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In fact, mxd expression in both mutants resembles the expression

In fact, mxd expression in both mutants resembles the expression level observed in logarithmically growing wild type cells, indicating a possible role for BarA/UvrY in starvation response. Methods Strains and media Strains used in this study are listed in Table 1. E. coli strains were grown at 37°C in lysogeny broth (LB) medium. Where necessary medium was solidified by 1.5% (w/v) agar and supplemented with 50 μg/mL CP673451 kanamycin or 100 μg/mL ampicillin. S. oneidensis Selleckchem PF-2341066 MR-1 strains were grown at 30°C in LB medium, lactate medium (LM) [0.02% (w/v) yeast extract, 0.01% (w/v) peptone, 10 mM (wt/vol)

HEPES (pH 7.4), 10 mM NaHCO3 ] with a sodium lactate concentration of 50 mM or in minimal medium (MM) [1.27 mM K2 HPO4, 0.73 mM KH2PO4, 5 mM sodium 4-(2- hydroxyethyl)-1-piperazine-ethane-sulphonic acid (HEPES), MGCD0103 mouse 150 mM NaCl, 485 mM CaCl2, 9 mM (NH4)2SO4, 5 mM CoCl2, 0.2 mM CuSO4, 57 mM HBO, 5.4 mM FeCl, 1.0 mM MgSO4, 1.3 mM MnSO4, 67.2 mM Na2 EDTA, 3.9 mM Na2MoO4, 1.5 mM Na2SeO4, 2 mM NaHCO3, 5 mM NiCl2 and 1 mM ZnSO4, pH 7.4] amended with 50 mM sodium lactate as electron donor. Where necessary medium was solidified by 1.5% (w/v) agar and supplemented with 25 μg/mL kanamycin, 10 μg/mL tetracycline, 10 μg/mL gentamycine and 60 μg/mL 5-bromo-4-chloro-3-indolyl-beta- D-galactopyranoside (X-gal). Biofilms of S. oneidensis MR-1 were grown in LM amended with 0.5 mM sodium lactate (pH 7.4) or MM amended with 1.5 mM

sodium lactate (pH 7.4). Where necessary medium was supplemented with 12.5 μg/mL kanamycin. Construction of mxd transcriptional reporter strains S. oneidensis

MR-1 mxd reporter strains were constructed by transcriptionally fusing various-length fragments of the mxd upstream region to lacZ and gfp. A promoterless copy Dimethyl sulfoxide of either lacZ or gfp in the appropriate vector served as a control. LacZ -reporter strains To obtain a strain reporting on the transcriptional activity of mxd, a 450 bp fragment upstream of the mxdA translation initiation site was amplified with primers Pmxd-fw-SphI and Pmxd-rv-XbaI (Table 2) using S. oneidensis MR-1 genomic DNA as template. The lacZ gene was amplified from E. coli MG1655 genomic DNA using primers LacZ-fw-XbaI and LacZ-rv-PstI (Table 2). Subsequently, the two PCR products were purified from an agarose gel, restriction digested with XbaI and ligated. The fusion product was PCR amplified with primers Pmxd-fw-SphI and LacZ-rv-PstI (Table 2), purified from an agarose gel, restriction digested with XbaI and PstI and ligated into vector pME6031 (pJM1). Truncations of the mxd promoter region were generated by amplification from pJM1 with the following primer combinations and subsequent ligation into pME6031 as described above: 150 bp upstream region: Pmxd-fw-150-SphI and LacZ-rv-PstI. 250 bp upstream region: Pmxd-fw-250-SphI and LacZ-rv-PstI. 300 bp upstream region: Pmxd-fw-300-SphI and LacZ-rv-PstI.

The control GFP sequence [30] was used to design oligos for makin

The control GFP sequence [30] was used to design oligos for making a shRNA MK-2206 in vivo control construct. Sense strand sequences chosen to make the Igl, URE3-BP and EhC2A shRNA constructs

successfully transfected into trophozoites are shown in Table 1, and PCR oligos used to amplify these sequences to generate shRNAs via PCR are shown in Table 2. PCR conditions for generating shRNAs Initially, E. histolytica genomic DNA was used as a template for the first round of Igl shRNA PCRs. For the URE3-BP and EhC2A shRNA PCRs, the cloned U6 promoter was used as the PCR template: the Igl shRNA plasmids were digested with HindIII and ApaI and the U6 promoter was gel-purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). Two rounds of PCR were used to generate the shRNA constructs. The first PCR round BAY 11-7082 order generated the sense strand of the hairpin and the loop. Reaction volumes of 40 μl were set up, each consisting of 0.6 μl SAHARA™ DNA polymerase (Bioline USA Inc., Taunton, MA, USA), 4 μl 10× SAHARA™ PCR buffer, 3.2 μl 50 mM MgCl2, 2 μl dNTP mix (stock 10 mM each), 0.4 μl U6 HindIII forward oligo (100 μM stock), 0.4 μl R1 oligo (100 μM stock), 1 μl (200 ng E. histolytica genomic DNA or 25 ng gel-purified digest Combretastatin A4 nmr of HindIII/ApaI U6 promoter), and 28.4 μl sterile water. Cycling conditions were as follows: 95°C for 8 minutes, 10 cycles of 95°C 45 sec, 40°C 1 min, 68°C 1 min 30 sec;

25 cycles of 95°C 45 seconds, 52°C 1 min, 68°C 1 min 30 sec, and a 5 min final extension Mirabegron at 68°C. 5 μl of each PCR product was subjected to agarose gel electrophoresis to check that the products were ~380 bp. In the second PCR round, the first round PCR product was used as a template to add the antisense strand of the hairpin, the terminator sequence and the NotI site. Each 100 μl-volume reaction contained 2 μl SAHARA™ DNA Polymerase (Bioline USA Inc., Taunton, MA, USA), 10 μl 10× SAHARA™ PCR buffer, 8 μl 50 mM MgCl2, 5 μl dNTP mix (10 mM each), 0.8 μl U6 HindIII forward oligo (100 μM), 0.8 μl R2 oligo (100 μM), 2 μl PCR product from the first PCR round, and 71.4 μl sterile water. Cycling conditions were:

95°C for 8 minutes, 10 cycles of 95°C 45 sec, 18.5°C 1 min 30 sec, 68°C 1 min 30 sec; 30 cycles of 95°C 45 seconds, 55°C 1 min, 68°C 1 min 30 sec, and a 5 min final extension at 68°C. The low annealing temperature in the early cycles of the second PCR was used since the loop is the only overlap between the first round product and the second round reverse oligo. The second round PCR products were checked by agarose gel electrophoresis for products of the correct size (~420 bp). Sometimes a smaller product was present in addition to the correct size product in the final PCR product; this was ignored since it had no effect on the subsequent cloning steps. These final PCR products were ethanol-precipitated, then they and modified pGIR310 were digested with HindIII and NotI.

As the standard of care for stage G3b-5 CKD, we recommend that pa

As the standard of care for stage G3b-5 CKD, we recommend that patients be encouraged to lower their dietary protein intake to 0.6–0.8 g/kg·standard body weight (SBW)/day. Actual protein intake should be estimated by analyzing the urea content in the 24-h urine sample using the Maroni formula; it should then be evaluated by comparing it with the results of previously published studies, which showed that the achieved protein intakes were 0.75–0.9 g/kg/day in clinical trials with protein restriction of 0.6–0.8 g/kg/day. Several studies also demonstrated both the efficacy and potential hazards of a very low protein diet. Therefore, the potential

benefits Dabrafenib research buy and risks of severe protein restriction should be specifically assessed for each patient. Digestibility and the amino acid score of protein sources should be taken into consideration when prescribing protein restriction diets. For early CKD with the risk of progression, we suggest encouraging patients to lower their protein intake to 0.8–1.0 g/kg·SBW/day. The extent of protein restriction should be individualized in accordance with each patient’s specific clinical condition, including severity, risk of progression, nutritional status, and adherence. Bibliography 1. Pan Y, et al. Am J Clin Nutr. 2008;88:660–6. (Level 1)   2. Gansevoort RT, et al. Nephrol Dial Transplant. 1995;10:497–504. (Level 3)

  3. Williams PS, et al. Q J Med. 1991;81:837–55. (Level find more 2)   4. D’Amico G, et al. Nephrol Dial Transplant. 1994;9:1590–4. (Level 2)   5. Mircescu G, et al. J Ren Nutr. 2007;17:179–88. (Level 2)   6. Rosman JB, et al. Kidney Int Suppl. 1989;27:S96–102. (Level 2)   7. Cianciaruso B, et al. Am J Kidney Dis. 2009;54:1052–61. (Level 2)   8. Fouque D, et al. Cochrane Database Syst Rev. 2009:CD001892. (Level 1)   9. Pedrini MT, et al. Ann Intern Med. 1996;124:627–32. (Level 1)   10. Hansen HP, et al. Kidney Int. 2002;62:220–8. (Level 2)   11. Kasiske BL, et al. Am J Kidney Dis. 1998;31:954–61. (Level 1)   12. SP600125 order Robertson L, et al. Cochrane Database Syst Rev. 2007;CD002181.

Ribonucleotide reductase (Level 1)   13. Koya D, et al. Diabetologia. 2009;52:2037–45. (Level 2)   14. Feiten SF, et al. Eur J Clin Nutr. 2005;59:129–36. (Level 2)   15. Jungers P, et al. Kidney Int. 1987;22(Suppl):S67–71. (Level 2)   16. Cianciaruso B, et al. Nephrol Dial Transplant. 2008;23:636–44. (Level 2)   17. Malvy D, et al. J Am Coll Nutr. 1999;18:481–6. (Level 2)   18. Di Iorio BR, et al. Kidney Int. 2003;64:1822–8. (Level 2)   19. Ihle BU, et al. N Engl J Med. 1989;321:1773–7. (Level 2)   20. Levey AS, et al. Am J Kidney Dis. 1996;27:652–63. (Level 4)   21. Zeller K, et al. N Engl J Med. 1991;324:78–84. (Level 2)   22. Klahr S, et al. J Am Soc Nephrol. 1995;5:2037–47. (Level 3)   23. Walser M, et al. Am J Kidney Dis. 1996;28:354–64. (Level 5)   24. Chauveau P, et al. J Ren Nutr. 2007;17:250–7.

Identification of confirmed Pectobacterium spp isolates to speci

Identification of confirmed Pectobacterium spp. isolates to species and subspecies was conducted

on the basis of biochemical tests (indole production from tryptophan, lecithinase activity and acid production from α-methyl glucoside, trehalose, sorbitol, melibiose, lactose). All tests were carried out at 27°C for 24 h and compared with the standard strains (see Additional file 1 Table S1 for the fourteen strains used only in this study) [2, 10]. DNA extraction and PCR amplification Bacterial cultures from frozen stocks were grown onto LPGA medium and suspended in sterile H2O. The concentration was adjusted to 108 CFU.ml-1. DNA was extracted from bacterial suspension as described by Terta et al. [2]. The precipitated Epoxomicin DNA then was quantified using a NanoDrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), adjusted to Caspase Inhibitor VI solubility dmso 100 ng.μl-1 and stored at 4°C. All PCR amplifications were performed using the following primers:

pmr A F0145 (5’-TACCCTGCAGATGAAATTATTGATTGTTGAAGAC-3’) and E2477 (5’-TACCAAGCTTTGGTTGTTCCCCTTTGGTCA-3’) as described by Hyytiäinen et al. 2003 [16]. A 25 μl PCR mix contained: 1 μl DNA, 0.5 U Taq DNA polymerase, 2.5 μl 10 × PCR buffer, 2.5 mM each of dNTPs, 2.5 mM MgCl2, 0.5 μM of each primer. DNA amplification was performed on Veriti® Thermal Cycler (Applied Biosystems) under the Exoribonuclease following conditions: 5 min at 94°C for initial denaturation, 35 cycles of 1 min at 94°C for, 1 min at 55°C and 2 min 72°C, followed by a final elongation step of 10 min at 72°C. PCR products (6 μl) were separated by gel electrophoresis in 1.8% agarose gels in TBE buffer. Following staining with ethidium bromide, the gels were viewed and photographed

under UV Transilluminator. Fragment sizes were determined by comparison to a 100 bp DNA Ladders. Sequencing of pmrA and phylogenetic analysis The PCR-amplified fragments of pmrA were purified and the sequencing reactions were performed with a Big-Dye Terminator v3.1 (Applied Biosystems). The pmrA sequences which we determined and the sequences of the reference strains of members of the family Enterobacteriaceae obtained from the GenBank databases were analyzed. The pmrA sequences were first aligned by using the Clustal W program [34], and then the alignments were corrected by hand. Evolutionary trees for the data set were inferred by using the Neighbor-Joining program of MEGA [31, 33]. The stability of relationships was assessed by performing bootstrap analyses of the Neighbor-Joining data based on 500 resamplings. The entire sequences corresponding to positions 4317866-4318532 of the reference Vemurafenib order sequence of the subspecies. Nucleotide sequence accession numbers The pmrA sequences which we determined have been deposited in the GenBank database under the accession numbers shown in Table 1.

Sty, Salmonella enterica serovar typhimurium Ype, Yersinia pesti

Sty, Salmonella enterica serovar typhimurium. Ype, Yersinia pestis. Pfl, Pseudomonas fluorescens. Lpn, Legionella pneumophila. Hpy, Helicobacter pylori. Ara, Agrobacterium radiobacter. Mtu, Mycobacterium tuberculosis. Figure 2 The growth curves #INCB018424 molecular weight randurls[1|1|,|CHEM1|]# of L. pneumophila wild-type JR32, the Lp ΔclpP mutant, both harboring the vector pBC(gfp)Pmip, and the complemented strain Lp ΔclpP -p clpP. Overnight cultures of mid-exponential bacterial cells were diluted into fresh medium and then incubated at (A) 25°C, (B) 30°C, (C) 37°C, and (D) 42°C, respectively. Growth was monitored by OD600 at various time points. Points indicate mean values

and error bars indicate standard deviations of three experiments. clpP homologue is required for stress tolerance in stationary phase L. pneumophila can respond to various environmental

stresses and cope with harsh conditions while entering eukaryotic hosts [12, 41]. To assess whether clpP homologue may be involved in stress response, the above three strains were grown to logarithmic or stationary phase and exposed to various stress conditions. When the logarithmic-phase cells were exposed respectively to low pH, hydrogen peroxide, potassium chloride, and heat shock, the survival S3I-201 rates of all three strains were similar and lower than those of the stationary-phase cells (data not shown). When treated with pH 4.0 citric acid for 30 minutes, WT JR32 cells in stationary phase exhibited approximately 70% survival rate. However, only about 10% of LpΔclpP mutant Celastrol cells survived (Figure 3A). Such a deficiency was rescued in the LpΔclpP-pclpP strain (Figure 3A). This result indicated that the deletion of clpP impairs the ability of L. pneumophila to respond to low-pH conditions. Similar results were also obtained in oxidative stress assay (Figure 3B). When the cells were treated with 1 mM hydrogen peroxide for 30 minutes, the survival rate of the LpΔclpP mutant was 10 ± 2.0%, much lower than that of WT cells (56 ± 8.6%; Figure 3B). In contrast, LpΔclpP-pclpP cells displayed

a CFU closely resembling that of WT cells (Figure 3B). Likewise, when cells were incubated in 57°C water bath for 20 minutes or treated with 0.3 M potassium chloride for 1 hour, the survival rate of LpΔclpP mutant was lower than that of WT and the complementation strain (Figure 3C and 3D), indicating that clpP is also required for responses to heat shock and osmotic stress. Collectively, these results indicate that ClpP homologue is involved in tolerance to multiple stresses in stationary-phase L. pneumophila. Figure 3 Impaired stress tolerance of the L. pneumophila Lp ΔclpP mutant during stationary phase. Overnight cultures of different strains were inoculated into fresh medium and grew to stationary phase (OD600 from 3.5 to 4.5), and the cells were then treated with (A) 1 mM H2O2 for 30 minutes. * p < 0.05, (B) pH 4.0 citric acid for 30 minutes. * p < 0.01, (C) 57°C heat shock for 20 minutes. * p < 0.05, or (D) 0.3 M KCl for 1 hour. * p < 0.05.

J Pathol 2001, 194 (1) : 15–19 CrossRefPubMed 9 Hainsworth AH, B

J Selisistat purchase Pathol 2001, 194 (1) : 15–19.CrossRefPubMed 9. Hainsworth AH, Bermpohl D, Webb TE, Darwish R, Fiskum G, Qiu J, McCarthy D, Moskowitz MA, Whalen MJ: Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex. J Cereb Blood Flow Metab 2005, 25 (8) : 1030–1040.CrossRefPubMed 10. Wang W, Wang S, Song X, Sima N, Xu X, Luo A, Chen G, Deng D, Xu Q, Meng L, et al.: The relationship between c-FLIP expression and human papillomavirus E2 gene disruption in cervical carcinogenesis. Gynecol Oncol 2007, 105 (3) : 571–577.CrossRefPubMed 11. Wong

SCC, Lo ESF, Cheung MT: An optimised protocol for the extraction of non-viral selleckchem mRNA from human plasma frozen for three years. J Clin Pathol 2004, 57 (7) : 766–768.CrossRefPubMed 12. Zhou Y, Pan Y, Zhang S, Shi X, Ning T, Ke Y: Increased phosphorylation of p70 S6 kinase is associated with HPV16 infection in cervical cancer and esophageal cancer. British Journal of Cancer 2007, 97 (2) : 218–222.CrossRefPubMed Competing interests The authors declare that they have no competing interests.

Authors’ contributions XJH: study design, data analysis, experimental studies, manuscript review. YZZ: the guarantor of integrity of the entire click here study, study design, experimental studies, data analysis, manuscript preparation. XL: clinical studies, manuscript review. LHM: experimental studies. YBQ: study design, manuscript editing.”
“Review The concept that a vaccine could be useful in the treatment of cancer diseases is a long-held hope coming from the observation that patients with cancer who developed bacterial infections experienced remission of their malignancies. In 1896, New York surgeon William Coley locally injected streptococcal broth cultures to induce erysipelas in a patient with an inoperable neck sarcoma, obtaining a tumour regression. Although the therapy was toxic, the patient’s

tumour ultimately regressed, and he lived disease-free for 8 years before succumbing to his cancer [1]. During the century since Coley’s first experiments, immensely more is understood about tumour immunology: the validation of the theory of cancer immunosurveillance, the definition of a large number of tumour antigens as targets for immune recognition, the prognostic significance of immunological Thalidomide parameters, such as the different sub-classes of T cell infiltrating human tumours, and therapeutic benefits of immune-related therapies from BCG to anti-CTLA-4 are the major achievements that pose the theoretical basis to test the validity of cancer vaccines. In particular some characteristics of HNSCC render these tumours susceptibly to explore efficacious immunotherapy: the presence of well characterized Tumour Associated Antigens (TAA) and the possibility to perform clinical trials as adjuvant cancer therapy to eradicate local regional microscopic and micrometastatic disease with minimal toxicity to surrounding normal cells.

DMSO served as a solvent control To investigate whether inhibiti

DMSO served as a solvent control. To investigate whether inhibition of HDAC8 might be counteracted by concomitant upregulation of other class I-HDACs (HDAC1, HDAC2 and HDAC3) their

expression levels were compared by real-time PCR and western blot analysis (Figures 11 and 12). In brief, HDAC1, HDAC2 and HDAC3 mRNA levels exhibited variable changes after siRNA-mediated knockdown of HDAC8. Both significant up-and downregulation of specific HDACs were observed. In LDN-193189 supplier particular, either HDAC1 or HDAC2 seems to become PCI-32765 mw upregulated after HDAC8 knockdown (Figure 11A). Western blot analysis shown in Figure 11B revealed a decrease of HDAC2 protein in RT-112 cells and HDAC3 protein in UM-UC-3 cells after siRNA mediated HDAC8 knockdown. No significant AS1842856 deregulation of other class I-HDACs took place (Figure 11B). Figure 11 Compensation mechanism after HDAC8 knockdown in RT-112, VM-CUB1,

SW-1710, 639-V and UM-UC-3 cells. Effects of siRNA mediated HDAC8 knockdown on (A) mRNA and (B) protein expression levels of the class I histone deacetylase HDAC8, HDAC1, HDAC2 and HDAC3 (72 h) in comparison to untreated and irrelevant control. The mRNA expression values were measured by quantitative RT-PCR analysis and were normalized to TBP as a reference gene. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. Protein expression levels were analyzed by western blotting, and α-tubulin was stained on each blot as a loading control. Figure 12 Compensation mechanism after specific HDAC8 inhibition in RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells. Effects of HDAC8 inhibitor treatment on (A) mRNA and (B) protein expression of the class I histone deacetylases HDAC8, HDAC1, HDAC2 and HDAC3, compared

to DMSO solvent control (compound 2, compound 5, compound 6; IC50, 72 h). The mRNA expression values were measured by quantitative RT-PCR analysis and were normalized to TBP as a reference gene. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances of the treated Benzatropine value refer to the DMSO solvent control. Protein expression levels were analyzed by western blotting, and α-tubulin was stained on each blot as a loading control. Measurements of mRNA expression after pharmacological inhibition of HDAC8 showed significant, but overall slight decreases or increases of the expression of several HDACs in the UCC (Figure 12A). Apart from a slightly reduced expression of HDAC1 and HDAC2/3 in SW-1710 and VM-CUB1 cells, no changes of protein expression were observed after c5 and c6 treatment (Figure 12B).

Selection criteria for enrolment in the study were vaginal delive

Selection criteria for enrolment in the study were vaginal delivery at term and uncomplicated perinatal period. Questionnaires were collected with data on the GDC-0973 ic50 parents, including demography, smoking and asthma.

Data of the child on demography, respiratory symptoms and risk factors for asthma were collected by postal questionnaires sent every 6 months starting at the age of 3 weeks until the age of 36 months. The question on the presence of wheezing referred to the period between two questionnaires, e.g. the presence of wheezing in the questionnaire at 6 months referred to the time period between 3 weeks and 6 months. The study protocol was approved by the medical ethics committees of the participating institutes. All parents gave written informed consent. Symptoms of wheeze were assessed see more by International Study of Asthma and Allergies in Childhood core questions [9]. Information about doctor’s diagnosed parental asthma was collected

by the following question: ”Did a doctor ever diagnose asthma?”. Based on the longitudinal questionnaire data on wheeze symptoms in the first 3 years of life, children were classified according to the ‘loose’ Asthma Predictive Index (API) into an API positive and an API negative group. According to the ‘loose’ index a positive API included wheezing during the first three years of life and eczema or parental history of asthma [10]. Approximately 2 g of stools was collected into a sterile recipient by the parents at 3 weeks of age. The sample was sent to the laboratory under anaerobic conditions where it was stored immediately at -70°C until analysis. DNA was extracted from fecal samples based on the method of Pitcher et al. [11] modified by Vanhoutte et al. [5]. A saline suspension of feces was made by diluting Clostridium perfringens alpha toxin 1 g of wet feces in 10 ml of sterile saline solution and homogenized using a stomacher. Of this fecal sample suspension, 1 ml was centrifuged at 20,000 g for

5 min. After removal of the supernatant, the pellet was resuspended in 1 ml TE LY333531 purchase buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and was again centrifuged at 20,000 g for 5 min. The pellet was resuspended in 150 μl enzyme solution (6 mg lysozyme powder [Serva] and 40 μl mutanolysine [Sigma] dissolved in 110 μl TE (1 ×) per sample) followed by incubation at 37°C for 40 min. Next, 500 μl GES reagent (Guanidiumthiocyanate-EDTA-Sarkosyl; 600 g l-1 guanidiumthiocyanate [Sigma], 200 ml l-1 0.5 M EDTA, 10 g l-1 sarkosyl) was added to complete all lysis, after which the solution was put on ice for 10 min. In the following step, 250 μl ammonium acetate (7.5 M) was added and the mixture was put on ice for 10 min. Subsequently, two chloroform-iso-amylalcohol extractions were performed with 500 μl chloroform/iso-amylalcohol solution (24/1). Finally, DNA was precipitated by adding 0.54 volumes of ice-cold isopropanol.

Mycologia 97:1365–1378PubMedCrossRef Jaklitsch WM, Komon M, Kubic

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