Bilateral insufficiency fracture of coracoid process: A rare case report.

A 69-year-old man was admitted to the hospital for a left femoral neck fracture. A preliminary chest computed tomography scan showed no coracoid process fracture. The patient had no history of trauma during his hospitalization. However, subsequent in-hospital computed tomography scan revealed bilateral coracoid process fracture. The patient underwent hip replacement surgery for femoral neck fracture, while conservative treatment was administered for the bilateral coracoid process fracture. After 1-year follow-up, the patient was diagnosed with bilateral insufficiency fracture of coracoid process after ruling out other types of fractures. The fractures did not heal while functions in both shoulders were adequate. Insufficiency fracture should be considered when fractures occur without trauma, especially in the presence of associated risk factors such as chronic renal failure and osteoporosis. For bilateral insufficiency fracture of coracoid process, conservative treatment is acceptable.

Effects of blanching treatments on acrylamide, asparagine, reducing sugars and colour in potato chips.

This study aimed to determine the effect of combinations of blanching parameters, including blanching temperatures ranging from 65 to 85 °C and duration times ranging from 2 to 10 min, on reducing sugars, asparagine, acrylamide, and color levels of fried potato chips. Response surface methodology was used to develop response surface equations to estimate these effects. These latter were evaluated before and after a 3-month storage period of potato tubers at 10 °C. It was found that certain blanching parameters resulted in optimal maximum reductions of 64.2, 49.8, and 61.3% for reducing sugar, asparagine, and acrylamide, respectively. Analysis of variance (ANOVA) determined that blanching time had a more significant impact than blanching temperature. The blanching time that resulted in maximum reductions of asparagine, reducing sugars-and ultimately acrylamide-were in the range of 8.8-9.7 min at 68.7-75.0 °C. ANOVA also determined that after the 3-month storage period of potato tubers, variations in blanching time and temperature did not result in any significant differences in acrylamide formation in fried chips. Blanching consistently improved the appearance of the fried chip products, indicated by increases in L* value and decreases in a* values. The relationship between acrylamide formation and a* value was linear (R2 = 0.839), while the relationship between acrylamide formation and L* value was not (R2 = 0.375).

Alternative splicing results in a lack of starch synthase IIa-D in Chinese wheat landrace.

We evaluated the SGP-1 protein composition of 368 Chinese wheat landraces using SDS-PAGE. The SGP-D1 null type was identified in three accessions (Xiaoqingmang, Pushanbamai, and P119). An 18-bp deletion and 9-bp variation were found at the junction region of the 7th intron and 8th exon, leading to deletion of the intron-exon junction recognition site AG when aligned the 8261-bp DNA sequence of TaSSIIa-D in Pushanbamai with that of Chinese Spring. Four cDNA types with mis-spliced isoforms were subsequently detected through amplification of TaSSIIa-D cDNAs. Among these, nine type II cDNAs with a 16-bp deletion in the 8th exon were detected, indicating that the major transcriptional pattern of TaSSIIa in Pushanbamai is type II. In the type IV cDNA, a 97-bp sequence remains undeleted in the end of the 5th exon. The amylose content in Pushanbamai was significantly higher than that in all control lines under field conditions, which suggested that deletion of SGP-D1 has an efficient impact on amylose content. As the TaSSIIa gene plays an important role in regulating the content of amylose, it is anticipated that these natural variants of TaSSIIa-D will provide useful resources for quality improvement in wheat.

Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

Characterization and expression analysis of WOX2 homeodomain transcription factor in Aegilops tauschii.

The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding.

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

New biotechnology enhances the application of cisgenesis in plant breeding.

Cisgenesis is genetic modification to transfer beneficial alleles from crossable species into a recipient plant. The donor genes transferred by cisgenesis are the same as those used in traditional breeding. It can avoid linkage drag, enhance the use of existing gene alleles. This approach combines traditional breeding techniques with modern biotechnology and dramatically speeds up the breeding process. This allows plant genomes to be modified while remaining plants within the gene pool. Therefore, cisgenic plants should not be assessed as transgenics for environmental impacts.

Characterization of high-molecular-weight glutenin subunits from Eremopyrum bonaepartis and identification of a novel variant with unusual high molecular weight and altered cysteine residues.

We characterized two high-molecular-weight glutenin subunit (HMW-GS) variants from Eremopyrum bonaepartis, determined their complete open reading frames, and further expressed them in a bacterial system. The variants have many novel structural features compared with typical subunits encoded by Glu-1 loci: 1Fx3.7 and 1Fy1.5 exhibit hybrid properties of x- and y-type subunits. In addition, unusual molecular mass and altered number and distribution of cysteine residues were unique features of HMW-GSs encoded by Glu-F1 from E. bonaepartis. The mature 1Fx3.7 subunit has a full length of 1,223 amino acid residues, making it the largest subunit found thus far, while 1Fy1.5 is just 496 residues. In addition, the mutated PGQQ repeat motif was found in the repetitive region of 1Fx3.7. Although it has a similar molecular mass to that previously reported for 1Dx2.2, 1Dx2.2* and 1S(sh)x2.9 subunits, 1Fx3.7 appears to have had a different evolutionary history. The N-terminal and repetitive regions have a total of four additional cysteine residues, giving 1Fx3.7 a total of eight cysteines, while 1Fy1.5 has only six cysteines because the GHCPTSPQQ nonapeptide at the end of the repetitive region is deleted. With its extra cysteine residues and the longest repetitive region, features that are relevant to good wheat quality, the 1Fx3.7 subunit gene could be an excellent candidate for applications in wheat quality improvement.

Characterization and expression analysis of WOX5 genes from wheat and its relatives.

The WUSCHEL (WUS)-related homeobox (WOX) gene family plays an important role in coordinating gene transcription in the early phases of embryogenesis. In this study, we isolated and characterized WOX5 from common wheat and its relatives Triticum monococcum, Triticum urartu, Aegilops speltoides, Aegilops searsii, Aegilops sharonensis, Aegilops longissima, Aegilops bicornis, Aegilops tauschii, and Triticum turgidum. The size of the characterized WOX5 alleles ranged from 1029 to 1038 bp and encompassed the complete open reading frame (ORF) as well as 5' upstream and 3' downstream sequences. Domain prediction analysis showed that the putative primary structures of wheat WOX5 protein include the highly conserved homeodomain besides the WUS-box domain and the EAR-like domain, which is/are present in some members of the WOX protein family. The full-length ORF was subcloned into a prokaryotic expression vector pET30a, and an approximate 26-kDa protein was successfully expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The WOX5 genes from wheat-related species exhibit a similar structure to and high sequence similarity with WOX5 genes from common wheat. The degree of divergence and phylogenetic tree analysis among WOX5 alleles suggested the existence of three homoeologous copies in the A, B, or D genome of common wheat. Quantitative PCR results showed that TaWOX5 was primarily expressed in the root and calli induced by auxin and cytokinin, indicating that TaWOX5 may play a role related to root formation or development and is associated with hormone regulation in somatic embryogenesis.

Structure and expression of barley starch phosphorylase genes.

The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes (Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter's cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, Aegilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon-intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.

Characterization of x-type high-molecular-weight glutenin promoters (x-HGP) from different genomes in Triticeae.

The sequences of x-type high-molecular-weight glutenin promoter (x-HGP) from 21 diploid Triticeae species were cloned and sequenced. The lengths of x-HGP varied from 897 to 955 bp, and there are 329 variable sites including 105 singleton sites and 224 polymorphic sites. Genetic distances of pairwise X-HGP sequences ranged from 0.30 to 16.40% within 21 species and four outgroup species of Hordeum. All five recognized regulatory elements emerged and showed higher conservation in the x-HGP of 21 Triticeae species. Most variations were distributed in the regions among or between regulatory elements. A 22 bp and 50 bp insertions which were the copy of adjacent region with minor change, were found in the x-HGP of Ae. speltoides and Ps. Huashanica, and could be regarded as genome specific indels. The phylogeny of media-joining network and neighbour-joining tree both supported the topology were composed of three sperate clusters. Especially, the cluster I comprising the x-HGP sequences of Aegilops, Triticum, Henrardia, Agropyron and Taeniatherum was highly supporting by both network and NJ tree. As conferring to higher level and temporal and spatial expression, x-HGP can used as the source of promoter for constructing transgenic plants which allow endosperm-specific expression of exogenous gene on higher level. In addition, the x-HGP has enough conservation and variation; so it should be valuable in phylogenetic analyses of Triticeae family members.

Characterization and expression analysis of waxy alleles in barley accessions.

Granule Bound Starch Synthase I (GBSS I) encoded by the waxy gene plays an important role in accumulating amylose during the development of starch granules in barley. In this study, we isolated and characterized waxy alleles of three waxy (GSHO 908, GSHO 1828 and NA 40) and two non-waxy barley accessions (PI 483237 and CIho 15773), estimated the expression patterns of waxy genes via Real-time quantitative PCR (RT-qPCR), investigated promoter activity by analyzing promoter-GUS expression, and examined possible effects of waxy alleles on starch granule morphology in barley accessions by scanning electron microscopy (SEM). A 193-bp insertion in intron 1, a 15-bp insertion in the coding region, and some single nucleotide polymorphic sites were detected in the waxy barley accessions. In addition, a 397-bp deletion containing the TATA box, transcription starting point, exon 1 and partial intron 1 were also identified in the waxy barley accessions. RT-qPCR analysis showed that waxy accessions had lower waxy expression levels than those of non-waxy accessions. Transient expression assays showed that GUS activity driven by the 1,029-bp promoter of the non-waxy accessions was stronger than that driven by the 822-bp promoter of the waxy accessions. SEM revealed no apparent differences of starch granule morphology between waxy and non-waxy accessions. Our results showed that the 397-bp deletion identified in the waxy barley accessions is likely responsible for the reduction of waxy transcript, leading to lower concentrations of GBSS I protein thus lower amylose content.

Characterization of HMW-GSs and their gene inaction in tetraploid wheat.

In this study, we report the expression of HMW-GSs in 87 accessions of tetraploid wheat, the characterization of three inactive and one active HMW glutenin genes, and the functional verification of HMW-GSs by promoter-GUS expression. SDS-PAGE profiles revealed that tetraploid wheat has many different combinations of HMW-GSs and the number of subunits varies from 1 to 4. HMW glutenin genes at the Glu-A1x, Glu-A1y and Glu-B1y loci exhibited different frequencies of inaction while the Glu-B1x allele was expressed in all 87 accessions. Gene cloning showed that only 1Bx (Tdu-e) could express a full-length protein and its deduced protein sequence has the typical primary structure but with fewer cysteine residues. The expression of the other three HMW glutenin genes has been disrupted by stop codons in their repetitive domains. Besides short indels or mutations of one or more bases, an 85-bp deletion and a 185-bp insertion were found in the promoter regions of 1Ay (Tdu-s) and 1Bx (Tdu-e). The transient expression of promoter-GUS constructs indicated that the 1Ay promoter can drive expression of the GUS gene. We conclude that defects (stop codons or the insertion of large transposon-like elements) in the coding regions may be the most probable cause for the inaction of the HMW glutenin genes.

Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding.

BACKGROUND: High molecular weight glutenin subunits (HMW-GSs), encoded by the genes at Glu-1 loci in wheat and its related species, are significant in the determination of grain processing quality. However, the diversity and variations of HMW-GSs are relatively low in bread wheat. More interests are now focused on wheat wild relatives in Triticeae. The genus Aegilops represents an important germplasm for novel HWM-GSs and other useful genes for wheat genetic improvement. RESULTS: Six novel Glu-1 alleles and HMW-GSs were identified and characterized from three species of Aegilops section Sitopsis (S genome). Both open reading frames (ORFs) and promoter regions of these Glu-1 alleles were sequenced and characterized. The ORFs of Sitopsis Glu-1 genes are approximately 2.9 kb and 2.3 kb for x-type and y-type subunits, respectively. Although the primary structures of Sitopsis HMW-GSs are similar to those of previously reported ones, all six x-type or y-type subunits have the large fragment insertions. Our comparative analyses of the deduced amino acid sequences verified that Aegilops section Sitopsis species encode novel HMW-GSs with their molecular weights larger than almost all other known HMW-GSs. The Glu-1 promoter sequences share the high homology among S genome. Our phylogenetic analyses by both network and NJ tree indicated that there is a close phylogenetic evolutionary relationship of x-type and y-type subunit between S and D genome. CONCLUSIONS: The large molecular weight of HMW-GSs from S genome is a unique feature identified in this study. Such large subunits are resulted from the duplications of repetitive domains in Sitopsis HMW-GSs. The unequal crossover events are the most likely mechanism of variations in glutenin subunits. The S genome-encoded subunits, 1Dx2.2 and 1Dx2.2* have independent origins, although they share similar evolutionary mechanism. As HMW-GSs play a key role in wheat baking quality, these large Sitopsis glutenin subunits can be used as special genetic resources for wheat quality improvement.

Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress.

The pre-mRNA processing (Prp1) gene encodes a spliceosomal protein. It was firstly identified in fission yeast and plays a regular role during spliceosome activation and cell cycle. Plant Prp1 genes have only been identified from rice, Sorghum and Arabidopsis thaliana. In this study, we reported the identification and isolation of a novel Prp1 gene from barley, and further explored its expressional pattern by using real-time quantitative RTPCR, promoter prediction and analysis of microarray data. The putative barley Prp1 protein has a similar primary structure features to those of other known Prp1 protein in this family. The results of amino acid comparison indicated that Prp1 protein of barley and other plant species has a highly conserved 30 termnal region while their 50 sequences greatly varied. The results of expressional analysis revealed that the expression level of barley Prp1 gene is always stable in different vegetative tissues, except it is up-regulated at the mid- and late stages of seed development or under the condition of cold stress. This kind of expressional pattern for barley Prp1 is also supported by our results of comparison of microarray data from barley, rice and Arabidopsis. For the molecular mechanism of its expressional pattern, we conclude that the expression of Prp1 gene may be up-regulated by the increase of pre-mRNAs and not be constitutive or ubiquitous.

Starch synthesis and programmed cell death during endosperm development in triticale (x Triticosecale Wittmack).

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source. Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.

Structural variation and evolutionary relationship of novel HMW glutenin subunits from Elymus glaucus.

High molecular weight (HMW) glutenin subunits (GS) are important seed storage proteins relevant to the end-use quality of wheat and other cereal crops. Here we report the isolation and characterization of two novel HMW-GS alleles (1St 1.4 and 1St1.1) from the perennial Triticeae species Elymus glaucus. The amino acid (aa) sequences of E. glaucus 1St1.4 and 1St1.1 were predicted as 434 aa and 358 aa, respectively. Both subunits comprise a signal peptide with a conserved N-terminal domain, a central repetitive domain and a C-terminal domain. Elymus glaucus 1St 1.4 and 1St1.1 exhibit several distinct characteristics different from other known HMW-GSs. The lengths of repetitive domains in E. glaucus 1St 1.4 and 1St1.1 are substantially smaller than those of other known HMW-GSs, in which 1St1.1 (only 358 aa) is the smallest subunit identified so far. The N-terminal domains of E. glaucus 1St 1.4 and 1St1.1 are homologous to y-type subunits, whereas their C-terminal domains are similar to x-type subunits. Our results indicate that E. glaucus 1St 1.4 and 1St1.1 are novel HMW-GS variants or isoforms, and the characterization of both subunits can enhance our understanding on the structural differentiation and evolutionary relationship of HMW-GSs in Triticeae species.

Isolation and characterization of the rye Waxy gene.

Complete genomic and cDNA sequences of the Waxy gene encoding granule-bound starch synthase I (GBSSI) were isolated from the rye genome and characterized. The full-length rye Waxy genomic DNA and cDNA are 2767 bp and 1815 bp, respectively. The genomic sequence has 11 exons interrupted by 10 introns. The rye Waxy gene is GC-rich, with a higher GC frequency in the coding region, especially in the third position of the codons. Exon regions of the rye Waxy gene are more conserved than intron regions when compared with the homologous sequences of other cereals. The mature rye GBSSI proteins share more than 95% sequence identity with their homologs in wheat and barley. A phylogenetic tree based on sequence comparisons of available plant GBSSI proteins shows the evolutionary relationship among Waxy genes from rye and other plant genomes. The identification of the rye Waxy gene will enable the manipulation of starch metabolism in rye and triticale.

Adult plant resistance-related gene expression in 'Camp Remy' wheat inoculated with Puccinia striiformis.

The French wheat variety 'Camp Remy' (CR) possesses a durable, adult plant resistance to yellow rust (YR), caused by the pathogen Puccinia striiformis. Using cDNA-AFLP on different sets of heterogeneous inbred families (HIFs) derived from the cross CR x Récital, we compared gene expression profiles during one seedling and two adult developmental stages following inoculation with P. striiformis. Transcripts differentially expressed in response to YR infection were isolated and cloned. Sequence analysis of the resultant clones revealed several classes of putative genes, including those related to resistance/defence responses, transcription and signal transduction, and primary metabolism. The expression profiles of seven selected genes were obtained using real-time PCR in CR leaves at the same three stages of development. The results confirmed the stage-specific expression of the genes at one or two specific stages in response to P. striiformis infection and demonstrated that CR modifies the expression of some resistance/defence-related genes during its transition from the seedling to adult growth stages. These results provided the first clue to understand the molecular basis of quantitative trait loci for adult plant resistance to YR and connect it with durability.

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins.

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.