Glaciozyma-related taxon and Phenoliferia psychrophenolica were the dominant species. Unexpectedly, amplicon sequencing uncovered sequences of Chytridiomycota in all samples and Rozellomycota in sea water, lake water, and tap water. Sequences of Malassezia restricta and of the extremely halotolerant Hortaea werneckii were also found in subglacial habitats for the first time. Overall, the fungal communities within a glacier and among glaciers were diverse and spatially heterogenous. Contrary to this, there was a large overlap between the bacterial communities of different glaciers, with Flavobacterium sp. being the most frequently isolated. In amplicon sequencing Actinobacteria and Proteobacteria sequences were the most abundant.The composition of fungal and bacterial communities in three polythermal glaciers and associated aquatic environments in Kongsfjorden, Svalbard was analysed using a combination of cultivation and amplicon sequencing. 109 fungal strains belonging to 30 mostly basidiomycetous species were isolated from glacial samples with counts up to 10 Analyses of more than one of these groups from the same samples and particularly the investigation of fungal diversity with molecular methods have not been published so far.The archipelago of Svalbard is located in the Arctic Ocean between Norway and the North Pole. It is one of the best studied locations in the Arctics and thus considered a “hot spot” of Arctic microbiological research. So far, studies of microbial glacial communities have focused on either Bacteria11 are defined by the presence of water at the subglacial drainage system at their basis12. They are entirely warm-based, except at the margins, meaning that liquid water present at the glacier bed is generated by surface flow, friction, pressure melting, and groundwater infiltration13. This base consists of a relatively thin layer of debris-rich ice called subglacial ice or basal ice formed by subsequent freezing and melting of subglacial waters and entrainment of subglacial sediment and debris15. For a long time these environments were considered abiotic, but later research has shown that they represent an important share of global microbiological activity that has received some attention only in the last two decades19. A few years after the first systematic investigations of subglacial bacteria, fungi were also found to be able to survive in these extreme environments, exemplified by large populations of Penicillium spp.6, basidiomycetous4 and ascomycetous yeasts20.Glaciers with a polythermal basal regime24. So far all studies of the microbial communities of the subglacial environments of these glaciers were based on cultivation. Here we focused on fungal and bacterial communities found in the subglacial ice of these glaciers and in the associated aquatic environments such as clear ice floating in sea water, glacial meltwater, moraine lake water, sea water and lake-derived tap water. The microbial communities were investigated using traditional culturing and, for the first time in case of subglacial ice fungi, high-throughput amplicon sequencing methods. Our findings show a considerable diversity (particularly for fungi) amongst local microbial communities and include the presence of a number species not expected in the Arctic environment.This report focuses on three polythermal Svalbard glaciers located on the southern side of Kongsfjorden Fig.  near Ny-Fungal and bacterial diversity of subglacial ice of three polythermal glaciers and their surrounding aquatic environments were analysed by a combination of cultivation and molecular methods.. Overall the lowest counts were obtained on the two low aw media, on MY10–12 only up to 88 CFU/100 ml, while on DG18 they ranged from 4.8 CFU/100 ml up to 500 CFU/100 ml. On the enumeration medium DRBC the highest counts were somewhat higher, from 0 to 540 CFU/100 ml. Highest counts were obtained on two low-nutrient media, where up to 1900 CFU/100 ml (MM) and up to 2400 CFU/100 ml (SNA) were detected.In cultivation experiments using several different media on which the filtered samples were inoculated, fungal counts differed considerably depending on the media used Overall, subglacial ice and glacial meltwater counts were comparable between samples. Glacier Pedersenbreen had both the highest (in sample S-ice B2) and the lowest (in the sample S-ice B1) fungal abundance. S-ice B2 counts were up to 2400 CFU/100 ml, while counts for S-ice B1 did not exceed 37 CFU/100 ml. Vestre Brøggerbreen samples (S-ice C1 and S-ice C2) also differed considerably in fungal abundance. While in S-ice C1 they ranged from 45 to 990 CFU/100 ml (MM), in S-ice C2 they were in the range from 5 to 200 CFU/100 ml. Values obtained for Midtre Lovénbreen  were similar between the samples, and ranged from 0 to 120 and 72 CFU/100 ml, respectively.w media, but on oligotrophic media the number of fungi reached up to 530 CFU/100 ml.No CFU were observed for some glacial meltwater (G-wtr A) samples on the low aIn total 3,938,443 paired-end reads were obtained from 12 samples. The reads corresponded to 740 different operational taxonomic units (OTUs). The number of reads per sample ranged from 174,770 (L-wtr) to 503,852 (S-ice-C1). Shannon indices ranged from H′ = 1.25 (S-ice-C1) to H′ = 3.63 (S-ice-B2), revealing high differences in diversity between the samples. Rarefaction curves suggested that Illumina sequencing depth was sufficient to capture the dominant phylotypes in all samples . To test for differences in the fungal taxa as determined by QIIME2 between sample types PERMANOVA multivariate diversity analyses were performed. Significant overall differences were found between the three major types of samples . However, the pairwise differences between the sample types as analysed by PERMANOVA were not statistically significant, possibly due to the small numbers of samples of each sample type.Although subglacial ice samples differed substantially in their diversity both between and within the glaciers, they were nevertheless all dominated by Basidiomycota (from 61% in S-ice-A1 to 96% in S-ice-C1), with Microbotryomycetes and Agaricomycetes being the most abundant classes of fungi Fig. . Also, aVestre Brøggerbreen glacier (S-ice-C1) was totally dominated by Agaricomycetes (95%), primarily the family Schizoporacea (90% sequences), while Pedersenbreen glacier S-ice-B2 was dominated by Microbotryomycetes, with a relative frequency of 75%. A large part of these sequences were placed in the order Leucosporidiales (53%), present in lower abundance also in S-ice-B1, S-ice-A2, S-ice-C1. The order Pleosporales was present in S-ice-B2 (16%), S-ice-B1 (9%), and S-ice-A2 (2%).Hortaea werneckii , and the giant polypore Meripilus giganteus   and a large proportion of unassigned fungi (28%).Both Midtre Lovénbreen glacier samples were substantially different the other two glaciers. S-ice-A1 surprisingly contained an abundance of sequences identified as the ascomycetous black yeast Schizopora flavipora (81%) belonging to the class Agaricomycetes, unidentified Chytridiomycota (7%) and unidentified species (5%).Clear ice samples were again very different from one another: C-ice-1 was dominated by Ascomycota (74%), the class Dothideomycetes (70%), primarily represented by the black yeast of the orders Dothideales (21%) and Capnodiales (15%). On the other hand C-ice-2 was characterised by the species Moraine lake water, glacial meltwater, harbour sea water, and indoor tap water were largely characterised by unidentified Chytridiomycota  Fig. . Phylum Hortaea werneckii and Malassezia restricta were among the species present in all the subglacial samples.Core microbiome analysis showed that 28 OTUs were shared between all subglacial ice samples, of which 13 belonged to Basidiomycota, 7 to Chytridiomycota, 5 to Ascomycota, and 3 to unassigned Fungi. The principal component analyses (PCoA) of ordination patterns revealed four separate sample clusters based on phylogenetic distance metrics (weighted uniFrac distance) Fig. . The thrGlaciozyma-related taxon, Mrakia sp., Glaciozyma watsonii, and Rhodotorula svalbardensis pro. tem. and more sporadically an unidentified basidiomycete. Amongst filamentous fungi Pseudogymnoascus sp., and Penicillium bialowiezense prevailed. Less frequent were Botrytis cinerea, Cladosporium sp., Geomyces sp., unidentified Helotiales sp. 2, Leptosphaeria sp., Mortierella sp., Penicillium swiecickii, Phialocephala sp., Pseudeurotium sp., P. hygrophilum, Tetracladium sp. and Thelebolus sp. The other Vestre Brøggerbreen sample, S-ice C2, displayed in comparison a much lower diversity. Isolates were identified as Glaciozyma-related taxon, Pseudogymnoascus sp., unidentified Helotiales, sp. 1 and unidentified Ascomycota sp. 4.From subglacial ice and glacial meltwater, 113 fungal isolates were obtained, belonging to 19 genera and 30 different species of filamentous fungi and 10 genera and 11 species of yeasts. Vestre Brøggerbreen S-ice C1 sample harboured the most diverse fungal community that contributed the most to the overall dissimilarity, as confirmed by SIMPER analysis . The most frequently isolated yeasts were basidiomycetous Glaciozyma-related taxon isolates prevailed, followed by Phenoliferia psychrophenolica, Rhodotorula svalbardensis pro. tem., and Basidiomycota sp. 225. Filamentous fungi were represented by unidentified Helotiales sp. 1, Lecanicillium sp., unidentified Ascomycota sp. 2, and unidentified Ascomycota sp. 3. S-ice A2 was again dominated by yeasts: Filobasidium magnum, Naganishia albida, and Rhodotorula svalbardensis pro. tem., Phenoliferia psychrophenolica, unidentified Ascomycota sp. 1, filamentous Cladosporium pseudochalastosporoides and unidentified Venturiaceae.Midtre Lovénbreen glacier (S-ice A1 and S-ice A2) samples were dominated by yeasts. In S-ice A1 Penicillium crustosum as the dominant species and occasional isolates of Venturia sp. The most frequently isolated yeast was Glaciozyma-related taxon, followed by Filobasidium magnum, Phenoliferia psychrophenolica, Ph. glacialis and Sporidiobolus sp. S-ice B2 had the highest yeast abundance (500 CFU/100 ml)  and was characterized in particular by Glaciozyma-related taxon, Glaciozyma watsonii, Mrakia sp. and Phenoliferia psychrophenolica. More sporadic yeasts/fungi were Leucosporidiella muscorum, Vishniacozyma victoriae, unidentified Ascomycota sp. 1, unidentified Helotiales sp. 3, and Paraleptosphaeria dryadis.The two subglacial ice samples (S-ice B1 and S-ice B2) of Pedersenbreen glacier were again strongly dissimilar. In S-ice B1 penicillia (12 CFU/100 ml) prevailed, with Helotiales sp. 2, sporadic basidiomycetous yeasts Glaciozyma-related taxon, Glaciozyma watsonii, Mrakia sp. and Phenoliferia psychrophenolica and filamentous fungi Collybia cirrhata sp., and Venturia sp.Glacial meltwater (G-wtr A) enviro Filamentous fungi were represented by unidentified nment harboured primarily unidentified Penicillium crustosum, unidentified Basidiomycota closely related to Peniophora sp., Cladosporium sp. and Cladosporium pseudochalastosporoides, Helotiales sp. 3, Lecanicillium sp., R. svalbardensis pro. tem., and Thelebolus sp.Analysis of the shared diversity (comparison between ITS2 sequences extracted from culture-sourced ITS sequences and from the amplicon sequencing) between the cultivation and the amplicon sequencing dataset showed that out of 30 species found by cultivation, 8 were also found by amplicon sequencing (27% of the diversity found with both approaches) . These species were Counts of bacteria on R2A medium were calculated . Overall, subglacial ice and glacial meltwater samples had similar bacterial counts, with values spanning from 300 CFU/100 ml (S-ice B1) up to 3300 CFU/100 ml (S-ice B2).After subtraction of chloroplast sequences, the dataset was composed of 1,793,737 assembled sequences , corresponding to 712 different features OTUs. Number of reads per sample ranged from 280,884 (C-ice-2) to 62,627 (Sea-H). Shannon indices for bacteria varied over a broad range from H′ = 2.5 (C-ice-1) to H′ = 5.5 (Sea-F) indicating large differences in the diversity of individual samples. Samples with lower bacterial diversity indices were both samples of clear ice , as well as four of the six subglacial ice samples . Nevertheless, significant differences were found between all three types of samples . However, no statistically significant differences were found in the pairwise PERMANOVA comparisons of sample types. Rarefaction curves suggested that Illumina sequencing depth was sufficient to capture the dominant phylotypes in all samples .The common bacterial phyla across all 12 samples were Actinobacteria and Proteobacteria, with wide ranges of relative abundances   was present at up to 58%, while Proteobacteria reached only 1% . Other sequences belong to Acidobacteria (37%) and Chloroflexi (3%). In S-ice-A2 Proteobacteria  were the most abundant, followed by Actinobacteria (7%), Planctomycetes  and Firmicutes (2%). The phylum Planctomycetes was additionally identified in lake water (3%) and tap water (2%).Midtre Lovénbreen subglacial ice samples were characterised by the above-mentioned phyla, but in different proportions. In S-ice-A1 Actinobacteria , Proteobacteria , Chloroflexi . Additionally S-ice-B1 contained Acidobacteria (6%) and Gemmatimonadetes (4%).Microccocus, 57%), followed by Proteobacteria  and Cyanobacteria (17%).The only sample from Vestre Broggerbreen glacier, S-ice-C1, was dominated by Actinobacteria (genus Micrococcus) with frequencies higher than 99%. In glacial meltwater Actinobacteria sequences prevailed with a relative abundance of 67%, but it contained additional characteristic taxa, such as OD1 (15%), TM7 (9%) and OP11 (2%). Lake water also contained phylum OD1 (15%), together with Bacteroidetes (9%), Proteobacteria (9%) and Actinobacteria (58%). Sea water samples, Sea-F and Sea-H, were dominated by Proteobacteria . Tap water was dominated by the phylum Proteobacteria (49%), followed by Actinobacteria (12%), Bacteroidetes (11%), Cyanobacteria (8%), Acidobacteria (10%) and Chloroflexi (3%).Clear ice samples were entirely dominated by Actinobacteria  of ordination patterns revealed five separate sample clusters based on phylogenetic distance metrics (weighted uniFrac distance) Fig. . The thrFlavobacterium sp., while S-ice A2 showed the highest bacterial subglacial diversity. Although Flavobacterium sp. again prevailed, isolates belonging to Cryobacterium sp., Hymenobacter sp., Massilia sp., Polaromonas sp., Pseudomonas graminis, Spirosoma sp., and an uncultured species belonging to Oxalobacteraceae family, were also retrieved.52 bacterial isolates were obtained from subglacial ice and glacial meltwater samples. In Midtre Lovénbreen all isolates from S-ice A1 belong to Flavobacterium sp., while S-ice B1 additionally harboured Cryobacterium sp., Massilia sp., Paenibacillus antarcticus and an uncultured species belonging to Oxalobacteraceae family, closely related to Actimicrobium antarcticum.In Pedersenbreen glacier a similar pattern was observed. Sample S-ice B2 was monopolized by Cryobacterium sp., Massilia sp., and Pseudomonas sp., while S-ice C2 contained as Flavobacterium sp., Herminiimonas sp., and unidentified Oxalobacteraceae clustering with S-ice A2 isolate.Vestre Brøggerbreen S-ice C1 isolates were identified as Pseudomonas sp. (including P. frederiksbergensis and P. graminis), followed by Flavobacterium sp., and Massilia sp. Among sporadically isolated species were Polaromonas sp., Sphingomonas sp., unidentified Burkholderiaceae, and Sphingorhabdus sp.Glacial meltwater had the most diverse cultivable bacterial community. The most frequently isolated species were 27, while others remain metabolically active and possibly multiply in the veins of brine between ice crystals28. Microbial communities also develop in deep ice or subglacial ice at the base of polythermal glaciers, where melting occurs due to gravitational pressure resulting in thin layers of liquid water, often with high concentrations of salts and enriched with the inorganic material of the glacier bed. These glacial populations likely represent an important share of global biological activity19 that has so far been poorly characterized. A deeper understanding of the microbial biodiversity of this unique and rapidly disappearing ecosystem would contribute to our understanding of the composition and dynamics of subglacial microbial communities and their roles in the Arctic ecosystems. Therefore, fungal and bacterial diversity of oligotrophic subglacial and glacial ice and meltwater and nearby marine water, moraine lake water and tap water were investigated both by cultivation and with molecular methods, the latter being applied on samples of subglacial ice for the first time.Glacier ice is one of the most challenging natural environments for life. Nevertheless, glacial habitats harbour a wide diversity of microorganisms, both prokaryotes and eukaryotes. Some microbes deposited on the glacial surface gradually travel to deeper ice layers. They can survive in ice as viable, frozen “living fossils”4 reported even higher yeast counts in Svalbard glacial ice (up to 4000 CFU/ml), while Turchetti et al.29 reported similar counts in Alpine subglacial sediments (100 to 1000 CFU/g). Both studies reported several orders of magnitude lower numbers of yeast cells in supraglacial ice and sediments indicating an enrichment of yeast in subglacial environments. Such enrichment could be supported by the presence of organic carbon in subglacial sediments17, deriving from permafrost soils that are overridden by the advancing glaciers and finely ground by subglacial abrasion processes. This organic material consists of cyanobacterial mats, plant material, and roots, which are readily biodegradable by microbial activity17.Fungi were abundant in all samples as shown by both cultivation and amplicon sequencing. Frequencies in the three glaciers differed considerably. Midtre Lovénbreen harboured the lowest number of cultivable fungi (up to 100 CFU/100 ml), followed by Vestre Brøggerbreen (up to 990 CFU/100 ml) and Pedersenbreen (1000 CFU/100 ml) including yeasts at 500 CFU/100 ml) . The large proportion of yeast in Pedersenbreen was confirmed by amplicon sequencing, where the highest number of sequences belonged to Microbotryomycetes (75%). Sixty-eight % of the cultured yeast species belonged to the Microbotryomycetes and the remaining 32% to the class Tremellomycetes . A previous cultivation studyet al.30. In contrast, meltwater flowing from under the glacier was very similar in fungal composition to the nearby subglacial ice sample . Differ31, Antarctic soils33, Patagonian glacier ice, meltwater and sediments35. In Arctic marine environments they are even the dominant fungi41. This is to the best of our knowledge the first report of Chytridiomycota in Arctic glacial ice and tap water. Chytridiomycota in household tap water probably originate from the water source itself, which in case of Ny-Ålesund water supply system is a surface lake filled by the snow meltwater42, and are possibly enriched during transportation to the final user. Tap water and to a lesser extent moraine lake water (from a different lake than the one providing the tap water) were also populated by unidentified Rozellomycota, an early diverging hyper-diverse lineage of fungi also known as Cryptomycota43 that have been documented almost exclusively by NGS analyses of environmental samples44. These uncharacterized fungi dominate fungal communities in different aquatic ecosystems, such as temperate freshwater lakes48, coastal and marine environments51, snow31 and polar aquatic systems53. Their abundant presence in glacial and related samples is in agreement with these findings, since the nature of samples analysed in this study is also aquatic, and/or is possibly linked to the ability of Rozellomycota to mycoparasitise chytrids55.Analysis of the ITS2 amplicon discovered sequences assigned to the phylum Chytridiomycota in all samples, with the highest abundance in tap water, followed by harbour sea water, lake water, and glacial meltwater. In the past isolations of chytrids from extreme habitats were rare. However, recent metagenomic studies revealed their widespread occurrence in a broad range of extreme habitats such as snow in Europe and AmericaLibkindia masarykiana was abundantly present in the sample S-ice-B2, but was present in very low numbers in the other samples. C-ice-1 was characterized by Dothideomycetes, while C-ice-2 was dominated by Schizopora flavipora otherwise known as a tea plant pathogen. Also, fungi in the sea water sampled in the harbour were different from the seawater fungi sampled in the middle of fjord .Hortaea werneckii (38%) in Lovenbreen glacier (S-ice-A1), never reported before in any glacial environment. The fungus was present also in all the other subglacial samples, but in lower frequencies. This extremely halotolerant black yeast is the dominant fungus in hypersaline waters of salterns worldwide56, and is additionally found in other marine related environments 59 and deep sea62. Furthermore, H. werneckii is known as the etiological agent of human tinea nigra, a superficial infection of salty soles and palms in humans, occurring in tropical and subtropical areas64.Among the most unexpected results of our study was the presence (as shown by NGS) of the black yeast Malassezia restricta (39%), the most common human skin-related fungus after M. globosa67. Initially Malassezia spp. were thought to be specifically associated with mammalian hosts, but culture-independent studies revealed their presence in very diverse marine and terrestrial ecosystems, such as cone snails68, Antarctic soils70, deep-sea sediments71, deep-sea hydrothermal vents72, nematodes74, corals75, and sponges76. Amend72 found that ribosomal DNA sequences of Malassezia in the above-cited studies are nearly identical to sequences of human-associated isolates, suggesting that they either diverged in their habitats very recently or that they are capable of large adaptability and high stress tolerance enabling them to colonize environments as different as human skin and subglacial ice. The fact that this species was not detected by cultivation may be due to its slow and fastidious growth and lipophilic nature, usually not taken into consideration in standard growth media77.The second sample of Lovenbreen glacier (S-ice-A2) was characterized by the presence of Phenoliferia psychrophenolica (former Rhodotorula psychrophenolica) and were similar to Glaciozyma antarctica, both frequently recovered from persistently cold habitats79. Glaciozyma-related taxon isolates differed from Glaciozyma antarctica CBS 5842 by 27 nucleotides in the LSU marker and additionally contained a deletion of 5 nucleotides towards the end of the sequence, suggesting the possibility of a new taxon. Glaciozyma species are obligate psychrophiles, with a maximum growth temperature below 20 °C78 and found in cold habitats such as Alpine and Apennine glaciers79, Antarctic sea water and soil81, and Greenland glacial ice81. Glaciozyma antarctica, formerly Leucosporidium antarcticum78, has been widely studied due to its biotechnologically important production of antifreeze proteins and cold-active enzymes84. The genome of Glaciozyma antarctica was sequenced and analysed only recently85, revealing systems of psychrophilic response that are present only in this species and might be associated with temperature variations. The higher expression of such unique genes associated with cold adaptation unique to G. antarctica, together with other physiological strategies such as modelling its membrane lipid composition86, could explain the high abundance of the genus in cold environments and its superior ability, compared to other species, to overcome stressful conditions associated with extreme habitats. Genus Phenoliferia has been proposed by Wang et al.79 to accommodate a clade of species segregated from the genus Rhodotorula, characterised by the ability to assimilate phenol as the sole carbon source at 10 °C87. In particular P. psychrophenolica and P. glacialis can degrade up to 12.5 and 5 mM concentrations of phenol, respectively86. P. psychrophenolica has so far only been isolated from Alpine and Apennine glaciers86 and from the Gulkana Glacier in Alaska25 and the glacier Austre Broggerbreen on Svalbard88.Cultivation of various samples confirmed some of the similarities between sample sites as observed by NGS: the most frequently isolated yeast species belonged to the basidiomycetous Rhodotorula, Mrakia, Filobasidium and Naganishia. Rhodotorula is a ubiquitous genus commonly isolated from cold habitats29. Rhodotorula spp., belonging to the same class as Glaciozyma spp. (Microbotryomycetes), are also known to be part of the core yeast communities in cold environments89, with some species recovered exclusively from cold habitats and for this reason considered endemic and other species ubiquitous90. The ability to grow over a wide temperature range, its membrane fluidity stability, marked adaptability, nutritional versatility and polyextremotolerance92, together with the abundant production of photo-protective pigments93, make Rhodotorula spp. adept in withstanding extreme climates. R. svalbardensis is a novel psychrophilic species recovered from Svalbard glacier cryoconite holes7 and Greenland glacial ice82. We refer to the species as R. svalbardensis pro. tem.  as suggested by Turchetti et al.94 due to its uncertain phylogenetic placement. Psychrophilic genus Mrakia has so far been restricted to the coldest areas of the world, such as Greenland, Antarctica, Siberia, Patagonia and Alaska96. The Principal Component Analysis based on cultivation data  also share ecological traits: they are psychrophilic yeasts unable to grow at temperatures higher than 17 °C, with distribution consequently restricted to habitats with persistently low temperatures98. Further explanation of the co-occurrence of Mrakia and Glaciozyma-related taxon will be possible once the metabolic and other characteristics of the latter have been characterised further. Filobasidium species are widely spread, from very arid environments99 to Italian glacial meltwaters100 and basal ice of the High Arctic4. Species of the genus Naganishia are among the most resistant organisms to UV radiation101: they are found in many extreme environments such as high elevation soil of the Atacama Desert102, of the Antarctic Dry Valleys81, and of subglacial ice in Svalbard4.Additionally, many different yeasts species were isolated from our samples. These belonged mainly to genera ata Fig.  revealedPenicillium crustosum, P. bialowiezense, and Pseudogymnoascus sp. prevailed. The frequent presence of Penicillium spp. in cold environments has been documented in various habitats, from soil to basal ice in the Arctic and Antarctic104. Sampling of subglacial ice of Pedersenbreen in 20016 showed a high abundance of P. crustosum and this was confirmed in our study by cultivation and NGS from sample S-ice B1 originating from the same glacier. Also, amplicon sequencing showed a higher presence of Penicillium (1.3% of sequences) in this subsample compared to all other samples and glaciers (<0.2%). On the other hand, Pseudogymnoascus sp. was isolated exclusively from one glacier (Broggerbreen)  Fig. , while nLecanicillium, Thelebolus, Tetracladium, Leptosphaeria, and Mortierella, all of which have previously been isolated from Antarctic soil samples105. Mortierella sp. was described as a decomposer of granite after glacier retreat88. Isolates in our study from S-ice B1 and G-wtr A closely related to Venturia (GenBank accession no. AB916509) were also isolated from feathers of a Barnacle goose on Svalbard106. Isolates of Thelebolus sp. (S-ice C1) closely related to Thelebolus strain with the GenBank accession no. AB916508 were also isolated from bird feathers106. Genus Thelebolus has been described initially as an aquatic taxon from lake microbial mats in Eastern Antarctica107. Now it is recognized as one of the most common genera in polar soil crusts adjacent to glaciers108.Other sporadically isolated filamentous fungi belonged to the genera 82.The overlap of the cultivation and amplicon sequencing results was modest, with only 8 out of 30 species found by cultivation also present in the amplicon sequencing results, again showing that each of the two approaches has its strengths and that cultivation and non-cultivation methods complement each other, as noted before by several authors investigating other environments112. Subglacial habitats are characterized by higher cells numbers compared to englacial habitats and can reach abundances up 105 cells/ml113. Canadian subglacial meltwater samples yielded more than 103 CFU/ml17 or 100-fold more than our samples (up to 103 CFU/100 ml).The presence of viable bacteria in subglacial ecosystems  has been intensively studied for several years115 culture independent results identified Proteobacteria and Actinobacteria as the most abundant phyla in almost all samples. On the one hand the composition of the bacterial populations varied between samples from the same glacier, but were on the other hand similar between some sample pairs collected from different glaciers. For example, subglacial ice samples S-ice-A1 (Lovenbreen) and S-ice-B2 (Pedersenbreen) were both dominated by Sulfuricurvum sp., a novel genus isolated from an underground crude-oil storage cavit116. Sulfuricurvum is a facultatively anaerobic, chemolithoautotrophic bacterium previously isolated from subglacial lakes in Iceland118. It uses elemental sulphur, sulphide and thiosulfate as electron donors during microaerobic (1% O2) and anaerobic respiration118. Sulphur is common in subglacial bedrocks, favoring microbial sulphide oxidation, and sulphate reduction19. Thus Sulfuricurvum spp. might play a role in the biogeochemical cycling of sulphur in the subglacial ice, which is in direct contact with the underlying bedrock.In line with previous studiesMicrococcus (Actinobacteria) being the most abundant species in all samples. Micrococcus has been previously found in ice, for example in 20.000 years old Bolivian core ice samples119 and Malan Glacier ice cores120.While the fungal diversity of different glacial samples differed considerably even between samples from the same glacier, the differences in bacterial diversity was much smaller, with Flavobacterium sp. (Bacteroidetes), which represented less than 4% of sequenced amplicons. Members of this genus are commonly isolated from aquatic ecosystems121. Flavobacterium species are considered psychrotolerant, since they have been isolated from several polar habitats including Antarctic soil, lakes, marine sediment and sea ice125, high Artic snow cover1, and frozen soil in China126. The genus contains the causative agent of bacterial cold water disease, F. psychrophilum127. Among other isolated bacterial species were Massilia sp. and Polaromonas sp. . Massilia members were originally isolated from clinical samples128 and characterized as aerobic and facultatively psychrophilic129. Later they were found in various habitats such as freshwater130, aerosols, air, and house dust133, soil135, and phyllosphere137 and also from glacier ice cores in China from which two novel species, M. eurypsychrophila and M. glaciei, were recently described139. Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria) have also been recognised as significant components of glacial microbiomes before140.Cultivation dependent results for bacteria overlapped to some extent with the results of the amplicon sequencing: 17 species of bacteria were isolated, 12 of them belonging to the phylum Proteobacteria. However, the most commonly isolated species was Subglacial ice of polythermal glaciers harbours a rich and spatially heterogenous fungal and bacterial diversity.Glaciozyma and Rhodotorula.Several as yet undescribed species were isolated, including strains related to the genera Hortaea werneckii and the skin pathogen Malassezia restricta.The first amplicon sequencing of the subglacial ice mycobiota uncovered species previously not reported from the Arctic ecosystems, such as the extremely halotolerant Of additional interest was the high abundance of the elusive phylum Chytridiomycota and their parasites Rozellomycota in the snow-derived tap water, but of Chytridiomycota also (and even more surprisingly) in the glacial ice.We investigated the microbial diversity of subglacial ice and nearby aquatic environments of three polythermal Arctic glaciers located in the Kongsfjorden area on Svalbard. The main findings of the study are:In summary, the first study combining the cultivation and amplicon sequencing to investigate the diversity of both fungi and bacteria in subglacial ice of polythermal glaciers has shown a rich diversity of species, an unexpected spatial heterogeneity of  communities and has found several species that are either new to science or have never been found in glacial ice before.Whirl-Pak® plastic bags. Glacial melt-water, moraine lake water, seawater and tap water were directly collected into sterile plastic bottles. All samples were transported to the lab for preparation within five hours of collection and processed at the NERC Arctic Research Station, Ny-Ålesund. Glaciers studied are small valley glaciers ending on land with similar surface areas (5–5.6 km2): Midtre Lovénbreen, Pedersenbreen, and Vestre Brøggerbreen. They are all non-temperate valley glaciers with polythermal characteristics141, and subject to substantial glaciological and ecological studies143. The glacier thermal regime is important in defining the presence of water at its bed and consequently exerts a firm influence on the subglacial drainage system development12.Fieldwork was conducted during the end of July and beginning of August 2017 in the Kongsfjorden area (Ny-Ålesund) of the Svalbard Archipelago, located at 79°N, 12°E, Norway. Subglacial ice, clear ice, sea water, lake water and glacial meltwater samples were collected from the locations shown in Table w) or low nutrient content. Since fungal diversity is usually neglected, greater effort in fungal isolation was attempted. The media used were therefore DRBC - a general-purpose enumeration medium144; MY10–12 - a medium for the isolation of xero- and halo-tolerant fungi with 10% glucose and 12% NaCl (aw = 0.880)145; DG-18 - a medium for detection of moderate xerophiles (aw = 0.946)146; SNA and MM – two nutrient-poor media for the isolation of oligotrophic fungi and R2A - a low nutrient enumeration medium for heterotrophic microorganisms (both bacteria and fungi)147. To prevent the growth of fast-growing prokaryotes, chloramphenicol (50 mg/l) was added to all media, except for R2A agar. Plates were incubated at 10 °C for up to 8 weeks at the University of Ljubljana, where subsequent procedures were carried out. For every medium the average number of colony forming units (CFU/100 ml) was counted and calculated.The surface layer of subglacial ice samples was melted in a sterile container at room temperature and discarded. The remaining ice was washed with sterile water, transferred to another sterile container and melted. Five, 20 and 50 ml aliquots of subglacial ice samples and glacial meltwater were filtered through Milli-pore membrane filters (0.45 μm pore size) in duplicate. Due to the high sediment content of the subglacial ice and glacial meltwater, the samples were vortexed and left to sediment for one minute before filtering. Filters were placed on two enumeration and four different selective agar media with either low water activity (a2 of mycelium according to Van den Ende and de Hoog (1999)148. For yeast-like and bacterial strains, DNA was extracted using PrepMan Ultra reagent (Applied Biosystems) according to the manufacturer instructions. For filamentous fungi a fragment of rDNA including ITS region 1, 5.8S rDNA and ITS region 2 (referred from here on as the ITS amplicon) was amplified using ITS5 and ITS4 primers149. Polymerase chain reactions (PCR) were performed using Thermo Scientific Taq DNA Polymerase according to the manufacturer’s protocol. Reactions were run in a PCR Mastercycler Ep Gradient (Eppendorf) with an initial denaturation of 2 min at 95 °C, followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 2 min, with a final elongation at 72 °C for 4 min. For identification of Penicillium spp. strains, the partial β-tubulin gene (benA) was amplified and sequenced with Ben2f and Bt2b primers150. Initial denaturation at 95 °C for 1 min was followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 1 min. Final elongation was at 72 °C for 10 min. Cladosporium strains were identified using partial actin (act) sequences, amplified with ACT-512F and ACT-783R primers151. Initial denaturation at 94 °C for 5 min was followed by 45 cycles of denaturation at 94 °C for 45 s, annealing at 52 °C for 30 s and elongation at 72 °C for 90 s. Final elongation was at 72 °C for 5 min. For yeasts domains D1 and D2 of LSU rDNA gene were amplified using NL1 and NL4 primers152. Initial denaturation at 95 °C for 5 min was followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 54 °C for 30 s and elongation at 72 °C for 2 min. Final elongation was at 72 °C for 4 min. For bacteria 16S rRNA gene was amplified with 27f-lane and 1492 R primers153 and a touchdown program. Initial denaturation at 95 °C for 5 min was followed by 5 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 1 min; 5 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 1 min; and 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and elongation at 72 °C for 1 min. Final elongation was at 72 °C for 7 min. The ITS, LSU, benA, act and 16S nucleotide sequences were determined by Sanger sequencing, performed by Microsynth AG, Switzerland. The resulting sequences of all the isolates were aligned using MUSCLE software154 implemented in MEGA5 package155 and compared against the GenBank database using the BLAST software156. Maximum likelihood methods implemented in PhyML 3.0157 were used to build phylogenetic trees after aligning the sequences with similar type and reference sequences from the GenBank database in MEGA5155 . Bootstrap values were calculated from 500 replicate runs. A principal component analysis (PCA) based on presence/absence data of fungal species in the studied environments was performed to determine which species best explained the variance among samples. The PCA was performed in R with the prcomp function158 and plotted with ggbiplot159.DNA was extracted from pure fungal and bacterial cultures up to one week after incubation on a malt extract agar (MEA) and R2A media, respectively. DNA from filamentous fungi was extracted by mechanical lysis of approx. 1 cmAll isolated strains have been deposited in the Ex Culture Collection of the Infrastructural Centre Mycosmo (MRIC UL) at the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.DNA was extracted from filtered biomass  of subglacial ice (S-ice), clear ice (C-ice), sea water (Sea), glacial meltwater (G-wtr) and moraine water (L-wtr). All filters were placed in 1.5 ml microcentrifuge tubes containing RNAlater®  and immediately frozen at −20 °C, until further analysis in the laboratory setting. DNA was extracted from filters using the PowerWater DNA Isolation Kit  and then from the same filters using the PowerLyzer PowerSoil DNA Isolation Kit (MoBio), according to the manufacturer’s instructions with a slight modification to increase the DNA yield and quality. To increase efficiency of fungal cells lysis an additional heating incubation at 65 °C for 10 min was used after adding PW1 solution. DNA from both isolation methods was pooled together and stored at −80 °C until PCRs were performed.160. Amplification was carried out in a PCR Mastercycler Ep Gradient (Eppendorf) with initial denaturation of 2 min at 98 °C, followed by 20 cycles of 10 s at 98 °C, 25 s at 54 °C and of 25 s at 72 °C, with a final elongation of 7 min at 72 °C.For the analysis of fungal diversity, Illumina Miseq V3  sequencing was carried out on the ITS2 region of the ITS rDNA gene amplified using the primers ITS4-Fun (5′- AGCCTCCGCTTATTGATATGCTTAART -3′) and 5.8S-Fun (5′- AACTTTYRRCAAYGGATCWCT -3′)161.For the analysis of bacterial diversity, Illumina Miseq V2  sequencing was carried out on the hypervariable V3 and V4 regions of the 16S rRNA gene by using the 341F_ill (5′- CCTACGGGNGGCWGCAG -3′) and 802R_ill (5′- GACTACHVGGGTATCTAATCC -3′) universal bacterial primersFor both fungi and bacteria, the first step polymerase chain reactions (PCR) were performed in house using the Phusion® High-Fidelity DNA Polymerase according to manufacturer’s protocol, due to the low amount of DNA. Reactions were run in a PCR Mastercycler Ep Gradient (Eppendorf) with initial denaturation of 3 min at 95 °C, followed by 20 cycles of 20 s at 95 °C, 30 s at 56 °C and of 30 s at 72 °C, with a final elongation of 5 min at 72 °C. S-ice-C2 was the only sample from which no amplicons (either ITS or 16S) could be amplified, despite several attempts and optimisation of the amplification protocol. The latter sample was therefore excluded from the amplicon sequencing analyses.162. Since the assembly of the paired end reads was largely unsuccessful, only single-end forward reads were used in the subsequent analysis. The reads were denoised, the tree was constructed by FastTree on a mafft alignment and rooted at midpoint and the alpha and beta diversity indices were calculated. For assigning the taxonomy to sequences, the 99% cut-off GreenGene database163 was used for training the feature classifier for bacteria, and the dynamically clustered UNITE ITS database164 was used for fungi. Abundances in each sample were normalized to the number of sequences in the least abundant sample. Due to the high amount of amplified chloroplast DNA in lake water samples and in order to investigate the bacterial diversity in all samples in a comparable way, chloroplast sequences were excluded with a taxonomy-based filtering. Shannon index was calculated to study the alpha diversity. The distance and dissimilarity matrices were determined through Unifrac distances to visualize the ordination and clustering of the bacterial and fungal community composition for beta diversity analyses. The ordination patterns based on phylogenetic distance metrics were evaluated using principal coordinate analysis (PCoA). Differences in microbial community composition between samples types were assessed by non-parametric permutational analysis of variance (PERMANOVA). All analyses above were performed in QIIME2 2018.8 software package 162. To evaluate community similarities between samples, a hierarchical clustering analysis of the taxa abundance in the communities was performed with the pheatmap and hclust packages in R165.Paired-end reads were quality checked and trimmed  and analyzed with QIIME2 2018.8 software package Bacteria GenBank MK670504-MK670553.Yeasts GenBank MK670448-MK670503.Filamentous fungi GenBank MK671591-MK671647.NGS fungi GenBank PRJNA 517070.NGS bacteria GenBank PRJNA 517069.Supplementary Dataset 1


Six risk factors for screw cutout after internal fixation of intertrochanteric fractures have been reported. The purpose of the present study was to evaluate and compare the impact of the 6 risk factors of screw cutout to clarify the most important one.We enrolled 8 consecutive patients who had screw cutout and 48 random control subjects after internal fixation of intertrochanteric fractures treated with proximal femoral nail antirotation systems at our institution. All of the patients were female. The group that had screw cutout and the control group were retrospectively evaluated and compared with respect to the OTA/AO classification, presence of a posterolateral fragment, types of reduction pattern on anteroposterior and lateral radiographic images, position of the screw, and the presence of a tip-apex distance (TAD) of ≥20 mm. The impact of each factor on screw cutout was assessed using backward stepwise multivariable logistic regression analysis with the Akaike information criterion. Risk stratification was assessed using classification and regression tree (CART) analysis.Among 6 risk factors, only a TAD of ≥20 mm had a significant impact on screw cutout, with an adjusted odds ratio of 12.4 . CART analysis also demonstrated that a TAD of ≥20 mm was the most important risk stratification factor (p < 0.001).Among the 6 previously reported screw cutout-related factors, only a TAD of ≥20 mm was associated with screw cutout after internal fixation of intertrochanteric fractures with proximal femoral nail antirotation systems.Level III. See Instructions for Authors for a complete description of levels of evidence.Therapeutic  The number of hip fractures and hip surgeries continues to increase, as osteoporosis of the bone and poor balance ability affect the growing worldwide elderly population9. Intertrochanteric fracture is one of the most important types of hip fracture. It is generally treated with internal fixation, which, in comparison with conservative treatment, relieves pain, rapidly improves the ability to resume activities of daily living, and results in the recovery of physical function and rehabilitation into society12.Hip fracture is defined as a femoral fracture that occurs in the proximal end of the femur and leads to serious physical and cognitive difficulties14. Cutout of the screw from the femoral head is defined as “the collapse of the neck-shaft angle into varus, leading to extrusion, or so-called cutout, of the screw from the femoral head,” which needs to be addressed with reoperation under general anesthesia15. Thus, the identification of risk factors and the prevention of screw cutout after internal fixation of intertrochanteric fractures are important for orthopaedic surgeons. Six risk factors for screw cutout after internal fixation of intertrochanteric fractures, including 2 patient-related and 4 operation-related factors, have been reported17. However, to our knowledge, there is no comprehensive evaluation available regarding which of the 6 factors has the largest impact on the prevalence of screw cutout. The purpose of the present study was to evaluate and compare the impact of the 6 screw cutout-related factors simultaneously, in order to clarify the most important screw cutout-related factor and to discuss possible preventive strategies.On the other hand, cutout of the screw from the femoral head has been reported as one of the most serious perioperative complications following internal fixation of intertrochanteric fractures, with an estimated prevalence of 1.9% to 3.2%Among the patients with intertrochanteric fractures who were admitted to Narita Red Cross Hospital and underwent surgical treatment with an intramedullary nail , 8 consecutive patients with screw cutout who were hospitalized between March 2010 and July 2014, and 48 random control subjects who were hospitalized between January 2013 and July 2014, were enrolled , or any partial or non-weight-bearing condition in postoperative rehabilitation . The position of the blade was stable when it was positioned in the central-central or the central-inferior zone of the femoral head by dividing the head into 3 zones on the anteroposterior radiograph and 3 zones on the lateral radiograph23. The position was plotted on the sagittal plane as seen on the postoperative radiograph to obtain better resistance against rotation of the femur after surgery23. Finally, we inserted a distal locking screw using the aiming arm device. Radiographic evaluations were performed preoperatively, immediately postoperatively, and once weekly for 2 months after surgery. After postoperative radiography confirmed the absence of displacement of the PFNA and the correct morphology of the fixed femur, we allowed early mobilization and full weight-bearing.The patients were positioned supine on a traction table and were placed under spinal or general anesthesia. We first attempted a closed reduction approach with the use of traction along the axis of the limb with the femur placed in internal or external rotation, and with an image intensifier (C-arm) used in the preoperative planning to obtain the correct alignment and morphology of the femur with reference to the healthy side18; (2) a posterolateral fracture fragment, which was defined using the Jensen classification as type III or V24; (3) a medial type of reduction pattern, in which the proximal fragment lay inward from the anatomical position in the postoperative anteroposterior radiograph25; (4) an intramedullary type of reduction pattern, in which the anterior cortex of the proximal part of the femur was located at the rear of the anterior cortex of the distal fragment in the postoperative lateral radiograph25; (5) an unstable position of the screw in femoral head zones other than the central-central and central-inferior zones ; and (6) a TAD of ≥20 mm23. The TAD is the sum of the postoperative distances from the tip of the screw to the apex of the femoral head on the anteroposterior and lateral radiographs. All calibrations were performed by referencing the PFNA blade, which was 10.3 mm in diameter. The first 2 factors were considered to be preoperative and the others were considered to be operative (Table I).We evaluated 6 previously reported cutout risk factors that included (1) an unstable intertrochanteric fracture, which was defined using the OTA/AO classification as type A2.2 or A2.3We set the postoperative cutout of the screw from the femoral head within 6 months after the operation as the primary end point. A cutout was defined as the collapse of the neck-shaft angle into varus, leading to extrusion of the screw from the femoral head. Continuous variables were summarized using medians and interquartile ranges (quartiles 1 to 3), and categorical variables were summarized by means of counts and percentages. We used the Wilcoxon rank-sum test for comparisons of continuous variables and the chi-square test for comparisons of categorical variables between cutout case and control groups. The impact of each risk factor on cutout was assessed using univariable and multivariable logistic regression analysis by estimating the odds ratio (OR) and its 95% confidence interval (CI). Covariates included all 6 previously reported cutout risks and age (Table II). For the multivariable model selection, we employed a backward stepwise method with the Akaike information criterion to improve model fit.27. All statistical analyses were performed using R software packages .In addition, classification and regression tree (CART) analysis was also used to evaluate the most important determinants of the primary end point. This analysis included age and the 6 risk factors of screw cutout as possible indices Fig. . CART isIn this retrospective case-control observational study, we enrolled 8 patients who had screw cutout and 48 control subjects. Patient characteristics and the frequencies of the 6 previously reported screw cutout risk factors are summarized in Table I. The median age of the patients with screw cutout and the control subjects was 83 and 85 years, respectively. All of the screw cutout cases occurred in female patients, and thus the control group was limited to women Fig. . With reThe impact of each risk factor on the frequency of cutout was determined using univariable and multivariable logistic regression analysis (Table II). Among the 6 previously reported risk factors, only a TAD of ≥20 mm had a significant impact on cutout, with an adjusted OR of 12.4 . Furthermore, CART also demonstrated that a TAD of ≥20 mm was the most important risk stratification factor . Considering the designs and results of previous studies, we speculated that this discrepancy between the results from previous studies and our study can be explained by the following possible mechanism. These preoperative risks could result in increased TAD if the ensuing difficulty in performing the reduction results in a remaining varus deformity. This is intuitively understandable for orthopaedic surgeons if there is difficulty in internal fixation of complex fractures with a higher OTA/AO classification or in the presence of a posterolateral fragment29. Since previous studies suggesting unstable intertrochanteric fractures and posterolateral fragments as risks of cutout did not use other operative risks, such as TAD, they could not exclude the confounding between preoperative and operative risks32. Thus, it is possible that the results from previous reports might be influenced by a potential confounding between the difficulty of the operation and operative risks such as a TAD of ≥20 mm as shown in the present study30.Historically, unstable intertrochanteric fractures and posterolateral fracture fragments have been reported as preoperative risks of screw cutout. These are known as 25. Among these 4 operative risk factors, only the intramedullary type of reduction did not show significant differences in the frequencies between the case and control groups. In addition, the medial type of reduction and unstable position of the screw did not have a significant impact on cutout in a multivariable model. As mentioned above, we speculated that these 3 operative risks could also lead to an increasing TAD, because the axis of the proximal fragment with an inserted lag screw was out of alignment with that of the distal fragment of the intertrochanteric fracture. The presence of mismatched axes would make it difficult for orthopaedic surgeons to achieve both a stable position of the screw and a TAD of <20 mm, resulting in positive results for medial and intramedullary types of reductions or an unstable position of the screw, as seen in previous reports25. Other possible reasons for the discrepancy between our results and previous reports include the relatively small number of patients and low statistical power used to detect the significant impact of multiple risk factors. However, since a TAD of ≥20 mm was identified as a major cutout risk in the multivariable logistic regression model and CART method, and since there was plenty of evidence regarding the TAD risk, it is clear that a TAD of ≥20 mm is the most important screw cutout risk factor after internal fixation of intertrochanteric fractures32.Medial or intramedullary types of reduction patterns, an unstable position of the screw in the femoral head, and a TAD of ≥20 mm have been reported as operative cutout risks17. Finally, as a reminder, surgeons should not seek to minimize the TAD further when it is <20 mm during insertion of the blade. These speculations should be evaluated in a randomized controlled trial to reach definite conclusions.On the basis of our findings, we believe that it is important for orthopaedic surgeons to consider the TAD so as to avoid a cutout during internal fixation of an intertrochanteric fracture. The most important and practical way to achieve this condition is by the nail insertion into the femoral shaft, because the position of the nail determines the position of the screw and the resultant TAD. If we insert the nail into the femoral shaft without imaging for an ideal position of the screw in the femoral head, then the nail position may force surgeons to place the screw in an inappropriate position, which can then lead to a wide TAD. With this point of view, sliding hip screws may have an advantage, compared with the PFNA, in that screw cutout could be avoided because the screw is placed first and with the best control, although this continues to be a controversial topic in this field17. Third, it is possible that low occurrence rates precluded any significance in the correlation between the fracture pattern and cutout. This is unavoidable, even in a large clinical study, because the frequency of cutout is very small17. The low frequency of cutout cases also led to very large 95% CIs as shown in Table II. However, the important point is that the impact of the TAD operative risk might exceed those of fracture patterns because the TAD risk showed significance even after multivariable adjustment or in a CART analysis. Fourth, only very limited data on patient characteristics were available, and bone quality might have been a characteristic of interest. Lastly, the timing of the control group differs from that of the case group, which could have introduced bias (Fig. There are several limitations that warrant mentioning. First, there might be a selection bias because we only enrolled patients who underwent PFNA implantation and because we excluded several patients, such as those with a partial or non-weight-bearing condition, even though cutout did not happen in these patients. Second, there were no male patients, which may be because the frequency of intertrochanteric fracture is higher in females than in malesAmong 6 previously reported cutout-related factors, only a TAD of ≥20 mm was associated with screw cutout after internal fixation of intertrochanteric fractures.


This finding fills a long-standing void in the structural chemistry of this classic antifungal compound, and suggests that prior theoretical models used to explain the spectroscopy, bioactivity, and mode of binding of tolnaftate to squalene epoxidase are probably inappropriate or suspect.Large flat colorless needles of tolnaftate have been obtained from Tinactin, and have been shown to contain a 50:50 mixture of the (+ v/v) acetone–water as large flat colorless needles in the centrosymmetric monoclinic space group P21/c. These crystals contain a 50:50 mixture of the  and  conformers. The bond lengths in the central CNOS unit are 1.3444 (19), 1.3556 (18) and 1.6567 (15) Å for C—N, C—O and C—S, respectively, and the CNOS and C3N moieties are flat and nearly coplanar with each other, consistent with the C—N bond possessing partial double-bond character. Tolnaftate and the four most closely related N,N-disubstituted thiocarbamates in the Cambridge Structural Database (CSD) all exist as E-conformational isomers in the solid state. Among these five compounds, tolnaftate is the only one in which the N-tolyl moiety is positioned trans to the S atom, i.e. the N-aryl substituent in each of the other compounds is positioned cis to their respective S atom. Notably, and more importantly, our experimental X-ray structure is unlike all prior theoretical models available for tolnaftate. The implication, either directly or indirectly, is that some of those theoretical models used in earlier studies to explain the spectroscopic properties of tolnaftate and to suggest which protein–ligand interactions are responsible for the binding of tolnaftate to squalene epoxidase are either inappropriate or structurally unreasonable, i.e. the results and conclusions from those prior studies are in need of critical reassessment.Tolnaftate, a classic antifungal compound, has been found to crystallize from 1:1 ( O-Naphthalen-2-yl N-methyl-N-(3-methylphenyl)carbamothioate, (I)et al., 1961et al., 1986et al., 1986Penicillium griseofulvum  with little to no side effects and holds the distinction of being the first globally successful synthetic topical antifungal agent  acetone–water and the capped vial of the resultant solution then placed in a freezer to effect supersaturation and nucleation. As soon as crystals were noted, the vial was removed from the freezer and allowed to warm to room temperature, at which point the vial cap was loosened and the acetone–water allowed to evaporate slowly further to yield large flat colorless needles suitable for a single-crystal X-ray diffraction experiment. No attempt was made to optimize the acetone–water ratio or to try other solvents since the task of obtaining crystallographic quality crystals had been achieved.A small vial was charged with 2 ml of Bayer Tinactin Liquid Spray followed by 2 ml of water and sealed. Upon standing at room temperature, extremely tiny colorless needles of tolnaftate formed and were harvested. These were redissolved in a minimum amount of 1:1 (Uiso(H) values set at 1.2Ueq(C) and 1.5Ueq(C) for the aryl and methyl H atoms, respectively. The m-tolyl methyl group was treated as rotationally disordered over two orientations. The refined site-occupancy factors were 0.76 (2) and 0.24 (2) for the major and minor components of that disorder, respectively.Crystal data, data collection and structure refinement details are summarized in Table 1i.e. for grades 6–12 pre-college students, homeschoolers, hobbyists, and amateur scientists. Chemistry is often introduced to this audience in the digestible and relatable form of common molecules and common household chemicals. One of the design criteria for our outreach project was to base it on a less commonly recognized common molecule. We believe that tolnaftate fits that criterion. It has been found in numerous households for over 50 years, yet most individuals have no notion of its structural identity and make-up. Another design criterion was cost, i.e. the chemical source was required to be relatively inexpensive and readily available to the targeted audience. The Bayer Tinactin Liquid Spray used in this study was purchased from a retail store for less than 6 US dollars. Other generic sprays can also be purchased for 3–4 US dollars. A single can of Tinactin can provide 2 ml aliquots for a class of roughly 75 students at a cost of 8 cents per student . We hope that the results and discussion provided below will be educational and simulating for those interested in STEM, and meaningful and entertaining for our academic and industrial colleagues as well.Some readers may have already surmised from §1sp2—O, Csp2=S, and Csp2—Nsp2 bonds, respectively  and 1.416 (18) Å, respectively. The sums of the bond angles at atoms C10 and N12 are 359.98 (13) and 359.97 (13)°, respectively, and are also consistent with those atoms being formally sp2-hydridized. Individually, however, the bond angles at C10 do exhibit significant deviations from the idealized sp2 value of 120°, e.g. the O9—C10—S11, O9—C10—N12, and S11—C10—N12 angles are 124.48 (11), 110.39 (13), and 125.11 (12)°, respectively. This pattern of two angles exceeding 120° and the third angle encroaching on the idealized sp3 value of 109.5° is commonly observed in thiocarbamates (and even carbamates).The molecular structure of (I)A/C5–C8/C8A and C13–C18 both being 0.0106 Å. The C2—O9—C10 angle is 119.25 (11) versus 120.0° for an idealized Osp2 atom, the C10/N12/C13/C20 moiety and the CNOS core are both planar, with r.m.s. deviations of 0.0060 and 0.0053 Å for the fitted atoms defining each plane, and the C2, C13, and C20 atoms are 0.091 (2), 0.009 (2), and −0.074 (3) Å off of the CNOS plane, respectively. The CNOS and C3N moieties are also nearly coplanar with each other, with the angle between their normals being 2.24 (11)°. These observations suggest that delocalization of π-electron density over the entire CNOS unit is not geometrically disallowed or, at least, that the core C—N bond possesses partial double-bond character.The three substituents attached to the CNOS core exhibit the expected structural metrics. The aromatic rings are flat, with the r.m.s. deviations for the planes defined by atoms C1–C4/C4E conformation in the solid state. The four most closely related N,N-disubstituted thiocarbamate structures in the CSD are GEHSAO R group, with X being either an O or S atom, and the aryl substitutent is either a tolyl or a phenyl group. The molecules in these prior structures are also present as E conformers. A wider comparison involving all relevant monosubstituted N-aryl thiocarbamates, i.e. with the methyl group replaced by an H atom, yields 46 such entries in the CSD with the ratio of E:Z stereochemistries being 40:6. Obviously, the N,N-disubstituted comparison suffers from both steric and electronic factors, e.g. the C(=X)R groups are significantly larger and more polar than methyl, and the monosubstituted comparison suffers from N—H being significantly more prone to hydrogen-bonding effects than N—CH3. Nevertheless, prior studies would seem to suggest that an E conformation is preferred, and that is indeed what is found for tolnaftate as well.As depicted in the Scheme and Fig. 1cis to the S atom, i.e. s–cis with respect to the C—O core bond, and that a more precise description of the tolnaftate molecule is that it has an E,Z or trans,cis conformation. While justifiably superior to the E-only description, this second approximation using two stereodescriptors rather than one also falls short of being fully descriptive. For example, the tolnaftate molecule shown is three-dimensional (3D) and chiral in the solid state, i.e. the conformer in Fig. 1i.e. it too is an E,Z or trans,cis conformer. Hence, one cannot differentiate between the two enantiomers with these stereodescriptors because the descriptors themselves are invariant on reflection in a mirror.For casual readers, this first approximation for describing the tolnaftate molecule is more than adequate. For others, additional stereodescriptors are required. Readers in the latter group will point out that Fig. 1i.e.https://doi.org/10.1351/goldbook.T06406  they divide torsional space into six 60° regions rather than two 180° semicircular sections, and (b) they are signed + or −. We will apply a few nonstandard conventions and further subdivide the +30 to −30° region into 0 to +30° and 0 to −30° and assign the descriptors +sp and −sp to them. Similarly, the +150 to −150° zone will be subdivided into 180 to +150° and 180 to −150° and assigned the descriptors +ap and −ap, respectively. This subdivision of torsional space into eight regions rather than six provides an even greater ability to distinguish one conformation from another. Lastly, we will expand the bonds of interest to be the core C—N, core C—O, N—Ctolyl, and O—Cnaphthyl bonds, and assign descriptors to each in that order. Thus, the tolnaftate molecule shown in Fig. 1ap,−sp,+ac,−ac) conformer, while its enantiomer would be uniquely described as the  conformer.A third approach for specifying the conformation is to use clinal and periplanar descriptors, sp2—H⋯S=C hydrogen bonds  x, −y + z + x, y + 1, z]. The observed H16⋯S11i and H18⋯S11ii distances are 2.98 and 2.93 Å, respectively, and are comparable to distances of 2.86–3.09 Å reported by others for Csp2—H⋯S=C hydrogen bonds sp2—H⋯π hydrogen bonds, i.e. C3—H3⋯X1 and C8—H8⋯X2. The first is an offset face-to-face hydrogen bond typically observed in π–π stacking, while the second is an edge-to-face hydrogen bond. The H3⋯X1 distance is 3.43 Å and is comparable to the value of 3.5 Å expected for a face-to-face hydrogen bond, while H8⋯X2 and C8—H8⋯X2 are 2.62 Å and 142°, and are in agreement with the values of 2.73 (13) Å and 148 (11)° expected for edge-to-face H⋯π and Csp2—H⋯π, respectively  networks of hydrogen bonds that are parallel to the (h00) family of planes at x = 0.2 and x = 0.8. Similarly, the Csp2—H⋯π interactions form a separate 2D network of hydrogen bonds also parallel to the (h00) family of planes but at x = 0.5. The end result is a packing structure reminiscent of an interdigitated lipid bilayer with the heteroatoms and polar bonds positioned near the outer surfaces and the nonpolar naphthyl substituents sandwiched in the interior of the bilayer.As shown in Fig. 2the world’s repository for small-molecule crystal structures with over 900,000 curated entries, its database of experimental 3D coordinates is miniscule compared to databases providing theoretical 3D coordinates. For example, the PubChem database currently contains 96,396,575 compounds and 3D coordinates for over 88.5 million molecules , , , and  for the 3DChem, ATB, Mol-Instincts, and PubChem models, respectively, while the experimentally observed conformation for (I)ap,−sp,+ac,−ac). The ATB and Mol-Instincts models are visually similar, but are strikingly different from the 3DChem and PubChem models. None of the theoretical models are a match to our X-ray structure. This is not unexpected since most theoretical models ignore intermolecular interactions and packing forces such as those described above in §3.2A visual comparison of online theoretical tolnaftate models et al., 2011The distances and angles in Table 3any 3D model or coordinates that they download from the web to be potentially suspect unless clearly demonstrated otherwise, i.e. we encourage them to critically examine the geometrical attributes  of those models or seek help from others to do so, if need be.Taken as a whole, all of these observations suggest that the current standards and guidelines for online theoretical 3D models and coordinates are rather loose, and that the validation methods used by website providers for assessing the structural reasonableness of their optimized molecules are less than fully adequate. Individuals in our targeted audience of nonscience professionals should therefore consider et al., 2011simulation of their model is shown in Fig. 4Avogadro 1.2.0n, downloaded from https://avogadro.cc/ , and not a match to our experimentally observed  conformation. Our X-ray results are particularly relevant to this 2011 paper since their IR spectrum was taken on a solid-state tolnaftate sample in KBr. Since our study unequivocally establishes that the solid-state conformation of tolnaftate is  and not , their vibrational analysis based on the latter conformation is unlikely to be valid. For their published results to be valid, their sample would have to be a tolnaftate polymorph with the molecules in the  conformation, which is highly improbable. To our knowledge, there is no powder diffraction or DSC evidence that tolnaftate polymorphs exist. Significant variations in powder pattern peak intensities have been observed, but such observations are completely attributable to preferred orientation effects without the need to invoke polymorphism. Hence, the calculated wavenumbers and all other computed quantities based on their use of a  model should be considered suspect.For completeness, we are aware of two additional theoretical tolnaftate models. The first of these was published by Joe and co-workers as part of a vibrational analysis of the tolnaftate IR and Raman spectra  and various other theoretical results are increasingly being showcased, should consider implementing policies that will safeguard against questionable theoretical models reaching print. Insisting that 3D coordinate files must be submitted as supplementary material for theoretical models (and not just for X-ray structures) would greatly facilitate the peer-review process by referees and editors, and any subsequent scientific inquiries by readers.What this comparison of our experimental model to peer-reviewed theoretical models reveals is that the standards and guidelines for publishing theoretical structures are currently less rigorous than those for publishing crystal structures. The take-home message for amateur scientists and science enthusiasts is that opportunities for scientific adventures and discoveries in a home setting do indeed exist and may even be publishable. All of the wet chemistry presented in this paper were done in a common household kitchen and all of the structure solution, refinement, and manuscript preparation were carried out with readily available freeware using public computers in local libraries. Exploring science in the 21st Century can be that simple. Also, be willing to investigate opportunities at local colleges and universities where specialized equipment like an X-ray diffractometer might be available through various outreach programs. Adult supervision and guidance are, of course, encouraged for any projects involving minors or other pre-college individuals.ap,−sp,+ac,−ac) and  conformers. The broad implication and significance of this experimental finding is that it calls into question, either directly or indirectly, the results and conclusions from prior theoretical models for tolnaftate. Notably, the vibrational analysis, natural bonding orbital (NBO) analysis, and predicted electronic absorption spectrum and associated computed quantities based on an alternate  conformation needs to be revisited and their actual numeric contributions to the bioactivity of tolnaftate reassessed. Similarly, our crystal structure suggests that a recent study on the binding mode of tolnaftate to squalene epoxidase by in silico methods is also in need of being revisited. It seems likely that the unusual tolnaftate (and liranaftate) conformation in that study was introduced at the ligand preparation stage. However, we cannot rule out with certainty whether the irregularities occurred at the docking stage, instead, or are due to other problems with the modeling methodology that was employed. Regardless, our kitchen-sink science argues that we do not yet know all that there is to know about the spectroscopy and enzyme-ligand interactions of this deceptively simple and historic antifungal compound.Our message for academic and industrial colleagues is that the long overdue X-ray structure of tolnaftate is now available, and that crystals of tolnaftate contain a 50:50 mixture of  global, I. DOI: 10.1107/S2053229618013591/yp3169Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2053229618013591/yp3169Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2053229618013591/yp3169Isup4.cmlSupporting information file. DOI: 1869447CCDC reference:


Corn appears to be an uncommon food source of allergens in dogs and cats. There is limited information on the nature of the corn allergens in dogs and cats and their presence in the various foodstuffs used in commercial pet foods. The aim of this study was to determine if serum IgE from corn-sensitized dogs and cats recognized proteins in corn flour and cornstarch, which are common sources of carbohydrates in pet foods.We selected archived sera from allergy-suspected dogs (40) and cats (40) with either undetectable, low, medium or high serum levels of corn-specific IgE. These sera were tested then by ELISA on plates coated with extracts made from corn kernels, corn flour, cornstarch and the starch used in the commercially-available extensively-hydrolyzed pet food Anallergenic . Immunoblotting was then performed on the same extracts with some of the sera from moderate-to-high corn-sensitized dogs and cats. Using ELISA, it is mostly the dogs and cats with moderate and high corn-specific IgE levels that also had IgE identifying allergens in the flour . In contrast, none of the tested sera had measurable IgE against proteins isolated from the cornstarch. Immunoblotting confirmed the existence of numerous major corn allergens in the corn kernel extract, fewer in that of the corn flour, while such allergens were not detectable using this technique in the two cornstarch extracts.In this study, ELISA and immunoblotting results suggest that IgE from corn-sensitized dogs are less likely to recognize allergens in cornstarch than in kernel and flour extracts. As corn is not a common allergen source in dogs and cats, and as its starch seems to be less allergenic than its flour, pet foods containing cornstarch as a carbohydrate source are preferable for dogs and cats suspected of suffering from corn allergy.The online version of this article (10.1186/s12917-018-1538-5) contains supplementary material, which is available to authorized users. Cutaneous adverse food reactions (CAFR) represent one of the most common diagnoses given to dogs and cats with pruritus or allergic skin diseases . To echoA recent critically appraised topic concluded that the most common allergenic food sources in dogs and cats are derived from mammalian, poultry, and fish meats, while grains are uncommon culprits . Among tRecent proteomic studies markedly expanded the list of possible IgE-targeted corn allergenic proteins for humans –7. An imTo date, there is only limited information available on the identity of corn allergens in dogs, while none has been reported yet in cats. Cornstarch is frequently added to commercial pet foods, including extensively-hydrolyzed ones. Whilst there is no clinical evidence to suggest its inclusion in such diets would be problematic for animals sensitized to corn, in a recent study evaluating the proteins present in three commercial hydrolysate-containing pet foods, granule-bound starch synthase 1  was the only corn protein identified as an allergen by serum IgE from a small fraction of dogs with suspected nonseasonal allergy ; of impoThe objectives of this study were to determine if IgE from corn-sensitized dogs and cats recognized proteins present in corn flour and starch. We will show herein that, presumably due to its very low protein content and limited proteome, cornstarch is hypoallergenic compared to its flour.We used canine and feline leftover sera submitted to Avacta Animal Health  for determination of food allergen-specific IgE serological testing.For these studies, sera from dogs and cats exhibiting signs suggestive of an allergic dermatitis were chosen based on their level of corn-specific IgE, but information whether or not these patients suffered from corn-induced clinical allergies was not available. All sera had been kept at − 80 °C until assayed.405 uncorrected for background) in the Sensitest commercially available ELISA (Avacta). The initial screening had been made with a standard corn extract .We selected canine and feline sera according to their original levels of serum corn-specific IgE  between 0.200 and 0.500 in the Sensitest assay.D1-LCoR: ten dogs with low corn IgE reactivity and a previous OD405 between 0.501 and 1.000)D1-MCoR: ten dogs with moderate IgE corn reactivity (OD405 > 1.000)D1-HCoR: ten dogs with high corn IgE reactivity (OD405 < 0.200).D2-NCoR: ten dogs with undetectable levels of IgE to corn  extract (SCE) from Greer laboratories (F102), and others made from regular corn flour (RCF), regular cornstarch (RCS) and the purified cornstarch used in Royal Canin Anallergenic ; the last three food sources were provided by Royal Canin .w/v) before being centrifuged at 15,000 g for 20 min. The supernatant was retained at 2–8 °C, and the pellet was further extracted with PBS overnight at 2–8 °C. Following the second centrifugation, the supernatant was collected again and combined with the previous one. The protein concentration of the extract was determined by measurement at 280 nm .All extracts but the SCE were prepared as follows: allergens were extracted with PBS, pH 7.4, for 4 h at 2–8 °C .Positive and negative serum controls, with moderate and negligible levels of corn specific IgE, respectively, were also diluted at 1:10 and included allergen-coated and uncoated (no-coat control) plates.The tested extracts were the same as those used for ELISA ; they were heated at 70 °C for 10 min in the presence of a reducing agent  before sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Approximately 5 μg of protein was deposited in each lane and, except for minor changes in reagent suppliers, SDS-PAGE and immunoblotting were otherwise performed similarly to our recent report [The protein yields for the RCF, RCS, and ACS averaged from three separate measurements, were 1.63, 0.13 and 0.27 mg/ml, respectively. These concentrations were sufficient for coating plates with a 5 μg/mL solution.405 plus three standard deviations of 95 canine sera without detectable anti-corn IgE.The canine ELISA standard curve had an accuracy of between 100 and 106% at concentrations varying between 3.7 and 100 AU. The inter-assay coefficient of variation of the positive control was 4%. We established the positive threshold of detection at 10.0 AU as the mean ODFor the feline ELISA, the standard curve was 92–113% accurate over the same concentration range as above. The inter-assay coefficient of variation of the positive control was 1.1%. The positive threshold, determined as for the canine ELISA, was calculated to be 12.7 AU.The percentages of IgE reactivity of the four groups of canine sera to the four different extracts are shown in Table All sera from the D1 group  had IgE against the SCE. Among these sera, all those with moderate and high corn-specific IgE levels  also had IgE identifying allergens in the flour (RCF). Importantly, none of the forty canine sera had positive levels of IgE against allergens present in the two cornstarch extracts (RCS and ACS).Similarly and as expected, none of the sera from the D2 group (corn-nonreactive sera) had measurable IgE against any of the four tested extracts.The percentage of reactivity of the feline sera to the various extracts are also compiled in Table Altogether, all 29 feline corn-reactive sera  had positive levels of IgE that recognized SCE proteins, 20/29 (69%) contained IgE-specific for allergens in the flour while none had measurable IgE against starch allergens.Sera from corn-nonreactive cats (C2-NCoR) did not have any positive IgE reactivity to any of the tested extracts.We used SDS-PAGE to separate the proteins according to their molecular weight under reducing and denaturing conditions. Multiple bands in the range of approximately 97–10 kDa were visible in the SCE lane  had detectable IgE that recognized RCF proteins of molecular weight between 4 and 103 kDa; major corn flour allergens were those of 93 , 61 (50%), 37 (70%), 30 (60%) and 22 kDa (70%) Fig. . The binIn contrast to the results above, there was no binding of SCE-positive dog serum IgE to RCS or ACS extracts Fig. . SimilarThe results from corn-sensitized feline serum IgE binding to proteins of SCE, RCF, RCS and ACS are shown in Fig. In the flour, feline serum IgE bound to proteins of molecular weight between 20 and 106 kDa, with all but one cat having a detectable reactivity to the RCF Fig. . Major cIn contrary to the results above and similarly to dogs, there was no binding of SCE-positive cat serum IgE to RCS or ACS extracts and no IgE reactivity in SCE-negative cat serum pool control blots.In this serological study using two different techniques, IgE detected allergens in corn flour only in dogs and cats having moderate and high IgE serum levels against a corn kernel extract. In contrast, none of the tested sera had detectable IgE against cornstarch proteins. As such, cornstarch should be considered hypoallergenic compared to its flour in dogs and cats with detectable serum corn-specific IgE.These results are not surprising, as they echo the clinical indings of experimental challenges in corn-sensitized dogs . In thishttp://nutritiondata.self.com; page last accessed October 31, 2017), the lower allergenicity that was seen with the starch is unlikely due to a difference in protein concentration between these foodstuffs. Indeed, we performed ELISAs and immunobloting with the same protein concentration (5 μg/ml) for all extracts. Consequently, the difference in the allergenicity of corn byproducts most likely resides in the different protein composition existing between flour and starch.Even though cornstarch contains approximately less than one-twentieth of the protein amount of corn flour  .www.allergen.org; site last accessed October 31, 2017) only lists three of these corn kernel allergens .Recent proteomic studies employed 2D-electrophoresis and mass spectrometry to characterize corn kernel allergens recognized by IgE from corn-allergic human sera –7. AltogMatching the recently identified corn kernel allergens –7 to proWhile the specific amounts of each of these allergenic proteins were not reported in the two cornstarch proteomic articles , 14, oneImportantly, the GBSS-I was reported recently to be an allergen for both corn-sensitized humans , 17 and That proteins like GBSS-I are recognized corn allergens does not mean that they could induce flares in corn-allergic individuals, be it an animal or a human. There are at least three factors that could reduce its allergenicity: 1) the nature of the targeted epitopes, 2) food processing and 3) the amount of allergen consumed.While most allergic patients have IgE that recognize epitopes on the peptidic moiety of proteins, others bind to glycans on complex glycoproteins (reviewed in ). The moFood processing is another reason why allergens would no longer be recognized by IgE from clinically-reactive patients, for example, if allergens were thermolabile. This phenomenon has been shown recently to also occur in dogs suspected of having a food allergy: raw proteins seemed more allergenic than cooked ones , 22. ConFurthermore, the clinical reactivity of food-allergic humans is mainly allergen-dose-dependent, with protein doses eliciting reactions varying markedly between children and adults or between food sources . For exaFinally, the immunoblotting experiments provided valuable data on the molecular weights of several major corn allergens recognized by corn-sensitized dogs and cats. It is hoped that the sequencing of these bands will help precisely determine the identity of such allergens and their respective amount in corn-derived products such as flour and starch.In this study, we established, using both ELISA and immunoblotting, that IgE from corn-sensitized dogs is less likely to recognize cornstarch proteins than those present in kernel and flour extracts. As corn is not yet known as a common allergen source in dogs and cats, and as its starch seems to be less allergenic than the flour, pet foods containing cornstarch as a carbohydrate source might be preferable for pets suspected of corn allergies.Additional file 1: SDS-PAGE. Extracts (~ 5 μg/lane) were separated in 4–12% gels by SDS-PAGE. Lane 1: molecular weight (M wt) markers; lane 2: standard corn extract (SCE); lane 3: regular corn flour (RCF) extract; lane 4: Regular cornstarch (RCS) extract; lane 5: Anallergenic cornstarch (ACS) extract. (TIFF 218 kb)