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Keywords:

  • clone library;
  • fungal biota;
  • International Space Station;
  • Malassezia

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

In addition to the crew, microbes also find their way aboard the International Space Station (ISS). Therefore, microbial monitoring is necessary for the health and safety of the crew and for general maintenance of the facilities of this station. Samples were collected from three sites in the Japanese experimental module KIBO on the ISS (air diffuser, handrail, and surfaces) for analysis of fungal biota approximately 1 year after this module had docked with the ISS. Samples taken from KIBO before launch and from our laboratory were used as controls. In the case of KIBO, both microbe detection sheet (MDS) and swab culture tests of orbital samples were negative. The MDS were also examined by field emission-scanning electron microscopy; no microbial structures were detected. However, fungal DNAs were detected by real-time PCR and analyzed by the clone library method; Alternaria sp. and Malassezia spp. were the dominant species before launch and in space, respectively. The dominant species found in specimens from the air conditioner diffuser, lab bench, door push panel, and facility surfaces on our laboratory (ground controls) were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. The fungi in the KIBO were probably derived from contamination due to humans, while those in our laboratory came from the environment (e.g., the soil). In conclusion, the cleanliness in KIBO was equivalent to that in a clean room environment on the ground.

List of Abbreviations: 
A. alternata

Alternaria alternata

CBEF

Cell Biology Experiment Facility

DDBJ

DNA Data Bank of Japan

ELM-ES

experiment logistics module, external section

ELM-PS

Japanese experiment logistics module, pressurized section

EMBL

European Molecular Biology Laboratory

FE-SEM

field emission-scanning electron microscope

HEPA

high efficiency particulate air

ISS

International Space Station

ITS1

internal transcribed spacer 1

JAXA

Japan Aerospace Exploration Agency

JEM

Japanese experimental module

KSC

John F. Kennedy Space Center

MDS

microbe detection sheet

NASA

National Aeronautics and Space Administration

OTU

operational taxonomic unit

PM

pressurized module

Several species of fungi have been found aboard the Russian orbital station Mir, among which microorganisms capable of colonization of the unique anthropotechnological niche of the environment of manmade objects in space were detected (1). The dominant species of fungi detected on Mir and the USA space station Skylab belonged to the genera Aspergillus, Penicillium, and Cladosporium (1, 2). On the ground, there have been some reports of cases with symptoms similar to the allergic disease sick building syndrome, which occurs in people living in places with heavy Aspergillus and Penicillium infestation (3). In a 56-day space stay simulation carried out by the USA NASA, the number and variety of fungal isolates obtained from the crew generally decreased, but the numbers of Candida albicans and Candida tropicalis increased markedly in the nasal and oral cavities (4). In the case of bacteria, following spaceflight the gene expression pattern of Salmonella typhimurium has been shown to change and the virulence of these bacteria to increase (5). Such variations may also occur in fungi. Therefore, it is necessary to monitor fungal biota onboard the ISS to prevent such health disorders and equipment problems. The JEM, known as the “KIBO” (pronounced key-bow), which means “hope” in Japanese, is the first Japanese human-rated space facility and the first contribution by JAXA to the ISS program (6). The new KIBO module is a suitable subject for investigating the fate of fungal biota in space. In the present study, we analyzed the fungal biota of KIBO before its launch in June 2008 and then again in September 2009, approximately 460 days after it was connected to the ISS. This paper is part of Microbe-I report, which is the first of three planned space microbiological experiments to investigate the fate of the fungal biota of KIBO.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Sampling

Samples were obtained using swabs (ITW Texwipe; ITW Texwipe, Kernersville, NC, USA) and MDS (Sani-ta kun for yeast/mold; Chisso, Tokyo, Japan). Swabs were moistened with 200 μL of sterilized water, vacuum-packed, and put in plastic bags before use. The surface of the CBEF (smooth powder-coated metal) of KIBO (7 × 7 cm, 49 cm2) was swabbed before launch at KSC (Orlando, FL, USA) on 11 June 2007. The Japanese ELM-PS and PM were launched on space shuttle missions Endeavour STS-123 (March 2008) and Discovery STS-124 (May–June 2008). The ELM-ES and final components were launched on space shuttle mission Endeavour STS-127 (July 2009). Sampling in orbit was performed by space shuttle mission Discovery STS-128 (August–September 2009). The samples were gathered from an air diffuser, handrail, and the surface of CBEF in the same way as before launch. Swabs were placed in activated charcoal agar (Seed swab γ-3; Eiken, Tokyo, Japan), transferred and stored at 2°C from ISS to the laboratory. MDS were cultured at room temperature (22°C) for 6 days (from flight day 03 to day 09) in the JEM PM, ISS, and transferred from ISS with storage at 2°C to our laboratory. In our laboratory the vertical surface of the facility (smooth powder-coated metal, untouched by human hands), door push plate (smooth, vertical resin routinely touched by people), laboratory bench (smooth horizontal surface), and air conditioner diffuser were sampled by the same methods to provide ground-based controls.

Culture

Swabs were streaked and MDS were stamped onto DG-18 (Oxoid, Cambridge, UK) and CHROMagar Malassezia/Candida (CHROMagar, Paris, France) (7) plates. The genus Malassezia consists of skin commensal yeasts that cause pityriasis versicolor, seborrheic dermatitis, atopic dermatitis, and Malassezia folliculitis (8). Therefore, Malassezia was the target of the culture. All samples were incubated at 28°C and 37°C.

Electron microscopy

After stereomicroscopic observation and stamping onto plates, MDS were fixed for electron microscopy as described previously (9) with some modifications as follows. All samples were subjected to critical point drying, sputter-coated with osmium, and observed using a FE-SEM (JSM-7500F; JEOL, Tokyo, Japan).

DNA extraction and clone library

All reagents were used according to the respective manufacturer's recommended protocols. Swabs were streaked onto plates, and vortexed for 5 min with 1 mL of 0.05% Tween 80. Aliquots of 30 μL of DNA solution were extracted from 200 μL of swab suspension with NucliSens magnetic extraction reagents (BioMèrieux, Marcy l’Etoile, France). Fungal ITS1 DNA was amplified and detected from swabs using a panfungal primer set and probe as described previously (10). Reactions were performed using Premix Ex Taq (Perfect Real Time, TaKaRa, Shiga, Japan). Real-time PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). DNA fragments were ligated into the pCR2.1 vector (Invitrogen, Foster City, CA, USA), and the plasmids introduced into competent Escherichia coli DH5 α cells (ECOS competent E. coli; Nippon Gene, Toyama, Japan). Clones were sequenced using the vector universal M13 reverse primer with a BigDye Terminator v3.1 cycle sequencing kit in an Applied Biosystems 3730xl DNA analyzer (Applied Biosystems). Sequencing was performed by the Dragon Genomics Center of TaKaRa Bio (Shiga, Japan). Rarefaction curves and Shannon diversity index estimates of OTU richness at over 97% similarity were calculated using the computer program EcoSim 7 (11).

Identification

The isolated strains and DNA clones were identified by comparison with DNA sequences registered in GenBank/EMBL/DDBJ as described previously (1).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Culture

Culture results are shown in Table 1. In the 2-month culture, all orbital samples and the surface of CBEF (before launch) were negative. Environmental fungi, such as Alternaria tenuissima (FJ755240), Aspergillus fumigatus (FJ358278), Aspergillus oryzae (HQ122941), Aureobasidium pullulans (GQ911487), Cladosporium cladosporioides (GU214409), Didymella bryoniae (EU167573), Eupenicillium crustaceum (AB479289), and Loratospora aestuarii (GU301838), were isolated from our laboratory as ground-based controls. GenBank/EMBL/DDBJ accession numbers with 100% similarity for pure isolates are shown in parentheses.

Table 1.  Isolated fungi of KIBO and ground controls (CFU/cm2)
MediaKIBOGround controls
Surface of CBEF (before the launch)Surface of CBEF (in orbit)Handrail (in orbit)Air diffuser (in orbit)Surface of facilitiesDoor push plateLab benchAir conditionerSwab (before use)
  1. †CHROMagar Malassezia/Candida (7). ‡Microbe detection sheet, Sani-ta kun for yeast/mold.

DG-18NegativeNegativeNegativeNegativeNegativeNegativeAlternaria tenuissima (0.02)Aspergillus fumigatus (0.04)Negative
       Cladosporium cladosporioides (0.14)Cladosporium cladosporioides (0.08) 
CHROMagar†NegativeNegativeNegativeNegativeNegativeNegativeAureobasidium pullulans (0.02)Aspergillus fumigatus (0.02)Negative
       Cladosporium cladosporioides (0.18)Cladosporium cladosporioides (0.02) 
       Didymella bryoniae (0.1)Eupenicillium crustaceum (0.06) 
MDS‡NegativeNegativeNegativeNegativeNegativeCladosporium cladosporioides (0.07) Loratospora aestuarii (0.13)Cladosporium cladosporioides (0.67)Aspergillus fumigatus (0.5)Negative
        Aspergillus oryzae (0.13) 
        Cladosporium cladosporioides (0.38) 

Electron microscopy

On FE-SEM observation, neither cells, fragments of microbes nor human dust, including hair or scurf, were observed on any of the MDS (data not shown).

Diversity and distribution of fungal internal transcribed spacer 1 sequences

Rarefaction curves (Fig. 1) were generated and the diversity indices at a 97% sequence similarity level were calculated for the available fungal ITS1 sequences in each clone library (Table 2). The rarefaction curves of each library tended to approach the saturation plateau. The sequences of the clones in each library were aligned and compared as shown in Table 3. Figure 2 shows the copy number and diversity of fungal DNA. The dominant species of CBEF before launching was Alternaria sp. However, as for all orbital samples, the dominant species were Malassezia spp. (mainly M. restricta). On the ground, Malassezia spp.was the dominant species on the door push plate. On the other hand, the dominant species in the ground-based controls from the air conditioner diffuser, lab bench, door push panel, and surface of facilities were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively.

image

Figure 1. Rarefaction curves (number of OTUs vs. number ineof clones) for clone libraries obtained from (a) KIBO and (b) ground-based controls with the operational taxonomic units defined by 97% sequence similarity. (a) ▴, surface of CBEF (before launch); ▴, surface of CBEF; ▪, handrail; ♦, air diffuser. (b) ▴, surface of facilities; ▪, door push plate; •, lab bench; ♦, diffuser of air conditioner; ○, swab (before use).

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Table 2.  Diversity indices of the fungal communities of KIBO and ground controls as represented in the ITS1 gene libraries
Diversity indexKIBOGround controls
Surface of CBEF (before the launch)Surface of CBEF (in orbit)Handrail (in orbit)Air diffuser (in orbit)Surface of facilitiesDoor push plateLab benchAir conditionerSwab (before use)
  1. †Confidence intervals for Shannon's indices are shown in parentheses.

Number of OTUs989491548223
Shannon's index†1.28 (1.13, 1.41)1.24 (1.04, 1.34)1.13 (1.01, 1.21)0.85 (0.69, 0.97)1.76 (1.67, 1.80)1.61 (1.51, 1.70)3.20 (3.05, 3.33)2.20 (2.08, 2.32)1.04 (1.01, 1.10)
Table 3.  Abundance of specific clones in the ITS1 gene libraries
 KIBOGround controls
Surface of CBEF (before the launch)Surface of CBEF (in orbit)Handrail (in orbit)Air diffuser (in orbit)Surface of facilitiesDoor push plateLab benchAir conditionerSwab (before use)
  1. †Surface of CBEF (before the launch): two copies of Plicaturopsis sp., Sporobolomyces sp., and Trichophyton sp., single copy of Limonomyces sp.; surface of CBEF (in orbit): single copy of Cystofilobasidium sp., and Periconia sp.; handrail: single copy of Sporidiobolus sp.; surface of facilities: two copies of Polyporus sp.; door push plate: single copy of Lophiostoma sp.; lab bench: two copies of Entoloma spp., Ganoderma sp., Hyphoderma spp., Microporus sp., and Tricholoma spp., single copy of Amanita sp., Amylostereum sp., Collybia sp., Cylindrobasidium sp., Gloiocephala sp., Heterobasidion sp., Hyphodontia sp., Hysterangium sp., Lentinellus sp., Lentinula sp., Lyophyllum sp., Oudemansiella sp., Panellus sp., Phialocephala sp., Physisporinus sp., Pleurotus sp., Ramaria sp., Resinicium sp., Stagonospora sp., Steccherinum sp., Strobilurus sp., and Thanatephorus sp.; air conditioner: two copies of Phellinus spp., single copy of Fuscoporia sp., Gymnopus sp., Phanerochaete sp., Phlebia sp., Phoma sp., Saccharata sp., and Talaromyces sp.. ‡uncultured fungus clone, <97% similarity.

Alternaria sp.42 (66.7)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)0 (0.0)0 (0.0)
Antrodiella sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)1 (1.6)0 (0.0)
Armillaria spp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (2.5)0 (0.0)0 (0.0)
Aspergillus sp.0 (0.0)0 (0.0)7 (9.2)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Bjerkandera sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (2.5)2 (3.1)0 (0.0)
Botryobasidium sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)0 (0.0)0 (0.0)
Candida sp.0 (0.0)0 (0.0)3 (3.9)0 (0.0)0 (0.0)3 (2.5)0 (0.0)0 (0.0)0 (0.0)
Cladosporium spp.5 (7.9)5 (13.5)2 (2.6)6 (13.3)0 (0.0)6 (5.0)35 (29.7)1 (1.6)0 (0.0)
Cryptococcus sp.5 (7.9)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Dothideomycete sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (2.5)0 (0.0)0 (0.0)
Epicoccum sp.2 (3.2)1 (2.7)1 (1.3)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Eurotium spp.0 (0.0)4 (10.8)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (1.6)0 (0.0)
Flavodon sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)7 (5.8)0 (0.0)0 (0.0)0 (0.0)
Hymenochaetaceae sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)1 (1.6)0 (0.0)
Hyphodontia spp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)5 (4.2)2 (3.1)0 (0.0)
Inonotus sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (0.8)30 (46.9)0 (0.0)
Junghuhnia sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)2 (1.7)3 (4.7)0 (0.0)
Leptospora sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)8 (10.4)0 (0.0)1 (0.8)0 (0.0)0 (0.0)
Malassezia spp.0 (0.0)24 (64.9)60 (78.9)36 (80.0)1 (1.3)93 (77.5)1 (0.8)0 (0.0)2 (28.6)
Penicillium spp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)17 (22.1)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Peniophora sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (2.5)0 (0.0)0 (0.0)
Pezicula sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)24 (31.2)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Pleosporales sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (2.5)0 (0.0)0 (0.0)
Rhodotorula spp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)6 (5.0)0 (0.0)0 (0.0)0 (0.0)
Skeletocutis sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)14 (18.2)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Toxicocladosporium sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)2 (1.7)1 (1.6)0 (0.0)
Trametes spp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)8 (6.8)6 (9.4)0 (0.0)
Trichaptum sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)2 (1.7)4 (6.3)0 (0.0)
Trichosporon sp.0 (0.0)0 (0.0)0 (0.0)3 (6.7)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)
Wallemia sp.0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)3 (42.9)
Others †7 (11.1)2 (5.4)1 (1.3)0 (0.0)2 (2.6)1 (0.8)32 (27.1)9 (14.1)0 (0.0)
Unknown ‡2 (3.2)1 (2.7)2 (2.6)0 (0.0)11 (14.3)3 (2.5)10 (8.5)3 (4.7)2 (28.6)
Total63 (100.0)37 (100.0)76 (100.0)45 (100.0)77 (100.0)120 (100.0)118 (100.0)64 (100.0)7 (100.0)
image

Figure 2. Number of fungal DNA and diversity of ITS1 gene sequences in clone libraries obtained from (a) KIBO and (b) ground-based controls. Values are means of triplicate real-time PCR. † and ‡ are same as in Table 3.

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Nucleotide sequence accession numbers

The internal transcribed spacer 1 partial sequences obtained in this study have been deposited in GenBank/EMBL/DDBJ with the accession numbers AB632592 to AB633198.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The ISS is a closed manned system exposed to microgravity and space radiation. It has HEPA filters to remove contaminants from the cabin atmosphere (12), and is cleaned weekly with a vacuum cleaner and 0.4% benzalkonium chloride antiseptic towelettes (BZK Antiseptic Towelette; PDI, New York, NY, USA). Cultures and FE-SEM observations were negative in all orbital samples. Therefore, the HEPA seem to function normally on the ISS. There was a significant difference in quantity of DNA obtained from the lab bench (horizontal surface) and the surface of the facility (vertical surface) of our ground-based laboratory. It was thought that the fungal biota were different on the two similar smooth surfaces because dust falls vertically, but can lie on horizontal planes, in the earth's gravity field. On the other hand, the fact that particles such as mold spores drift continuously under the conditions of microgravity on the ISS could explain why there were no significant differences between points of collection in the ISS biota. Alternaria sp. was the dominant species on the surface of the CBEF before launch. A. alternata is an environmental fungus that is especially common in the soil (13), and for which the temperature conditions at KSC in Florida are particularly suitable. Therefore, soil fungal DNA was dominant before launch.

With the exception of the door push plate, environmental fungi were also the dominant species in our laboratory. These observations suggest that environmental soil microbes influenced indoor fungal biota on the ground. On the other hand, Malassezia spp. was the dominant species in orbital samples and on the door push plate. Dust did not attach to the door push plate or the facility surfaces as both were smooth vertical planes. Malassezia restricta is lipophilic and colonizes the human skin (14). Whereas our colleagues routinely touch the door push plate, the facility surfaces are not exposed to such contact; this is thought to explain the variation in fungal biota between these two surfaces despite their similar smooth and vertical characteristics. The environmental fungi of the ISS are derived from astronauts or other carriers, such as the space shuttle; soil is obviously not a significant source of fungal contamination. Although the ISS has a capacity of six people, it held only three astronauts during the study period. Therefore, fungal activity would have been less than usual because of limitations in growth conditions, including humidity and nutrients such as scurf. Thus, Malassezia spp. derived from human occupants of the ISS would be expected to be the dominant species in this artificial space environment. Because they are human resident yeasts and would have been either dead cells or cell fragments, some of the Malassezia spp. detected in each sampling location were not valid. These results suggest that the fungal biota of orbital samples would be similar to those of the door push plate on the ground. Cultures have indicated that the dominant fungal species belong to the genera Aspergillus, Penicillium, and Cladosporium on both space station Mir and Skylab (1, 2). The results of the present study indicate that, after Malassezia, Cladosporium was the next most dominant genus in orbit. However, Cladosporium species were not detected by the culture method we used because of their low levels of activity. Although our lab bench is routinely exposed to contact with human skin, Malassezia sp. was a minority in these samples. Environmental fungi were active on the lab bench, and would digest the obligate Malassezia spp.; this species may therefore be useful as an index of the mycological hygiene of human living spaces.

In conclusion, the degree of cleanliness at KIBO, which had been connected to the ISS for about 460 days at the time of this study (June 2008–September 2009), was equivalent to that in a clean room environment on the ground. Fungi have already been detected from the HEPA filters of the US module by culture and mold-specific quantitative PCR methods (15). Because the ISS now houses six crew members, it will become a suitable environment for fungal growth. Thus, although no fungi were cultured in this Microbe-I mission from KIBO, fungal biota may be isolated in future. The further extension of our mission, Microbe-II & III, will be conducted in future and will clarify the state of the KIBO fungal biota.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

We thank Ms. Yoshiko Umeda, Ms. Yayoi Hasumi. and Ms. Mari Maeda for their technical assistance. This study was supported in part by a grant from JAXA and a grant for KIBO 2nd Stage Research Program for Space Utilization from the Japan Space Forum (K.M.).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors have no financial support or relationships that may result in conflict of interest with any company whose product figures prominently in the submitted manuscript or with any company making a competing product.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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