Biodiversity and biocatalyst activity of culturable hydrocarbonoclastic fungi isolated from Marac-Moruga mud volcano in South Trinidad.

Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.

Effects of dietary grape pomace on the intestinal microbiota and growth performance of weaned piglets.

Grape pomace (GP) is an abundant by-product from wine production and is rich in phenolic compounds, unsaturated fatty acids, dietary fibre and beneficial bacteria. In this study, weaned piglets were fed a basic diet supplemented with 5% GP for 4 weeks. Compared with those in the control (CON) group, it was found that the proportion of Lactobacillus delbrueckii, Olsenella umbonata and Selenomonas bovis in the caecum and the villus height and villus height/crypt depth ratio (VCR) of the jejunum were both significantly increased in the GP group (p < 0.05). Meanwhile, at the mRNA expression level, several proinflammatory cytokines (IL-1ß, IL-8, IL-6 and TNF-α) were significantly downregulated (p < 0.05) in piglet caecal tissue, and the short-chain fatty acid receptors (GPR41 and GPR43) were not significantly upregulated. In contrast, the levels of IgG was significantly increased (p < 0.05) in the sera of weaned piglets in the GP group. However, no difference in growth performance between the two groups of piglets was detected. These results show that GP had no adverse effects on the growth performance of piglets, but GP can promote the content of some beneficial bacteria in the caecum; this effect is conducive to improving the disease resistance potential of piglets.

Varieties of immunity activities and gut contents in tilapia with seasonal changes.

We performed 16S rDNA sequencing of tilapia fecal samples to analyze changes in tilapia gut contents after cultivation of the fish in the presence of sandwich-like floating beds of Chinese medicinal herbs (5 and 10% planting-areas; 5% Polygonum cuspidatum). The interactive effects between water quality and blood and hepatic pro- and anti-inflammatory concentrations were also assessed. Our results showed that the water quality (i.e., NO3--N, NO2--N, TP removal rates) improved, and the abundance of Chloroflexi and Cyanobacteria increased. The abundance of Bacteroidetes, Verrucomicrobia, Saccharibacteria, and Actinobacteria showed both significant seasonal decreases and increases in the presence of P. cuspidatum (increases in August and decreases in July). Fish blood and hepatic IL-10 and IFN-γ levels (together with fish sampled in September) significantly increased in the P. cuspidatum group sampled in August, while those of TNF-α (10% sandwich-like, P. cuspidatum), IL-1ß (P. cuspidatum), IL-8 (5% sandwich-like in September, S905S) significantly decreased. Heat shock proteins 60 and 70 levels significantly increased in the P. cuspidatum group, and complement C3 and C4 concentrations significantly increased in S905S. This study demonstrated that enhanced immunity through the regulation of pro- and anti-inflammatory proteins was sustained throughout development until harvest, particularly in fish grown with P. cuspidatum.

Effect of selenium supplements on the antioxidant activity and nitrite degradation of lactic acid bacteria.

Selenium (Se) is one of the essential trace elements in the human body, and Se-enriched lactic acid bacteria (LAB) can improve the biological utilization value of inorganic Se. The aim of this study was to isolate Se-enriched LAB and study their effects on antioxidant activity and nitrite degradation. The Se-enriched LAB L.P2, which was nitrite-tolerant and could grow in 30 µg/mL sodium selenite (Na2SeO3) medium, was isolated from the traditional fermented Chinese sauerkraut. L.P2 belonged to Lactobacillus plantarum according to the 16S rDNA analysis. The biomass and lactic acid production of L.P2 reached to a maximum (9.52 log CFU/mL and 16.99 mg/mL) when 2.0 µg/mL Na2SeO3 was supplemented in the medium. Additionally, the nitrite degradation rate reached 85.76% when the initial concentration of Na2SeO3 was 2.0 µg/mL. The Se-enriched LAB enhanced the scavenging capacity of hydroxyl radical and superoxide free radical of L.P2 and improved the lipid peroxidation and ion-chelating abilities. Moreover, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in Se 4 group (4.0 µg/mL Na2SeO3 was added) reached 48.49 and 50.35 U/mg, respectively. Thus, Se 4 concentration was significantly higher than that of Se 0 group (with no Se added). In particular, SOD and GSH-Px enzymes correlated with nitrite degradation (P < 0.01). Collectively, our results indicate that Se supplementation can enhance the antioxidant capacity of LAB, contribute to its nitrite degradation, and thus may have potential applications in functional foods.

AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis.

Studies on affecting factors and mechanism of treating decentralized domestic sewage by a novel anti-clogging soil infiltration system.

The effects of bore diameter and particle size of polyurethane (PU) foam on soil wastewater infiltration system as well as its anti-clogging mechanism were investigated in this study. Different types of PU were used to determine the effect of bore diameter and particle size on the chemical oxygen demand (COD) removal. The results revealed that bore diameter showed little effects and the optimal size of PU should be not less than 10 mm. The formation of strong hydrophilic group on the outer layer of hydrophobic PU foam was fixed with active ingredient Al2O3, leading to good anti-clogging effect. Denaturing gradient gel electrophoresis fingerprint profiles and cluster analysis showed that the microbial community in the bottom was different from that in other places of the normal column, while it in the top has obvious differences from that in other places of the clogging column. Furthermore, the dominant microbial species of the normal column was Betaproteobacteria while Alphaproteobacteria in the clogging column.

Metabolic Profiling of Alpine and Ecuadorian Lichens.

Non-targeted ¹H-NMR methods were used to determine metabolite profiles from crude extracts of Alpine and Ecuadorian lichens collected from their natural habitats. In control experiments, the robustness of metabolite detection and quantification was estimated using replicate measurements of Stereocaulon alpinum extracts. The deviations in the overall metabolite fingerprints were low when analyzing S. alpinum collections from different locations or during different annual and seasonal periods. In contrast, metabolite profiles observed from extracts of different Alpine and Ecuadorian lichens clearly revealed genus- and species-specific profiles. The discriminating functions determining cluster formation in principle component analysis (PCA) were due to differences in the amounts of genus-specific compounds such as sticticin from the Sticta species, but also in the amounts of ubiquitous metabolites, such as sugar alcohols or trehalose. However, varying concentrations of these metabolites from the same lichen species e.g., due to different environmental conditions appeared of minor relevance for the overall cluster formation in PCA. The metabolic clusters matched phylogenetic analyses using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences of lichen mycobionts, as exemplified for the genus Sticta. It can be concluded that NMR-based non-targeted metabolic profiling is a useful tool in the chemo-taxonomy of lichens. The same approach could also facilitate the discovery of novel lichen metabolites on a rapid and systematical basis.

Antileishmanial activity of compounds produced by endophytic fungi derived from medicinal plant Vernonia polyanthes and their potential as source of bioactive substances.

The purpose of this work was to evaluate the antileishmanial activity of endophytic fungi isolated from leaves of Vernonia polyanthes plant and their prospective use in the discovery of bioactive compounds. Sixteen endophytes were isolated by using potato dextrose agar medium and submitted to cultivation in rice medium. The fungal cultures were extracted with ethanol and used as crude extracts for testing their antileishmanial activity. The most active ethanol extract was obtained from P2-F3 strain, which was identified as Cochliobolus sativus by ITS rRNA gene sequence data. Followed by a bioassay-guided fractionation, the cochlioquinone A, isocochlioquinone A and anhydrocochlioquinone A compounds were isolated from the crude extracts and demonstrated to inhibit the parasites. From the present work, it is possible to conclude that endophytic fungi derived from medicinal plant V. polyanthes may be considered promising source for the discovery of bioactive compounds.

Efficiency of Indigenous Filamentous Fungi for Biodegradation of Petroleum Hydrocarbons in Medium and Soil: Laboratory Study from Ecuador.

The competence of two fungal isolates for degrading petroleum hydrocarbons was evaluated. The filamentous fungi were isolated from a crude oil-contaminated soil in northeastern Ecuador, and were 99 %-100 % similar in 18S rDNA sequence to the genus Geomyces. Their efficiencies of degradation were tested in vitro for 30 days, using medium and soil microcosm. Residual hydrocarbons were tracked by gas liquid chromatography with a flame ionization detector. The maximum removal percentages of total petroleum hydrocarbons were 77.3 % and 79.9 % for experiments in the medium and soil microcosm, respectively. The percent germination of cow pea (Vigna unguiculata) seeds was increased from 20 % to 100 % upon bioremediation. Isolates sporulated optimally on minimal salts agar medium at pH 5, 25°C temperature, 1 %-1.5 % substrate (crude oil) and 4-6 g L(-1) N-P-K. These findings suggest that these fungal isolates are potential degraders for bioremediation in crude oil-contaminated areas in Ecuador.

Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen "Candidatus Liberibacter solanacearum".

An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers.

Diversity and space-time dynamics of endophytic archaea from sugar beet in the north slope of Tianshan Mountain revealed by 454 pyrosequencing and T-RFLP.

Plants harbor complex and variable microbial communities. Using molecular-based techniques targeting the 16S rRNA gene, we studied the developmental stages and geographical location diversity of endophytic archaea in two locations (Shihezi and Changji) and four periods (the seedling growth, rosette formation, tuber growth and sucrose accumulation sampling periods) in the north slope of Tianshan Mountain, China. Community structure of mixed sample from 60 sugar beet plants was examined using PCR-based 454 pyrosequencing and terminal restriction fragment length polymorphism (T-RFLP). In total, 5290 archaea 16S rRNA sequences were obtained from all sugar beet samples. The most abundant archaea groups in all sugar beet were Methanococci, the miscellaneous Crenarchaeotic Group and Thermoplasmata. There was a marked difference in diversity of endophytic archaea in sugar beet for different growth periods. The greatest number of Operational T-RFLP Units (OTUs) was detected during sucrose accumulation (298) and rosette formation (282). Endophytic archaea diversity was reduced during seedling growth (128 OTUs) and tuber growth (55 OTUs). Nine OTUs were common to all four periods of growth. There were more OTUs in Shihezi than in Changji. Clustering analysis and principal component analysis of T-RFLP data revealed distinct shifts in endophytic archaea community profiles that corresponded to plant growth stage rather than geographical location. The dynamics of endophytic archaea communities were influenced by plant growth stage. To our knowledge, this is the first report that archaea has been identified as endophytes associated with sugar beet by the culture-independent approach. The results suggest that the diversity of endophytic archaea is abundant in sugar beet.

Invasive Trichosporon infection in solid organ transplant patients: a report of two cases identified using IGS1 ribosomal DNA sequencing and a review of the literature.

Trichosporon species are rare etiologic agents of invasive fungal infection in solid organ transplant (SOT) recipients. We report 2 well-documented cases of Trichosporon inkin invasive infection in SOT patients. We also conducted a detailed literature review of Trichosporon species infections in this susceptible population. We gathered a total of 13 cases of Trichosporon species infections. Any type of organ transplantation can be complicated by Trichosporon infection. Bloodstream infections and disseminated infections were the most common clinical presentations. Liver recipients with bloodstream or disseminated infections had poor prognoses. Although the most common species was formerly called Trichosporon beigelii, this species name should no longer be used because of the changes in the taxonomy of this genus resulting from the advent of molecular approaches, which were also used to identify the strains isolated from our patients. Antifungal susceptibility testing highlights the possibility of multidrug resistance. Indeed, Trichosporon has to be considered in cases of breakthrough infection or treatment failure under echinocandins or amphotericin therapy. Voriconazole seems to be the best treatment option.

Isolation and characterization of xanthan-degrading Enterobacter sp. nov. LB37 for reducing the viscosity of xanthan in petroleum industry.

A Gram-negative, straight rod and facultative anaerobic bacterium was isolated from soil sample. It exhibits the phenotypic characteristics consistent with its classification in the genus Enterobacter. The isolate ferment glucose to acid and gas. Arginine dihydrolase, ornithin decarboxylase and gelatinase but not deoxyribonuclease was produced by this isolate. There was no hydrogen sulfide production. On the basis of the phenotypic data, together with phylogenetic analysis based on 16S rDNA gene sequences, this strain should represent a novel species of the genus Enterobacter and was designated as LB37. The strain LB37 could degrade xanthan molecules resulting in the rapid decrease of the viscosity of xanthan solution used in oil drilling process. Endoxanthanase activity was also detected in the culture supernatant. To our knowledge, it is the first report on the microbes being involved in the xanthan degradation for oil industry. The isolate LB37 would be useful for potential application in enhanced oil recovery and oil drilling field.

Helicobacter canis bacteremia in a renal transplant patient.

Here we present a case report of a 41-year-old woman suffering from high fever and bacteremia due to Helicobacter canis, 11 months after kidney transplantation. Identification of H. canis was achieved by 16s rDNA sequence analysis of a positive blood culture. The patient was restored fully to health after antibiotics therapy (cefuroxime and ciprofloxacin). Until now, only 4 human clinical cases have been described with H. canis bacteremia. This study describes for the first time, to our knowledge, an infection with H. canis in a kidney transplant patient.

Phylogenetic examination of crude drugs derived from Yunnanese Swertia plants.

Aiming to examine whether the genetic background of the crude drugs derived from four Yunnanese Swertia plants and their chemical constituent profiles correlate, we analyzed the nucleotide sequences of their nuclear ribosomal DNA regions including ITS1, 5.8S ribosomal RNA gene, and ITS2, together with those of Japanese S. japonica and S. pseudochinensis from Hebei Province. The result that two of the Yunnanese Swertia plants, S. binchuanensis and S. punicea, were genetically similar may explain their similarity in chemical constituent profiles. On the other hand, in spite of differences in chemical profile, S. decora and S. pseudochinensis were genetically close. The other Yunnanese Swertia plants, S. delavayi, and S. japonica, stood at intermediate positions between these two genetically similar pairs. The result suggests that although genetic background would have an influence, environmental factors, e.g., soil and weather conditions, might be critical for their production of secondary metabolites.

Bacterial communities associated with lesions of two forms of shell disease in the American lobster (Homarus americanus, Milne Edwards) from Atlantic Canada.

Shell disease is a major threat to the American lobster (Homarus americanus, Milne Edwards) fishery. Here we describe the composition of microbial communities associated with lesions of 2 forms of shell disease in Atlantic Canada, (i) a trauma shell disease (TSD) characterized by massive lesions and (ii) an enzootic shell disease (EnSD) characterized by irregularly shaped lesions with a distinct orange to yellow color. The microbiology of the lesions was described by polymerase chain reaction and denaturing gradient gel electrophoresis of 16S rDNA amplified from scrapings of the shell lesions and was compared with communities of unaffected carapaces and previously described forms of shell diseases. Both TSD and EnSD lesions were dominated by members of Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria, all commonly detected in other forms of shell disease; however, unique members of Epsilonproteobacteria were also present. Two Vibrio spp. and 2 Pseudoalteromonas spp. were dominant in lesions of TSD and a Tenacibaculum sp. and Tenacibaculum ovolyticum were dominant in lesions of EnSD. The TSD and EnSD in this study contained similar taxa as other shell disease forms; however, their microbiology is mostly different and neither resembles that of epizootic shell disease.

Sphingomonas ginsengisoli sp. nov., isolated from soil of a ginseng field.

A Gram-reaction-negative, oxidase- and catalase-positive, non-gliding motile bacteriun, designated DCY58(T), was isolated from a ginseng field in Pocheon Province, South Korea. Colonies of strain DCY58(T) were circular, 0.5-1.5 mm in diameter, yellow, and convex on an R2A agar plate after 2 days. The 16S rRNA gene sequence analysis revealed that strain DCY58(T) belongs to the family of the Sphingomonadaceae and is most closely related to the species of genus Sphingomonas with similarity levels of 95.7-96.4%. Strain DCY58(T) contained Q-10 as the predominant ubiquinone. The major cellular fatty acids included Summed Feature 8 (containing C(18:1ω)6c and/or C(18:1ω)7c), C(14:0) 2-OH and C(16:0). The DNA G+C content was 65.1 mol%. Furthermore, strain DCY58(T) differed from other related Sphingomonas species by a number of phenotypic characteristics. On the basis of the evidence presented in this study, strain DCY58(T) is described as a novel species of genus Sphingomonas, for which the name Sphingomonas ginsengisoli sp. nov., is proposed. The type strain is DCY58(T) (=KCTC 23762(T) =JCM 18016(T)).

Anaerocella delicata gen. nov., sp. nov., a strictly anaerobic bacterium in the phylum Bacteroidetes isolated from a methanogenic reactor of cattle farms.

A strictly anaerobic bacterial strain (WN081(T)) was isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. Cells were Gram-staining negative, non-motile, non-spore-forming straight rods. The strain grew rather well on PY agar slants supplemented with a B-vitamin mixture as well as sugars (PYV4S medium) and made translucent and glossy colonies. Growth in liquid medium with the same composition, however, was scanty, and growth was not improved in spite of various additives to the medium. Strain WN081(T) produced small amounts of acetate, propionate, isobutyrate, butyrate, isovalerate and H(2) from PYV liquid medium. The strain did not use carbohydrates or organic acids. The pH range for growth was narrow (pH 6.8-8.2), having a pH optimum at 6.8-7.5. The temperature range for growth was 10-37°C, the optimum being 25-30°C. The strain was sensitive to bile, and did not have catalase or oxidase activities. Hydrogen sulfide was produced from L-cysteine and L-methionine as well as peptone. Indole was produced from L-tryptophan and peptone. The strain had iso-C(15:0) as the exclusively predominant cellular fatty acid (70%) together with some branched chain components (such as iso-C(15:0) DMA, iso-C(17:0) 3-OH and iso-C(15:0) aldehyde) as minor components. The genomic DNA G+C content was 32.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN081(T) in the phylum Bacteroidetes with rather low sequence similarities with the related species such as Rikenella microfusus (85.7% sequence similarity), Alistipes putredinis (85.5%) and Alistipes finegoldii (85.5%) in the family Rikenellaceae. Based on the phylogenetic, physiological and chemotaxonomic analyses, the novel genus and species Anaerocella delicata gen. nov., sp. nov. is proposed to accommodate the strain. The type strain is WN081(T) (= JCM 17049(T) = DSM 23595(T)).

[Evaluation and choice of PCR-DGGE primers in analyzing the microbial community structure of activated sludge in traditional Chinese medicine wastewater].

In the study of traditional Chinese medicine wastewater, it was discussed for the effect of different sets of 16S rDNA universal primers on DGGE fingerprinting and microbial community diversity of aerobic and anaerobic activated sludge from one traditional Chinese medicine wastewater treatment. The genome DNA of activated sludge was isolated, and eleven sets of primers were used to amplify the four variable regions of 16S rDNA, the resolution of DGGE fingerprinting and community diversity was analyzed. The results indicated that community diversity with different sets of universal primers by DGGE was obviously different. Separated patterns of the V3 and V6-V8 regions were better than of V1-V3 and V3-V5. In the DGGE profiles, bands and diversity from V3 were most, bands and diversity from V3-V5 and V6-V8 were a little worse than those of V3. According to the length of targeted sequence and the resolution of DGGE fingerprinting, V6-V8 (B968F/B1401R) are recommended to be used to do the DGGE analysis. Mix I341F/I534R and B341F/B534R PCR product equally to make DGGE analysis can get more community diversity information.

Localization of rDNA at nucleolar structural components by immunoelectron microscopy.

Fluorescence in situ hybridization (FISH) shows that DNA encoding ribosomal RNA cistron (rDNA) is localized to small speckles scattered in the nucleolus and the nucleolus-associated chromatin (NAC). This technique cannot however precisely locate rDNA in the nucleolar ultrastructural components such as the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). In situ hybridization at the electron microscopic level is suitable for localization of rDNA at the ultrastructural level. We have tried to determine the precise localization of rDNA in the nucleolus of a higher plant by electron microscopic (EM) in situ hybridization using biotin-labeled 18S rDNA and demonstrated that it is exclusively localized in the fibrillar centers (FCs) and the nucleolus-associated chromatin (NAC). A secondary antibody coupled to the smallest (5 nm) colloidal gold particles was used in this technique to increase the label. Another important factor to increase the label was pretreatment with proteinase. Convincing results are obtained when the samples are pretreated with 1 microg/mL proteinase K for 45 min at 37 degrees C before immunogold labeling.