also observed significant site differences in macroinvertebrate indicator metrics that are commonly used to evaluate the quality of aquatic life in Ozark streams. We utilized the same macroinvertebrate protocol and habitats included in these assessments, and our spring and fall data coincides with their recommended index periods for sampling . Total Click here for additional data file.


The isotopic niches were locally shaped by the different degrees of glacier retreat-related disturbance within the Cove. Wider isotopic niche widths were found at the site that has become ice-free most recently, and narrower niches at the older ice-free sites. At an intermediate state of glacier retreat-related disturbance (e.g. via ice-growler scouring) species with different strategies could settle. The site at the earliest stage of post-retreat development was characterized by an assemblage with lower trophic redundancy. Generally, the isotopic niche widths increased with increasing size spectra of organisms within the community, excepting the youngest assemblage, where the pioneer colonizer meiofauna size class displayed the highest isotopic niche width. Meiofauna at all sites generally occupied positions in the isotopic space that suggested a detrital-pool food source and/or the presence of predatory taxa. In general ice scour and glacial impact appeared to play a two-fold role within the Cove: i) either stimulating trophic diversity by allowing continuous re-colonization of meiofaunal species or, ii) over time driving the benthic assemblages into a more compact trophic structure with increased connectedness and resource recycling.The western Antarctic Peninsula is experiencing strong environmental changes as a consequence of ongoing regional warming. Glaciers in the area are retreating rapidly and increased sediment-laden meltwater runoff threatens the benthic biodiversity at shallow depths. We identified three sites with a distinct glacier-retreat related history and different levels of glacial influence in the inner part of Potter Cove , a fjord-like embayment impacted since the 1950s by a tidewater glacier retreat. We compared the soft sediment meio- and macrofauna isotopic niche widths (δ The West Antarctic Peninsula (WAP) has been one of the Earth’s most rapidly warming regions over recent decades –5, with Laternula elliptica, ascidians and the sea-pen Malacobelemnon daytoni )2; ). SEAs a2; . They ar2; . A Bayes2; [c) . Using Ses in R; ) within iolation . In “sizanalysis .13C ranges were the same [13C range (CR), indicative of niche diversification; (ii) δ15N range (NR), indicative of trophic length; (iii) mean distance from centroid (CD), indicative of the average trophic diversity; (iv) mean nearest neighbor distance (MNND), or a measure of the density of species packing; and (v) standard deviation of mean nearest neighbor distance (SDNND), or evenness of species packing. Together, the last two metrics are indicative of trophic redundancy: small MNND indicates higher trophic redundancy, where more species perform the same trophic function, and lower SDNND indicates more evenly distributed species with more species having similar ecological traits.In addition, we compared the three sites in terms of trophic structure using the community-wide metrics of Layman et al. . These mthe same . The sam“complete dataset”), where we merged all the available information and samples from the three sites, and ii) the analysis without “ambiguous” data excluding possible outliers or species not found during the qualitative sampling for stable isotope analysis but known to be present at the study sites [reduced dataset” we excluded from Isla D i) the harpacticoid copepod MT2 since this species was as an outlier  and ii) the two polychaete species Barrukia cristata (Polynoidae) and Aglaophamus trissophyllus (Nepthydae), since they were found during the qualitative sampling only at Creek station. Nevertheless, since a previous investigation [complete dataset” analysis.For the analysis we ran the SIBER analysis on two sets of data: i) the primary analysis based on the complete dataset  the results from the “reduced dataset” analysis are given in reduced dataset” analysis are given in 13C values is given as extra information in Results are expressed as mean±SD. In 13C: -25.03‰ ± 5.45) and SPM (mean δ13C: -26.08 ± 1.17 ‰), to the more enriched values of MPB (mean δ13C: -13.15‰ ± 0.35). SPM showed overlapping δ13C signatures with phytoplankton 200 μm and macroalgae. Macroalgae showed the widest range of δ13C values. In terms of δ15N signatures SPM showed the most depleted values (0.45‰ ± 1.17) whereas the other food TL ranged around 3–4‰. For the consumers, the deposit feeder/omnivore feeding group showed the widest range of δ13C values (-17.82‰ ± 2.57), followed by scavenger/omnivore (-16.76‰ ± 1.79), predator/omnivore (-16.70‰ ± 1.74), zooplankton (-23.80‰ ± 1.41) and filter/suspension feeder (23.51‰ ± 0.95 SD). The most depleted δ13C (-34.9‰) as well as δ15N (-2.6‰) values were recorded for the harpacticoid copepod MT2 which was the taxon present at Isla D colonized by epibionts (likely ciliates). δ15N values of consumers showed a similar pattern with deposit feeder/omnivore holding an intermediate position in the trophic level bi-plot (15N signatures (8.96‰ ± 2.18). The filter/suspension feeder occupied the lower trophic level , whereas scavenger/omnivore (10.17‰ ± 1.14) occupied the range of TL2-TL3 and predator/omnivore (11.86‰ ± 0.71) TL3-TL4 (The isotopic signature see  of the f bi-plot  represen TL3-TL4 .c) “by site” showed differences in shapes and sizes between the sites (2), followed by Faro (19.88‰2) and Creek (14.89‰2). The probabilities that the standard ellipse area (SEAb) of Isla D was larger than those of Faro and Creek were 0.84 and 0.98, respectively (b of Faro was bigger than that of Creek was 0.90. The overlap between Faro and Creek was larger (11.92‰2) than between either Creek and Isla D or Faro and Isla D . An SEAc of the “site by size class” (2) and “large macrofauna (11.38‰2) size classes. Faro displayed a rounder and more compact ellipse in the “large macrofauna size class (37.97‰2), whilst the meiofauna and small macrofauna ellipses resembled each other in shape, but differed in size . The three size classes at Creek showed similar SEAc ellipses, both in shape and size , although the overlap could not be compared due to small sample size. The probabilities that meiofauna ellipses were larger than small macrofauna ellipses were 0.38 at Creek, 0 at Faro and 1.00 at Isla D. The probabilities that “large macrofauna ellipses were larger than “small macrofauna ellipses were 0.84 at Creek and 0.99 at Faro and 0.98 at Isla D. In light of the possible “outlier effect” of some specimens . At Isla D the large macrofauna SEAc now showed very reduced trophic niches, and the meiobenthos occupied the highest position compared to the other two size classes  hosted the least compact and widest isotopic niche, whereas the site known to be ice-free for the longest time (Creek) had the most compacted SEAy theory .This trend indicates the potential ability of the Antarctic benthic assemblage to increase the degree of interconnectedness within the food chain during a relatively short (years to decades) time after an important environmental change (in this case the newly ice-free status). This would suggest these communities have a good capacity to adapt to the strong ongoing climate-driven changes.c ellipse at Isla D was mainly due to the meiofauna size class assemblage. Within this size group, the epibiosis trophic strategy identified in the meiobenthic harpacticoid copepod (MT2) was present only at this site. This morphotype stable isotope analysis showed negative δ15N and very depleted δ13C values, indicative of symbiosis and chemosynthetic relationships. At Isla D, cumaceans were also found to carry epibiont ciliates on their exoskeleton . This strategy was not observed in the other two sites during our sampling. In our earlier study we identified the relatively greater dominance of the pioneer coloniser nematode genus Microlaimus and the higher abundance of harpacticoid copepods at the site [Microlaimus is known to be a successful colonizer of fresh ice-scoured sediments (94) and to be present in intermediate/late stages of succession after the collapse of the Larsen B ice shelf [The very elongated shape of the SEAthe site . Microlace shelf . Similarediments  and to bce shelf  sedimentc and SEAb) were larger for Isla D and to a lesser extent also in Faro, compared to Creek. The additional resource pool represented by these primary producers could provide an explanation for the wider isotopic niche (wider primary producer isotopic niche) of the local assemblages found at these two sites.Isla D is the site which remained under the glacier tongue for the longest time. The local benthic assemblage here may have undergone a successional process that resulted in species impoverishment. This is observed on islands where the communities typically show an expanded niche width compared to their mainland counterparts . Terrest13C values of benthic consumers, e.g. deposit feeder/omnivore group, and values in c showed an intermediate position and a compacted shape, with relatively higher trophic levels  of meiofauna taxa. This may indicate that both the macrobenthos and the meiobenthos rely on a mixture of food sources  and meiofauna organisms may include organisms with likely predatory feeding habits in their diets.Strong benthic-pelagic coupling was suggested to sustain a detritus-based sediment community at Faro site  where mac ellipse of the Creek community overlapped by 80% with that of Faro, suggesting a similar “centralized” isotopic niche with a short food chain and efficient resource use.From the present data, the isotopic niche width at Creek was the smallest and showed the highest relative position in the bi-plot space. The older ice-free age, the high degree of disturbance resulting from the adjacent glacial meltwater stream and the likely frequent re-suspension and ice-scouring events characteristic for the location, may force the community to a more efficient use of the available resources which are finally transferred to the higher trophic levels. Not surprisingly, the SEAWhen species with different life strategies colonize a ‘new’ environment the functional diversity increases locally, and so the trophic niche of the community is expected to widen. With time an increase in trophic redundancy can be expected, since newcomers often share similar functional traits, as already observed for stream communities after glacial retreat in south-east Alaska . Our anac ellipses , lacking the higher TLs. Further, the size spectra ellipses , the lower the redundancy in trophic functions found within its community [reduced dataset” analysis, higher trophic diversity (SEAs) was apparent where the level of disturbance (e.g. ice scouring and inorganic sedimentation) was intermediate to low (Faro), a pattern already observed elsewhere in streams [Isla D large macrofauna appeared to exploit mostly the basal resources  by means of a reduced (Δδ15N = 1.8‰) invertebrate/plant based-diet fractionation step [Overall, the benthic food web at the three Potter Cove contrasting sites showed the presence of several levels of consumers spanning from the wide stable isotopic range of deposit feeder/omnivore to the more defined range of predator/omnivore and scavenger/omnivore. We identified a total of four consumer trophic levels  than those of the microalgae. Considering the most important meiobenthic group, the nematodes, a strong link is apparent to the local signature of sediment  when considering the water column and sediment as one benthic-pelagic trophic environment. Nevertheless, it is important to stress that the sampling protocol for phytoplankton collection used in this study may not have sampled those components smaller than 55 μm (the lower limit of our mesh size) and hence failed to find possible trophic connections between smaller phytoplankton and the filter feeders investigated.The food web in Potter Cove has long been suggested to very likely rely on macroalgal detritus  in view sediment . Nematod15N values. Organic matter recycling may have an important role in the Cove’s sediments due to the abundant bacterial community [15N values of these small metazoan taxa  and convex hull area (dashed lines) for all sites (see legend) for the “reduced dataset” analysis.SEA(TIF)Click here for additional data file.S2 Figc) is also presented as red squares.Mode (black dots) and probability of data distribution  for each site are presented. The standard ellipse area corrected for small sample size (SEA(TIF)Click here for additional data file.S3 FigFor each site the three consumer size classes (see legend for colors) are represented.(TIF)Click here for additional data file.S4 FigThe dark squares are the mode, the triangles the Bayesian probability and the bars represent the 95% credibility interval of the posterior probability distribution. NR = nitrogen range; CR = carbon range; CD = mean distance from centroid; MNND = mean nearest-neighbor distance; SDMNND = standard deviation MNND. See legend for colors.(TIF)Click here for additional data file.S5 FigYoldia eightsi is highlighted in green. The abbreviations in the list are Creek = C; Faro = F; Isla D = I; SPM = suspended particulate matter; POM = particulate organic matter; MPB = microphytobenthos; TL = trophic level. Specification of the net mesh size used to sample the phytoplankton is included in the list (55 μm or 200 μm mesh size). Symbols refer to trophic group designation as from In the list of species/taxa, where appropriate, the site where the organism was sampled is reported in parentheses. Meiofauna taxa have been highlighted in bold. The baseline organism (TIF)Click here for additional data file.S1 Tablecomplete dataset” analysis (left) and the "reduced dataset” analysis (right) for the “by site” analysis.Comparison of SIBER analysis results for the "(DOCX)Click here for additional data file.S2 Tablecomplete dataset’ analysis (left) and the "reduced dataset” analysis (right) for the “size by site” analysis.Comparison of SIBER analysis results for the "(DOCX)Click here for additional data file.S3 TablePairwise comparison for each site using Bayesian posterior probabilities . Results(DOCX)Click here for additional data file.