The Roma are a European ethnic minority threatened by several recessive diseases.MANBA cause a rare lysosomal storage disorder named beta-mannosidosis whose clinical manifestation includes deafness and mental retardation. Since 1986, only 23 patients with beta-mannosidosis and biallelic MANBA variants have been described worldwide.Variants in MANBA.We now report on further 10 beta-mannosidosis patients of Roma origin from eight families in the Czech and Slovak Republics with hearing loss, mental retardation and homozygous pathogenic variants in MANBA variant c.2158-2A>G screening among 345 anonymized normal hearing controls from Roma populations revealed a carrier/heterozygote frequency of 3.77%. This is about 925 times higher than the frequency of this variant in the gnomAD public database and classifies the c.2158-2A>G variant as a prevalent, ethnic-specific variant causing hearing loss and mental retardation in a homozygous state. The frequency of heterozygotes/carriers is similar to another pathogenic variant c.71G>A (p.W24*) in GJB2, regarded as the most frequent variant causing deafness in Roma populations.MANBA variant, is an important and previously unknown cause of hearing loss and mental retardation among Central European Roma.Beta-mannosidosis, due to a homozygous c.2158-2A>G  Beta-mannosidosis is an extremely rare metabolic storage disorder resulting from a beta-mannosidase deficiency that is involved in the degradation of glycoproteins. (MANSB MIM: 248510]). The rarity of the disease has been documented in only 23 reported affected patients from 19 families worldwide since the description of the disease in 1986 [0). The rSo far, only one family with beta-mannosidosis, confirmed by biochemical and enzymatic examination, was described in the Czech Republic in 1990 . The famBiochemical diagnostics confirmed the typical disaccharides (primarily Man (beta1 → 4) GlcNac) in the urine of affected patients and a decreased activity of the beta-mannosidase enzyme in different cells, particularly in leukocytes , 22. As MANBA gene encoding the beta-mannosidase enzyme was actually described in the same Czech Roma family in 1998 [The human The second family carrying the identical splicing variant c.2158-2A>G was recently reported in a Roma patient from Hungary who presented with hearing loss and mental retardation .Roma known formerly as Gypsies are an ethnic minority group with a high rate of endogamy resulting in a higher occurrence of specific homozygous variants and unique diseases –27. The GJB2 gene, c.71G>A (p.W24*), which is also found in India, and the other is c.1331+2T>C in MARVELD2, described originally in Pakistani families [The two most common variants known to cause hereditary hearing loss among Roma have been identified: the first is in the families .MARVELD2 is 0.5–2.24% for some European Roma groups [Both are ancestral founder variants originating in Asia before the Roma migrated to Europe. The average carrier rate of c.71G>A (p.W24*) among the European Roma ranges between 4 and 5% . The avea groups , 33.MANBA c.2158-2A>G pathogenic variant.No other frequent cause of deafness has been reported among Roma until now. Here we present a founder variant, causing the rare lysosomal storage disease beta-mannosidosis, presented with hearing loss and mental retardation in Czech and Slovak Roma patients. The detected carrier rate is 3.8% among Czech and Slovak Roma populations. In total, ten Roma patients of Central European origin were found to be homozygous for the 250 hearing-impaired Roma patients with unknown cause of hearing loss (9 Czech (CZ) and 241 Slovak (SK) patients) were examined. From Slovakia, 187 Roma hearing loss patients with unknown cause of hearing loss were collected from 102 families during the period from 2010 to 2019 and the samples originated from Ear, Nose and Throat departments, schools for hearing – impaired children and from isolated Roma communities. An additional 54 Roma patient samples from Slovakia were collected at schools for hearing-impaired children as part of an earlier research project [Altogether  project .GJB2 (NM_004004.5) exon 2 and 187 of them for the variant c.1331+2T>C (intron 4) in MARVELD2 (NM_001038603.2); no biallelic variants were detected.All hearing loss patients were previously tested by Sanger sequencing of the 59 hearing loss Slovak Roma patients from 39 families, with already known causal biallelic variants in other deafness genes, were also tested for the potential presence of the MANBA c.2158-2A>G pathogenic variant.An additional group of In order to detect the cause of their hearing loss, all subjects, or their legal representatives, gave informed consent for the genetic examination.MANBA variant c.2158-2A>G among hearing Roma population, 345 Roma samples originated from two cohorts were selected. The first cohort comprised 132 Czech unrelated Roma samples without hearing loss and was selected from families collected with the following diagnoses: congenital myasthenic syndrome; hereditary motor and sensory neuropathy type Lom and Russe; and anonymized. The second cohort of 213 anonymized unrelated Slovak Roma samples without hearing loss came mainly from isolated Roma settlements in different parts of Slovakia.To examine the carrier rate (frequency of heterozygotes) for the pathogenic Whole exome sequencing was performed in nine unrelated Czech patients with SureSelect Human all Exon XT v6 kit on Illumina Hiseq4000. The raw fastq data were analysed with SureCall  and vcf was uploaded to Ingenuity Variant Analysis  for annotation . First, MANBA gene (NM_005908.3) was examined.In the group of 241 Slovak Roma patients with unknown hearing loss and in the 59 Slovak Roma patients with other known causes of hearing loss, the real time PCR allelic discrimination TaqMan Assay  for c.2158-2A>G variant in MANBA gene was tested with custom designed probes for real time PCR allelic discrimination TaqMan Assay .In controls, the variant c.2158-2A>G in MANBA variant c.2158-2A>G detected by WES or real time PCR was confirmed by Sanger sequencing of exon 16 and an adjacent intronic part of the MANBA gene. In all detected hearing loss heterozygotes for the MANBA variant, all 17 coding exons of the MANBA gene were Sanger sequenced in order to exclude a second possible pathogenic variant.The presence of the The clinical investigations were made up from findings sent by the referring physicians. The examination of hearing loss was carried out by various methods (see the results section), with respect to the subjects age and their limited ability to cooperate during the examination.MANBA gene. Interestingly, one additional homozygote for MANBA c.2158-2A>G was also detected in a group of 59 Roma patients with an already clarified cause of hearing loss, namely in a homozygote for GJB2 c.71G>A (p.W24*) variant. The MANBA c.2158-2A>G variant in homozygous patients with hearing loss was also found to cause moderate mental retardation, in line with findings in previously reported beta-mannosidosis patients.Among the 250 Roma patients with an as yet unclarified cause of hereditary hearing loss, nine patients from seven families were found to be homozygous for the pathogenic variant c.2158-2A>G in the MANBA in all three patients. No other variant either previously reported or classified as pathogenic/likely pathogenic could explain the cause of hearing loss in these patients.In the initial three patients from three different Czech families  of 3.79%. In the group of 213 Slovak Roma controls without hearing loss, eight heterozygotes and no homozygotes were detected with an allele frequency of 1.88% and a carrier rate (frequency of heterozygotes) of 3.76%  and independent metabolic disease screening followed by biochemical examination confirmed the disease in her older brother (CZ1 - II/8) long before a genetic examination. In both siblings delayed motor milestones were reported. Both started to walk independently at two years and say their first words at four and a half. Table . The boyIn family CZ2, four siblings are affected by hearing loss, but only one (CZ2 - II/4) was available for genetic testing Fig. . PrelingHearing loss in patient (II/2) from family CZ3 was detected at three years of age and is classified as moderate . First neurological examination at the age of 7 revealed mental retardation and patient is in dispensary care for mentally-retarded patients.In family SK4 with confirmed consanguinity between the parents, at least three out of five siblings suffer from early onset sensorineural hearing loss. Their mother is mentally retarded, as is the patient (SK4 – II/1) and his younger sister though to a moderate degree. Only two subjects were available for DNA analysis. The patient (SK4 – II/1) is a homozygous boy with reported developmental and growth delay, but with a normal birth size ; recurrent respiratory and gastrointestinal infections, urinary incontinency of 3rd degree until at least six years of age, strabismus, abnormal appetite without obesity, severe to profound hearing loss, and moderate degree of mental retardation with autistic behaviour. His brother (SK4 – II/2) has moderately-severe hearing loss and thought to have mild intellectual disability without any other reported disease symptoms. The c.2158-2A>G variant was not detected in this subject. Based on available data, children from this family are dispersed among several orphanages due to the poor social-economic conditions of the parents.The 26-year-old female patient from family SK5 (II/1) has prelingual hearing loss with gradual progression based on serial audiograms and flat to gently sloping audiometric curves  has prelingual hearing loss of moderate degree based on absent otoacoustic emissions and audiometric evaluation Fig.  b. HowevMANBA variant he is also a homozygote for the c.71G>A (p.W24*) variant in GJB2. His hearing-impaired mother (SK7 – II/2) is a heterozygote for the c.2158-2A>G variant and she had several other relatives suffering from hearing loss, who were not available for genetic testing. This family case confirms and highlights the high frequency of both pathogenic variants in the Roma population.In family SK7, recruited in an isolated Roma community in eastern Slovakia (different from the previous case), we identified a mother and child affected by hearing loss  or c.-249-3818G>C in the HK1 gene (NM_033498.2) are an important causes of hereditary neuropathy. Similarly, the variant c.863 + 389C>T in the CTDP1 gene (NM_004715.4) is responsible for Congenital cataract facial dysmorphism and demyelinating neuropathy , but all these diseases are found only in the Roma population [GJB2 gene, which is responsible for a substantial number of hearing loss cases in the Roma population, and in Indian cases as well [MANBA gene belongs to this group, as the c.2158-2A>G variant also shows ethnic specificity and is prevalent in the Roma population.Due to various reasons, cultural, behavioural and social habits, the Roma population is very endogamous and may explain the various prevalent and ethnic-specific variants responsible for several different diseases that exist in this population. Some genes are known to be only affected in the Roma ethnicity. Variants c.442C>T (p.Arg148*) in the pulation , 27. Oth as well . On the  as well . We may GJB2 and the c.1331+2T>C in MARVELD2. The detected carrier frequency 3.8% for c.2158-2A>G in MANBA is very similar to the average carrier rate of c.71G>A (p.W24*) in GJB2 among the European Roma, which is in the 4–5% range [MANBA variant seems to be a more important and more frequent cause of hearing loss compared to MARVELD2. The c.1331+2T>C in MARVELD2 has an average carrier rate of 1% for Czech Roma compared to 2.24% for Slovak Roma and 0.5% for Hungarian Roma [If we focus on the most prevalent variants causing hearing loss in the Roma population, there are only two other variants known so far, the c.71G>A (p.W24*) in ian Roma , 33.MANBA variant, we have to consider the following facts: our findings show the variant c.2158-2A>G to be an important cause of hearing loss in the Czech and Slovak Roma, but not in non-Roma European populations. This suggests that the variant did not arise from an admixture with the majority European population such as with c.35delG in GJB2, but either originated in India before the Roma left, or arose among the isolated Roma minority on the European continent. According to Schrauwen et al. the multidimensional scaling analysis (MDS) and admixture analysis showed a more pronounced South Asian background for two individuals carrying the GJB2  and MANBA (c.2158-2A>G) variants, based on the similarity with individuals from the Punjab province (SAS – South Asian). Unfortunately, no patient with simultaneous MARVELD2 variant was detected in the paper and therefore it could not be added to the admixture and MDS analysis [MANBA) was detected in the SAS population, compared to 134 heterozygotes for c.71G>A (p.W24*) (GJB2) and six heterozygotes for c.1331+2T>C in MARVELD2. The almost opposite is true for the NFE  population, where two heterozygotes for c.2158-2A>G in MANBA, six heterozygotes and one homozygote for c.71G>A (p.W24*) (GJB2) and three heterozygotes for c.1331+2T>C in MARVELD2 were detected , the frequency of SAS is by 1–2 orders higher compared to NFE. Therefore, we assume that the prevalent MANBA variant arose among the Roma population only after they left India. Only the frequency investigation of this variant in individual Roma subpopulations over Europe can answer the commonality of this variant in other European countries. Moreover, the detected carrier frequency of MANBA in the Czech and Slovak Roma population is 925x higher compared to the carrier frequency in the NFE population gnomAD [If we try to trace the origin of the analysis . Howeveranalysis  gave difMANBA gene. There is probably and even greater incidence of beta-mannosidosis in the patients (8 families) included in this study, however, samples were not available for all family members with hearing loss. The most prominent and common clinical manifestations are mental retardation reported in ten patients and hearing loss mentioned in 13 patients  and objective (ASSR) methods are compared. The higher values of PTA thresholds compared to ASSR could be at least partially explained by limited cooperation due to mental retardation. Therefore, both subjective and objective methods for audiometric examination should be used in MANBA, c.71G>A in GJB2). A condition for recruitment into this study was the presence of hearing loss in Roma patients; hearing loss is a heterogeneous disease, where over 100 genes have been reported [Interestingly, in several patients with hearing loss from SK families (Fig. reported . We haveThe clinical manifestation of beta-mannosidosis is variable and genotype/phenotype correlation cannot be completely traced. Bedilu et al. tried to identify clinical signs in null variants causing beta-mannosidosis in two families . In concMANBA is another important ethnic-specific variant threatening the Roma population in Central Europe. This variant, in a homozygous state, causes the disease beta-mannosidosis and is an important and previously unknown cause of hereditary moderate to severe hearing loss in Roma patients associated with mental retardation. Based on the high frequency of carriers, we expect a higher frequency of those affected by beta-mannosidosis among the Roma. Due to the main feature of the disease, i.e. mental retardation, most of the affected subjects may suffer from an even higher degree of social isolation than the rest of the Roma minority and, with poor access to adequate healthcare, go undetected. The second important clinical feature that could be overlooked is hearing loss, especially when it is only moderate; it is a result of either poor patient compliance or a failure to associate hearing loss with this phenotype. Beta-mannosidosis is an extremely rare disease that can be detected easily, biochemically and genetically, yet there is no treatment. Due to the high frequency of disease carriers in the Roma population, we suggest offering carrier screening to interested Roma individuals. Ultimately, we recommend routine genetic testing, at least in Central Europe, of this ethnically-specific pathogenic variant in all Roma with unexplained hearing loss and mental retardation.The detected c.2158-2A>G variant in Additional file 1. Supplementary file – table 1_ genes included in the virtual gene panel for early/prelingual hearing loss.


TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N-methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N-methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of  Tea plant roots have stronger degradation activity on both caffeine and theophylline. Caffeine feeding to tea plant shoot tips and roots resulted in feedback inhibitions in the biosynthetic gene expression and accumulation of theobromine, whereas theophylline feeding resulted in both caffeine biosynthesis and theophylline degradation through 3-methylxanthine. Both metabolite profiling, transcriptome analysis, and enzyme characterization supported the interconversions among these xanthine alkaloids in a complex metabolic network.Camellia sinensis (L.) O. Kuntze] and owns diverse and pleasant flavors and health benefits. The tea flavors and health functions are mostly attributable to several characteristic secondary metabolites, including catechins, caffeine, theanine, and terpenoids in tea plant leaves , averagely 35 mg/g in dry mass basis , and then further methylated at 1-N into caffeine (N-methyltransferases (NMTs), including xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT), and tea caffeine synthase (TCS), which all use S-adenosyl-L-methionine (SAM) as a methyl donor  → adenine → AMP → IMP → XMP → xanthosine pathway; NAD route using the AMP (degraded from NAD by NAD pyrophosphatase) to synthesize xanthosine; and GMP route by a GMP → guanosine → xanthosine pathway  or 7-methylxanthine methyltransferase (MXMT) converting 7-methylxanthine to theobromine (TCS1a–f) and TCS1a is the primary allele among section Thea plants, ICS1 and PCS1 are TCS1b and TCS1c respectively  enzyme present in tea plants and 1-N and 3-N MT loci were segregated. However, little is known about them  is a key enzyme involved in caffeine biosynthesis pathway by catalyzing 3-on steps . PCS1 anobromine . Structuobromine . Four hiobromine . The amiobromine  and aminobromine . TCS1 haectively . Based oarieties . Howeverout them .2 + NH3 + glyoxylic acid via urea and ureidoglycolic acid  and roots of hydroponically cultured tea cuttage seedlings. We then also conducted degradation experiments with these shoot tips and roots absorbed and accumulated purine alkaloids to high concentrations. By monitoring purine alkaloid metabolites and transcriptomic analyses of shoot tips and roots fed with these purine alkaloids, we observed strong feedback inhibition on caffeine synthesis and inter-conversion between caffeine, theophylline, theobromine, and 3-methylxanthine. This work provides special insight on the understanding of caffeine biosynthesis and catabolism in tea plants. It also lays a foundation for further study of the catabolism of xanthine alkaloids at molecular levels.Camellia sinensis L., cv. Baihaozao) were plucked from the experimental field of Anhui Agricultural University at April in Hefei, China , two treatments to tea plant shoot tips were that feeding them with 15 mM caffeine and 15 mM theophylline respectively. After feeding for 48 h, shoot tips were rinsed, removed the surface water and transferred to a chamber with high humidity (90%–95%) under light at room temperature for examination of caffeine or theophylline degradation inside the shoot tips.Shoot tips  of tea plant  attained from a tea garden of Tangchi, Hefei, China  were cultured by Shigeki Konishi (SK) solution in greenhouse with day and night temperatures of 26°C and relative humidity of 60%, the SK solution was updated once a week. Some tea plants were fed with 3 mM theobromine totally 72 h for examination of its metabolism in roots. Some tea plants were also fed with 15 mM caffeine and 15 mM theophylline independently, after feeding for 72 h, roots were cutted, rinsed, removed the surface water and transferred to a chamber with high humidity (90%–95%) under dark at room temperature, which was considered as the beginning of caffeine or theophylline degradation in roots.Two-year-old tea cuttage seedlings  powder was extracted with 1.5 ml 80% (v) methanol in water and sonicated for 30 min at 12°C. All samples were mixed up and down every 10 min and centrifuged at 10,000 rpm for 10 min, then the supernatants were filtered through 0.22 µm membranes before UPLC analysis .Xanthine alkaloids in the extracts were measured using an ACQUITY UPLC H-Class instrument  and separated on a ACQUITY UPLC BEH C18 Column  at a flow rate of 0.3 ml/min at 35°C with 1 μl sample volume, the mobile phase was selected as 0.2% (v) acetic acid in water (A), and methanol (B), and the gradient elution was as follows: B 6% from 0 to 2 min, to 8% at 3.5 min, to 10% at 4.5 min, to 18% at 7.5 min, to 26% at 10.5 min, to 80% at 13.5 min and kept at 80% for 1 min, to 6% at 16 min and kept at 6% for 1 min, the detection wavelength was set to 278 nm . XanthinShoot tip and root samples were also used for RNA-Seq analyses. Total RNAs were extracted by using RNA extraction and purification kit  according to the manufacturer’s protocols. RNA integrity and concentration were detected by Agilent 2100 Bioanalyzer . cDNA libraries construction and RNA-Seq performed on an Illumina HiSeq 4000 sequencing platform were carried out professionally by BGI .http://www.ccb.jhu.edu/software/hisat) was used to map the clean reads to the tea plant genome (http://cpc.cbi.pku.edu.cn) was used to predict the protein-coding potential of the novel transcripts.Hierarchical indexing for spliced alignment of transcripts . Differentially expressed gene (DEG) was analyzed by using DEGseq method based on Poisson distribution . In orde™ RT Master Mix kit . QRT-PCR was performed in a 20 μl volume containing 7.6 μl ddH2O, 0.8 μl forward primer, 0.8 μl reverse primer, 0.8 μl cDNA, and 10 μl TB Green®Premix Ex Taq™ II . The tea β-actin gene was used as an internal reference gene (GenBank: HQ420251.1), its forward primer and reverse primer (both from 5’ end to 3’ end) were designed as GCCATCTTTGATTGGAATGG and GGTGCCACAACCTTGATCTT respectively. Other qRT-PCR primers were designed by Primer Premier 6.0 software  and listed in ™ Real-Time System . The cycling profile was 95°C, 30 s; 40 cycles of 95°C, 5 s and 60°C, 30 s. Relative gene expression was calculated with the 2-ΔΔCT method  was used to search for all structural genes involved in caffeine biosynthesis pathways with characterised homologue protein sequences from other plants as queries. Unrooted phylogenetic trees were constructed following the Neighbour-Joining method involving 500 replicates with the bootstrap test in MEGA 7.0  genes used for building the phylogenetic tree (TCS-1 to TCS-8) from tea plant genome CSS  were from various tea plant varieties (MXMT-2 to MXMT-7 genes from tea plant genome (XMT-1 to XMT-13) genes from tea plant genome (O-methyltransferase genes from tea plant genome (Coffea arabica xanthosine methyltransferase (CaXMT1), and 11 Camellia MT genes published previously.Among 49 nome CSS , six allarieties , six MXMt genome , 13 xantt genome , four int genome , which aTCS-4 and TCS-8 were cloned from the cDNA of tea plant  buds and ligated into pGEX-4T-1 vector which could induce the synthesis of GST (Glutathione S-transferase)-fusion proteins. The forward and reverse primers were designed with an EcoRI and a NotI restriction site at their 5’ ends respectively and are shown in E. coli Transetta (DE3) chemically competent cell . The positive clones were chosen and shaken in 3 ml LB (Luria-Bertani) medium with 50 μg/ml ampicillin for 12 h at 37°C, then 500 μl E. coli cultures were added into 50 ml LB medium with 50 μg/ml ampicillin and shaken at 200 rpm and 37°C till OD600 value were between 0.6 and 0.8, then 0.5 mM final concentration IPTG  was added to induce the expression of these two genes at 16°C, 200 rpm. For SDS-PAGE analysis, 5 μg crude proteins from uninduced cells and induced cells were boiled with loading buffer and subjected to 10% SDS-PAGE.The open reading frames (ORFs) of E. coli cultures individually, the concentrations of the substrates are all 1 mM. Then the cultures were incubated continually at 16°C for 20 h, after that they were incubated at 30°C for appropriate time. E. coli cultures were sampled at various time points and centrifuged at 10,000 rpm for 10 min, and supernatants were filtered with 0.22 μm membranes before the detection of xanthine alkaloids using above-mentioned UPLC method.After induced at 16°C for 4 h, eight substrates  were added to Heatmaps were constructed with the R package. Multiple sequences alignment analysis was performed using the ClustalW program in the software MEGA 7.0. Phylogenetic trees were constructed for caffeine synthetic enzymes with homologue protein sequences using the NJ (Neighbor-Joining) statistical method in MEGA 7.0, with parameters of bootstrap method (500 replications), p-distance model, Uniform rates, and Partial deletion (50% site coverage cutoff).All the data presented in this study were obtained from at least three independent biological replicates, including xanthine alkaloids feeding experiments, metabolite profiling, RNA-Seq, and qRT-PCR analysis. Statistical analyses were conducted using the Minitab 16.0 statistical software package . Data were analyzed by one-way analysis of variance (ANOVA) and a Fisher’s least significant difference (LSD) test at the 5% level.Camellia sinensis L., cv. Baihaozao) contain about 35.87 mg/g caffeine (on dry mass basis). After 72 h of feeding, the caffeine contents in shoot tips increased to 109 mg/g, indicating the caffeine was taken up by shoot tips  with Shigeki Konishi (SK) solution containing 15 mM caffeine. These shoot tips from tea plants   was almost not detected, however, it increased to 47.85 mg/g after being fed with theophylline for 72 h , including xanthosine methyltransferases (XMT), 7-methylxanthine methyltransferases (MXMT), and tea caffeine synthases (TCS), which all use S-adenosyl-L-methionine (SAM) as a methyl donor, were also identified in tea genome  and ICS2 (BAE79731.1), the other 47 MT genes were simply classified to five groups  (Camellia sinensis xanthine dehydrogenase (CsXDH), urate oxidase (CsUOX), allantoinase (CsALN) and urease (CsURE) are all multi-gene families  in the shoot tips and roots fed with caffeine or theophylline with their controls revealed a large number of genes were altered in their expression levels significantly  and tea caffeine synthase (TCS) catalyze the last two MXMTs and TCSs in tea plant shoot tips are much higher than in roots. Except for MXMT-7 (TEA030024.1), the transcripts of all other MXMTs and TCSs in shoot tips were significantly down-regulated upon caffeine or theophylline feeding for 48 h. These transcripts of MXMTs and TCSs continuously decreased in caffeine- or theophylline-fed shoot tips after set for caffeine or theophylline degradation for 3 days (TCS-6 (TEA022575.1) which was not changed drastically, other five TCS genes were all down-regulated significantly in caffeine degradation shoot tips for 3 days as compared with caffeine degradation for 0 day. These results indicated that feeding with high concentrations of caffeine or theophylline could feedback repress the expression of the most MXMTs and TCSs. Thus from both metabolite profiling and two key gene expression patterns, it is posited that caffeine or theophylline feeding inhibited caffeine synthesis through a negative feedback in tea plant shoot tips.Expression levels of TCS-4 (TEA015791.1) transcript level increased significantly by about 2.5-fold and decreased by about 2-fold in tea plant roots after fed with caffeine for 12 and 48 h, respectively, as compared with that of control (feeding for 0 h) (TCS-4 and TCS-8 (TEA028050.1) are the most abundant TCS genes in tea plant shoot tips, both genes were expressed by 3.81 and 3.04 times lower, respectively, in caffeine-fed shoot tips for 48 h than in normal shoot tips. In addition, these two major TCS genes were also down-regulated in tea plant shoot tips fed with theophylline for 48 h, by 3.19 and 2.61 times, respectively, compared with these in non-fed control shoot tips. These data indicated that caffeine feeding to shoot tips drastically repressed major TCS genes more severely than theophylline feeding did (MXMT-2 (TEA010123.1), MXMT-3 (TEA012581.1), MXMT-4 (TEA028049.1), and MXMT-5 (TEA028051.1) were all down-regulated significantly in shoot tips by caffeine feeding for 48 h compared with non-fed control (feeding for 0 h), also in caffeine degradation shoot tips for 3 days as compared with caffeine degradation for 0 day. Except for MXMT-2, which was not changed significantly in theophylline degradation in shoot tips for 3 days compared with theophylline feeding for 48 h, these observations were also true when caffeine was replaced with theophylline at the same concentration for feeding or degradation experiments (XMT-10 (TEA021081.1) and XMT-13 (TEA021334.1) were down-regulated significantly after caffeine or theophylline feeding for 48 h in shoot tips compared with feeding for 0 h. By contrast, XMT-5 (TEA010250.1), XMT-7 (TEA014697.1), and XMT-9 (TEA014705.1) were significantly up-regulated  was downregulated significantly after caffeine feeding for 48 h compared with feeding for 0 h, and its expression in shoot tips fed with caffeine for 48 h was 1.7 times higher than that in caffeine degradation for 3 days (IMPDH-1 (TEA015896.1) and IMPDH-2 (TEA024175.1), and IMPDH-2 expression is significantly higher than IMPDH-1 in control tea plant shoot tips and roots. IMPDH-1 expression in shoot tips decreased gradually during caffeine or theophylline feeding and degradation, while IMPDH-2 expression increased a little at caffeine or theophylline degradation for 3 days compared with caffeine or theophylline feeding for 48 h (S-adenosyl-L-methionine) is known as the important methyl donor during the methylations of caffeine synthesis, and SAM synthetase (SAMS) catalyzes the conversion of methionine to SAM. SAMS-1 (TEA006735.1), SAMS-2 (TEA012514.1), SAMS-4 (TEA020895.1) and SAMS-5 (TEA026890.1) expressions all decreased significantly after caffeine or theophylline feeding for 48 h in shoot tips compared with the control (caffeine or theophylline feeding for 0 h) (S-adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the conversion of SAH (S-adenosyl-L-homocysteine) to homocysteine in SAM cycle pathway, and releasing adenosine in this step which may participate in caffeine biosynthesis as is referred to in ‘introduction’ part. SAHH-1 (TEA006264.1), SAHH-2 (TEA008283.1), and SAHH-4 (BGI_novel_G002551) expressions decreased significantly after theophylline feeding for 48 h (SAM (ADK-1 (TEA001315.1) expression is the most abundant in control shoot tips and it decreased significantly after caffeine or theophylline feeding for 48 h (5’-Nase-1 (TEA000333.1) and 5’-Nase-11 (TEA002062.1) are the most abundant in control shoot tips, which are both downregulated after caffeine or theophylline feeding for 48 h in shoot tips compared with the control (feeding for 0 h) (APRT-1 (TEA007061.1), APRT-2 (TEA015248.1), APRT-4 (TEA017916.1), and APRT-6 (TEA022940.1) decreased significantly after caffeine or theophylline feeding for 48 h in shoot tips compared with the control (feeding for 0 h) (Adenosine kinase (ADK) catalyzes the conversion of adenosine to AMP. TCS, MXMT, XMT, 5’-Nase, ADK, SAMS, and SAHH significantly decreased in shoot tips after fed with caffeine or theophylline for 48 h (SAMS-4 (TEA020895.1) expression had an increase from theophylline feeding for 48 h to theophylline degradation for 3 days rather than decrease as presented in transcriptomic data, other gene expression changes from caffeine or theophylline feeding for 48 h to caffeine or theophylline degradation for 3 days in tea plant shoot tips were consistent with the transcriptomic data and these genes expressions all decreased during this period. These findings in qRT-PCR could further verify that caffeine or theophylline feeding to tea plant shoot tips significantly inhibited gene expression involved in caffeine biosynthesis pathway and SAM cycle pathway, the latter is crucial to synthesize the methyl donor that is SAM, thus caffeine or theophylline feeding had stimulated a regulation of negative feedback to caffeine biosynthesis in tea plant shoot tips.To validate the transcriptomic data in our research, relative expressions of 12 selected genes in shoot tips during caffeine or theophylline feeding and degradation were analyzed by qRT-PCR. These genes included nine structural genes involved in caffeine biosynthesis pathway and three genes involved in SAM cycle pathway. Consistent with the transcriptomic datasets, the expression of TCS-4 (TEA015791.1) and TCS-8 (TEA028050.1) are the two MT genes with the most abundant gene expression in tea plant shoot tips. As elucidated above, the expression levels of TCS-4 and TCS-8 decreased significantly in shoot tips fed with caffeine or theophylline, suggesting that these two genes played a crucial role in caffeine biosynthesis. We thus investigated the functions of TCS-4 and TCS-8. The GST (Glutathione S-transferase) tagged TCS-4 and TCS-8 were expressed in E. coli, using TCS1 (GenBank: AB031280.1) gene in the same tags expressed in E. coli as a positive control.E. coli and partial purification were performed, and results showed that GST-TCS-4 and GST-TCS-8 fusion proteins were ~ 68 KDa, including ~ 42 KDa TCS-4 or TCS-8 plus 26 KDa of GST tag, suggesting that these fusion proteins were clearly expressed and partially purified  to theobromine, 1mX (1-methylxanthine) to theophylline, or paraxanthine  to caffeine, as well as 1-N-methyltransferase activity that converted 3mX (3-methylxanthine) to theophylline or theobromine to caffeine , TCS-8 (TEA028050.1), MXMT-5 (TEA028051.1), and MXMT-3 (TEA012581.1). Evidence is also shown that theophylline feeding resulted in faster degradation into 3mX, which was accumulated to the higher levels in theophylline-fed shoot tips than caffeine-fed ones  , expression levels of most TCS and MXMT genes continuously decreased from the beginning of feeding to 48 h after feeding of caffeine and theophylline, to degradation for 3 days.While shoot tips had higher capacity to synthesize and accumulate more caffeine, theobromine, fed with 15 mM caffeine or theophylline caused much significant feedback inhibition effects than roots did. This was mainly reflected by more significantly repressed TCS and MXMT gene expression. Furthermore, such repression of TCS genes, TCS-4 (TEA015791.1) and TCS-8 (TEA028050.1) were the most highly expressed ones in shoot tips, apical buds, and young leaves. Compared with un-fed shoot tips, these two genes in caffeine-fed shoot tips were repressed by about 3.8- and 3-fold, respectively, at 48 h after feeding. TCS-4 and TCS-8 expression levels in theophylline-fed shoot tips were reduced by 3.2- and 2.6-fold, respectively, at 48 h after feeding. On the contrary, compared with that in non-fed control roots, TCS-4 expression in caffeine-fed roots even increased by 2.5-fold at 12 h after feeding, and then decreased by 2-fold at 48 h after feeding. TCS-8 was also repressed slightly in roots fed with caffeine or theophylline.For examples, two As caffeine is relatively stable in tea plant shoot tips, many green teas contain high levels of caffeine, and cause sleeping problems to tea drinkers. Consistent with previous studies , caffeinvia two mechanisms: demethylation and oxidation: the major demethylation metabolite formed in fungi is theophylline whereas theobromine is the major demethylation metabolite in bacteria , the National Key Research and Development Program of China (2018YFD1000601), the Changjiang Scholars and Innovative Research Team in University (IRT_15R01), and the Key Research and Development (R&D) Program of Anhui Province (18030701155).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Research using the UK Biobank data has shown ethnic inequalities in hearing health; however, the hearing test used may exhibit a disadvantage for non-native language speakers.To validate the results of the UK Biobank hearing test  against self-reported measures of hearing in the dataset and create classifications of hearing health. To observe if language proficiency and migration age have the same effect on hearing health classification as on the DTT in isolation. Our hypothesis is that language proficiency acts differently on the DTT, demonstrating that the DTT is biased for non-native speakers of English.Latent classes representing profiles of hearing health were identified from the available hearing measures. Factors associated with class membership were tested using multinomial logistic regression models. Ethnicity was defined as (1) White, native English-speaking, (2) ethnic minority, arrived in the UK aged <12 or (3) ethnic minority, arrived aged >12.The UK Biobank participants with valid hearing test results and associated covariates (N=151 268).DTT score, self-reported hearing difficulty, self-reported hearing difficulty in noise and hearing aid use.Three classes of hearing health were found: ‘normal’, ‘generally poor’ and ‘only subjectively poor’. In a model adjusting for known confounders of hearing loss, a poor or insufficient hearing test result was less likely for those with better language  or numerical ability  but more likely for those having migrated aged >12 .The DTT showed evidence of bias, having greater dependence on language ability and migration age than other hearing indicators. Designers of future surveys and hearing screening applications may wish to consider the limitations of speech-in-noise tests in evaluating hearing acuity for populations that include non-native speakers. To our knowledge, the first study comparing self-reported and psychophysical hearing outcomes for ethnic minorities using the UK Biobank data.Potential language bias in the UK Biobank’s hearing test was investigated for the first time.All available hearing outcomes from the UK Biobank were combined into three latent classes; the behaviour of these classes was substantively consistent with the literature.This study uses the available language proficiency data in the UK Biobank, which is limited.Hearing impairment is one of the most common chronic age-related health conditionset al while investigating the correlates of hearing loss using the UK Biobank data.6This research study is concerned with the ethnic inequality observed by Dawes Hearing acuity in the UK Biobank is measured using the Digit Triplet Test (DTT), a psychophysical test of speech recognition that involves perception of three spoken digits. Other UK health surveys collect hearing data using methods based on pure-tone audiometry (PTA),11Given the potential conflation of language ability and hearing acuity in the DTT, the present study divides the ethnic minority cohort into those likely to have native-level English proficiency, and those who migrated to the UK as an adolescent or older. It is hypothesised that, correcting for respondents’ self-reported hearing health and related factors, DTT scores for the older-migrating cohort will be worse due to their likely lower language proficiency.The present research therefore aims to validate the results of the DTT against three self-reported measures of hearing provided in the UK Biobank. These measures will be used to create classifications of hearing health. We will see if language proficiency and migration age have the same effect on the hearing health classifications as for the DTT in isolation.Our hypothesis is that the DTT is biased for non-native speakers of English. If the DTT is not biased, it would be expected that language test scores would have the same effect across every measure of hearing in the dataset. A different outcome would indicate a dependency on language ability. If, after controlling for language ability, the DTT still shows poorer outcomes for the ethnic minority groups, it can be concluded that inequalities are present regardless of any language bias.The study population was participants in the UK Biobank, a biomedical study of over 500 000 adults aged 40–69 years recruited in the UK from 2006 to 2010.13Four measures of hearing health were used to form the hearing health classifications: the DTT, two self-reported measures of hearing difficulty and self-reported hearing aid use. All data, including the psychophysical measure, were collected in a self-administered manner via the touchscreen questionnaire undertaken in Biobank test centres.et al.As a speech-in-noise test, the DTT was devised to detect impaired speech intelligibility in noisy conditions,Yes/No/I am completely deaf /Do not know/Prefer not to answer), and ‘Do you find it difficult to follow a conversation if there is background noise ?’ (Yes/No/Do not know/Prefer not to answer). The completely deaf category was amalgamated with the ‘Yes’ category. Respondents were asked if they currently use a hearing aid ‘most of the time’ (Yes/No/Prefer not to answer). Participants responding ‘Do not know’ or ‘Prefer not to answer’ to any of the hearing questions were excluded from the dataset.For the self-reported measures, respondents were asked: ‘Do you have any difficulty with your hearing?’ ; (2) ‘Bud is to Flower as Child is to?’ (Grow/Develop/Improve/Adult/Old). Given the association between hearing loss and cognitive performance,Language proficiency was evaluated according to two language-based questions included in the UK Biobank’s Fluid Intelligence (FI) assessment: (1) ‘Stop means the same as?’  was initially used to combine all four hearing health measures into a single latent dimension. This model exhibited unusual behaviour by way of inflated coefficients for factor variables. This unreliability suggested that latent hearing health was not well characterised by a single dimension; that is, self-reported hearing difficulties may not always correlate well with psychophysical measures of hearing acuity. This inconsistency could also have been due to external factors that act differently on each measure, for example, propensity to report health problems, or bias in the hearing test for certain groups.Latent Class Analysis was employed to overcome the constraints of an LFA by allowing for the existence of arbitrarily different ‘classes’ of latent hearing health. A latent construct of hearing health was formed using the four hearing measures. The suitability of this construct as a proxy to understand the latent trait of hearing health was evaluated by studying its association with known correlates of hearing loss. Analyses were performed in MPlus V.8 using the three-step approach to correct for classification uncertainty in the latent classes.31Although the UK Biobank participants were involved in the design of the biobank resource itself, neither they, or any member of the public, were involved in this secondary analysis.A total of 165 306 participants provided valid hearing test data, reducing to 151 268 after the removal of cases having missing data . Table 110.1136/bmjopen-2020-042571.supp2Supplementary dataA combination of exploratory and confirmatory approaches was used to determine the properties of the hearing health latent classifications. It was expected that there would be a particular group who exhibited insufficient or poor performance on the DTT, but did not self-report hearing problems or use a hearing aid. To test this, three-class and four-class confirmatory models were created with this group specified; other group thresholds were unconstrained, allowing the model to determine their properties. However, this class did not fit well with the data, having very low membership in both models.Accordingly, an exploratory approach was revisited: unconstrained models with two to seven classes were created, and the model with best fit was selected. A mixture of Bayesian Information Criterion (BIC) and Bootstrapped Likelihood Ratio Test (BLRT) is recommended for determining the optimum number of classes in a latent class analysis10.1136/bmjopen-2020-042571.supp4Supplementary data‘Normal’ : characterised by low levels of hearing aid use, poor/insufficient hearing on the DTT or reporting hearing difficulty more generally.‘Generally poor’ : those who reported hearing difficulties, the highest levels of poor/insufficient DTT performance and used hearing aids.‘Only subjectively poor’ : those who reported hearing difficulties but had a low probability of having poor/insufficient hearing on the DTT.A multinomial logistic regression was performed using class membership as the dependent variable. The latent classes were regressed on a range of demographic, socioeconomic and health-related predictors, as well as known biases in the dataset. Uncertainty in the latent class classification was compensated for using the manual ‘three-step’ procedure.10.1136/bmjopen-2020-042571.supp5Supplementary data10.1136/bmjopen-2020-042571.supp6Supplementary dataThe latent class regression models described above assumed complete mediation of the relationship between the observed predictor variables and the observed hearing health latent class indicators by the latent classes. This assumption did not tally with our hypotheses, which assumed that responses to the DTT in particular would be different for participants with different values on variables hypothesised to be related to language skills  and known biases (whether tested in a mobile centre). We therefore expanded the latent class regression models to include direct, non-mediated effects of these predictors on the DTT outcome in particular. These expanded models were similar in structure to psychometric models of (uniform) ‘differential item functioning’, with the difference that in psychometrics the latent mediator variable is usually a continuously distributed latent factor, whereas in our models it was a discrete latent class distribution. A non-zero estimate for these direct effects would indicate bias, whereby responses to the DTT variable were affected not only by hearing health, but also by other characteristics of the participant, such as language skill and ethnicity. Without the DTT direct effects, the BME NEEM group had significantly lower odds  of being in the generally poor compared with the normal hearing group; however, these odds were no longer significant  once direct effects were introduced. Higher numeric and language scores caused a significant reduction in the likelihood of having poor or insufficient hearing on the DTT, but did not have a significant impact on group membership. The later-migrating BME group were much more likely than the White NE group to have insufficient or poor hearing .et alThis study further investigated the ethnic differences in hearing health observed by Dawes By dividing the ethnic minority cohort according to migration age, the present research reveals intergenerational differences in DTT outcomes. As hypothesised, it is the later-migrating BME ethnic minority group who bear the burden of poorer hearing outcomes: the UK-born/early-migrating BME cohort had DTT results that were indistinguishable from the White NE cohort.Aside from the DTT, the LFA was constructed using self-reported measures, which are thought to be reliable measures of hearing health. This construct indicates that ethnic minority groups are most likely to have normal hearing. However, even after correcting for hypothesised sources of bias, the results from the DTT suggest poorer hearing outcomes for the later-migrating BME group. This is consistent with literature showing ethnic inequalities in other physical and mental health outcomes,33The DTT showed evidence of language bias, having greater dependence on language ability and migration age than other hearing indicators. The self-report questions did not appear to demonstrate a strong linguistic bias, despite being posed in English. This may mean that the concept of hearing difficulties is reasonably consistently understood, and not prone to misinterpretation due to language barriers.A secondary finding of this study regards mobile test centres, which caused the largest bias in the DTT, possibly due to elevated background noise levels in the test centres. Respondents who were tested at these centres were 5.52 times (95% CI 4.99 to 6.10) more likely to have poor/insufficient hearing on the DTT, but were not more likely to have any particular hearing classification. It is also of note that those who did not take the cognitive tests had worse DTT scores. It may be that a cognitive or language difficulty precluded participants from taking, or finishing, these tests.The LFA produced classes that were substantively consistent with the literature. The likelihood of being in the generally poor hearing group was increased by exposure to noise and most of the health conditions known to affect hearing. The strongest indicator for the only subjectively poor hearing group was exposure to noise, which would appear consistent with a cohort who are concerned about their hearing. Although it may seem unusual that this cohort would perform well on a speech recognition test yet report hearing difficulties in daily life, such phenomena are not uncommon in the literature; explanations include audiometric notches,36One limitation to this study regards language measures: proficiency was determined by responses to just two questions, so may be imprecise. Despite this, the measures are still able to provide evidence for the hypothesis of DTT bias, as the LFA outcomes did not exhibit any dependency on language. It is important to note that the DTT is a test of sensorineural hearing loss, detecting only hearing loss of cochlear origin. The DTT, being a test of signal-to-noise ratio, allows conductive losses to be overcome by increasing the volume of the stimuli, which participants were permitted to do in the UK Biobank test. No other hearing tests were conducted; as such, the UK Biobank does not contain any data on conductive pathologies, otoscopy findings or middle ear function measures.It must also be noted that the BME classes contain the ‘White other’ group, which may include participants from Western Europe who are not generally designated as minority ethnic. Regarding generalisability, the UK Biobank, while not employing a representative sample, is thought to be generalisable for disease-exposure relationships.37The authors of the Biobank study did not explicitly state why the DTT was used over the pure-tone average method, but the available information points to practical factors, such the ‘minimal impact on throughput’ by incorporating the test with the existing touchscreen questionnaire,Even after accounting for this bias, the results suggest that there are still ethnic inequalities in hearing health, although with contradiction between psychophysical and self-reported outcomes. Designers of future studies must take precautions to ensure that this inequality can be reliably analysed by ensuring that hearing test measures are not language dependent. The present study may also have implications for hearing screening applications, which many countries currently implement using the DTT.10.1136/bmjopen-2020-042571.supp3Supplementary data10.1136/bmjopen-2020-042571.supp7Supplementary data


Ganoderma lucidum originating from boreal forests. The substrates tested included sawdust and wood chips of Betula spp., Populus tremula, Picea abies, Pinus sylvestris and Larix sp. The suitability of the substrates and the ability of the strains to develop fruiting bodies and produce β-glucan were evaluated. Fruiting body formation was supported by applying two different cold shock treatments to substrate bags. The highest yields were observed with MUS192 strain and Betula spp. and P. tremula wood-based substrates. β-Glucan content in the fruiting bodies was highest with the MUS75 and P. tremula wood-based substrate. Based on these findings, the combination of P. tremula wood residues and the MUS192 strain is proposed to enhance the yield and β-glucan content of the fruiting bodies. A cold treatment of 5 °C is suggested to induce primordia formation and to increase fruiting probability. This is the first time that strains of G. lucidum originating from boreal forests have been compared and successfully cultivated simulating commercial indoor cultivation.Wood residues from forestry industries can be potential raw materials for specialty and edible mushroom production. The aim of this study was to evaluate the suitability of wood residues for the cultivation of  Pinus sylvestris and Picea abies) and birch (Betula pendula and Betula pubescens) [Populus tremula and Larix sp. The high availability of these resources along with new European policies towards a circular economy [High volumes of wood-based side streams are generated from wood processing industries in Europe. In Finland, sawmill and plywood industries alone produced in 2016 over 1.2 million cubic meters of side streams [bescens)  are used economy  have pusWood side streams have been found to be a potential raw material for specialty and edible mushroom production . MushrooGanoderma is a popular genus including pathogenic and wood-degrading species that have been studied in regard to their bioactivity [Ganoderma lucidum [G. lucidum has been cultivated in a wide variety of substrates including sawdust from Acacia sp., Alnus sp., Carpinus sp., Dalbergia sp., Dipterocarpus sp., Fagus sp., Pinus sp., Populus sp., Quercus sp. and Swietenia sp., among others [Ganoderma contains several compounds that define the bioactive and pharmacological properties of the fungus such as antiviral [Ganoderma genus [β-glucan is a dominant structural polysaccharide that defines the shape and rigidity of the cell, serving as a reservoir of carbohydrates [β-glucan has numerous applications in the pharmaceutical and food industries [Among cultivated fungal species, activity . The mos lucidum . G. lucig others ,15,16,17ntiviral , immunomntiviral . Polysacma genus . Among thydrates . This pohydrates . Due to dustries . To suppdustries ,25,26,27Ganoderma lucidum’ has been used to name the traditionally cultivated Reishi or Ling-Zhi mushroom [Ganoderma (P. Karst.) was described by Karsten [G. lucidum (W. Curtis) Karst. into the new genus. Curtis, the author of the epitheton, based his description on a specimen from the UK [G. lucidum’ differs from the originally described species from Europe [In Asian countries, ‘mushroom . Howevermushroom ,29,30. T Karsten , combinim the UK . Since tm the UK ,34. Recem Europe ,29,30.Ganoderma lucidum is naturally distributed in Finnish boreal forests. Its distribution is sporadic and it is commonly found in coastal areas [G. lucidum has been found in stumps of Alnus glutinosa, Betula sp., P. abies, Quercus robur and P. sylvestris [G. lucidum in Finland is still in its infancy. An attempt to cultivate Finnish strains of G. lucidum in vitro using wood residues has been carried [G. lucidum fruiting bodies and β-glucan using strains originating from boreal forests has not been investigated.al areas , but theal areas . In Finllvestris . Current carried . HoweverG. lucidum strains originating from boreal forests. The aim is to find the most suitable strain, wood substrate and cold treatment for the production of G. lucidum fruiting bodies and β-glucan. The strain selection and cold shock treatment were critical for successful fruiting. The wood substrate had the strongest effect on the yield and β-glucan content of the fruiting bodies. Betula spp. and Populus tremula wood side streams were found the most suitable substrates to enhance the yield of G. lucidum fruiting bodies. The highest content of β-glucan was obtained in the fruiting bodies growing in the P. tremula wood-based substrate.In this work, different wood side streams of sawmill and plywood industries were tested as substrate media for the cultivation of Betula spp., P. tremula, P. abies, P. sylvestris and Larix sp.) and cold treatments (5 °C and −20 °C) for the cultivation of G. lucidum were evaluated by their capability of producing fruiting bodies. From the total substrate bags (N = 300), only 31.7% the produced fruiting bodies (N = 95), of which 89.5% were exposed to 5 °C (N = 85) and 10.5% to −20 °C (N = 10). Substrate bags exposed to −20 °C did not produce fruiting bodies and were susceptible to contamination. Successful fruiting bags exposed to −20 °C were inoculated with the MUS19 strain into P. tremula and P. abies wood-based substrates.The suitability of strains , wood substrates  than P. abies, P. sylvestris and Larix sp. . Substrate colonization was completed within one month for most of the wood substrates except for P. sylvestris and Larix sp. wood-based substrates, in which colonization was slow or uncompleted.From a total of 50 bags per strain, strains MUS19, MUS192 and MUS6 were more capable of producing fruiting bodies  than MUS9, MUS12 and MUS75 . Regarding the wood substrates (60 bags per substrate), G. lucidum as a function of strains, wood substrates and cold treatments  indicating that cold treatment in +5 °C increased the probability of a successful fruiting body in comparison to treatment at −20 °C. The fungal strain , followed by wood substrate , significantly predicted the probability of the fruiting of G. lucidum. For instance, the inoculation of MUS75 or MUS12 would significantly decrease the probability of fruiting (using MUS19 as reference). On the contrary, the utilization of the wood substrate based on P. tremula would significantly increase the probability of fruiting (using P. abies as a reference).A logistic regression model (Equation (1)) was fitted to predict the probability of the successful fruiting of eatments . TreatmeG. lucidum was studied using the substrate bags that produced fruiting bodies. The linear regression model expressed in Equation (2) showed that the effect of strain and wood had a significant effect (p < 0.05) on the yield of the fruiting bodies of G. lucidum (p > 0.05). For example, the interaction of wood and strain was not significant  = 1.12; p = 0.361).The effect of strain, wood, treatment and their interactions on the fruiting body yield of  lucidum . Wood sup < 0.05). These strains presented fruiting bodies with the highest probability of success fruiting bodies in comparison with other strains . The yields per bag were 59.8 g and 52.1 g, respectively, for Betula spp. and P. tremula. However, the highest yield per bag (90.2 g) was observed in the P. tremula wood-based substrate inoculated with the MUS192 strain. The P. abies and P. sylvestris wood-based substrates were not as suitable as deciduous species for the yield of fruiting bodies. Larix sp. was found the least suitable for the yield of G. lucidum fruiting bodies .In G. lucidum fruiting bodies was studied using the substrate bags treated at +5 °C (Equation (3)). As expected, the strain and wood substrate had a significant effect (p < 0.05) on the contents of total glucan and β-glucan in the fruiting bodies of G. lucidum . Similarly to the results of fruiting body yield  even though it was the strain with the lowest probability of fruiting .In fruiting  and fruifruiting . MUS12 aβ-glucan content of the fruiting bodies of G. lucidum was affected by the tree species used as wood substrate . The P. tremula wood-based substrate also presented a significantly higher probability of fruiting .The ubstrate . The higfruiting  and fruiβ-glucan content of the fruiting bodies of G. lucidum was studied in this work. The strain selection and the cold shock treatment had the strongest effects on the probability of fruiting. The wood substrate had the strongest effect on the yield and β-glucan content in the fruiting bodies. The highest yields were obtained with Betula spp. and P. tremula wood side streams. P. tremula was found to be the most suitable substrate to enhance the β-glucan content in the fruiting bodies.The effect of strain, wood substrate and cold treatment on the probability of successful fruiting, yield and Pleurotus sp. [Cold treatment was needed after full colonization of the substrate bags to induce primordia formation. In this study, the cold treatment was adequate at 5 °C. When a lower temperature (−20 °C) was applied, the substrate bags did not produce fruiting bodies and were susceptible to contamination. In a previous study, a cold treatment at 5 °C decreased the time needed for fruiting and enhanced the biological efficiency of otus sp. . Fungal otus sp. . Due to G. lucidum was tested in agar media [Betula spp. and P. tremula sawdust-amended media enhanced the mycelial growth of G. lucidum in vitro. Therefore, it is not surprising to observe a higher probability of fruiting using wood residues of those tree species as substrate.Previously, the effect of wood sawdust from the same tree species on the mycelial growth of ar media . In accoBetula spp. and P. tremula in comparison with conifer species. The interaction of strain and wood did not have a significant effect on the probability of fruiting, yield or β-glucan production. Surprisingly, the maximum yield and biological efficiency of 90.2 g and 9% were achieved with the poplar wood-based substrate. In Finland, G. lucidum has not been reported growing in P. tremula in nature [Ganoderma fruiting bodies [The yield was found also higher in n nature . Higher n nature  and Erken nature . Biologin nature  reportedg bodies ,7,14,17.G. lucidum fruiting bodies had a very high content of β-glucan (47.5–55.9 g/100 g dm). The high content of β-glucan indicates that G. lucidum fruiting bodies may be a source for bioactive compounds for the food and pharmaceutical industries. Similar levels were obtained by McClearly and Draga [β-glucan content as high as other fungal species such as Trametes versicolor or Piptoporus betulinus  [β-glucan content (<20 g/100 g dm) for G. lucidum specimens originating from Korea, Japan, Taiwan, USA and Brazil. Considering the taxonomy confusion of G. lucidum, it is possible that these specimens differ from the European G. lucidum [β-glucan production could be expected between closely related species. Another possible explanation is the extraction method. For instance, Benito-Román et al. [β-glucan (range between 24.0 and 62.2%) in G. lucidum using pressurized hot water as a solvent. The material analyzed may have an effect on the β-glucan content. The fruiting bodies analyzed in this study consisted of a mixture of cap and stipe. A higher content of β-glucan was detected in the stipe than in the cap of several wild mushrooms [Lentinula edodes [β-glucan content within the fruiting body remain to be studied in G. lucidum.In this study, nd Draga  using thctively) . Howeverctively)  reported lucidum ; therefon et al.  tested dushrooms  and culta edodes . Differeβ-glucan is catalyzed by β-1,3-glucan synthase [β-1,3-glucan synthase activity in L. edodes has been hypothesized to be associated with the presence of phenolic compounds in the media [β-glucan synthases are activated in order to preserve the integrity of the cell wall. Therefore, β-glucan synthesis could be related to the defense mechanism of the cell wall [The synthesis of synthase . β-1,3-ghe media . The prehe media . In resphe media  suggesteell wall .β-glucan content. However, in this study, β-glucan content in the fruiting bodies was higher in the P. tremula wood-based substrate than in other species, especially when compared to the P. sylvestris wood-based substrate. Similar results were obtained by Kuhar et al. [P. tremula is not a typical host species for G. lucidum in Finland [G. lucidum. Other wood components such as lignin, cellulose and hemicellulose fractions [L. edodes [G. lucidum fruiting bodies and mycelia can be also due to differences pH levels and C:N ratio, carbon, nitrogen, phosphate and magnesium source of the media [Generally, conifer species contain higher levels of phenolic compounds than deciduous species; therefore, it would be expected that fruiting bodies grown in a conifer wood-based substrate presented higher r et al. . Moreover et al. . Alkalinr et al. . Since P Finland , it is practions ,27, fattractions  and resiractions ,46, amonhe media ,24,25.Ganoderma lucidum originating from boreal forest was successfully cultivated using wood side streams of plywood and sawmill industries. Cold shock treatment was required to trigger fruiting body formation. The P. tremula wood-based substrate enhanced β-glucan content in the fruiting bodies. The availability of wood residues and the potential of G. lucidum strains to produce fruiting bodies offer an opportunity to increase the valorization of wood side streams and to produce high valuable mushrooms and compounds such as β-glucan. This is the first study to simulate commercial indoor cultivation of G. lucidum strains originating from boreal forests.G. lucidum and identified based on the internal transcribed spacer (ITS) DNA region sequence data [P. abies and B. pubescens located in the Satakunta and Uusimaa regions, Finland . Wood residues from P. tremula, P. abies and P. sylvestris and Larix sp. were obtained from small sawmills companies located in North Karelia and South Savo, Finland. Polypropylene bags were filled with a mixture of wood chips (particle size from 20 to 50 mm) and sawdust (ca. 0.8 mm particle size). Dry weight bags (60% wood chips and 40% sawdust by weight) of 1 kg were prepared with P. tremula, P. abies, P. sylvestris and Larix sp. The substrate bags containing Betula spp. were filled entirely with wood chips (particle size from 20 to 50 mm). Ten replicates of 1 kg (dry weight) per each combination of wood type and strain were prepared. Wood moisture content on a dry basis [Wood residues from ry basis  was adjuSubstrate bags were autoclaved for 45 min at 125 °C and 1.2 bar. After cooling down, the substrate bags were inoculated with 0.3 L of fully colonized spawn. Substrate bags were sealed, shaken and kept at 22 °C in dark conditions until full colonization. After full colonization, substrate bags were transferred to a cropping greenhouse with a relative humidity of 65 to 75% and light exposure (8/24 h) to induce fruiting body formation.After several weeks, there was no observation of primordia formation. Because of the difficulties of strains to produce fruiting bodies, substrate bags were submitted to two different cold shock treatments. Half of the replicates were kept at 5 °C and the other half at −20 °C for three months. After the cold treatment, the substrate bags were maintained at 25 °C and a relative humidity of 90% until fruiting body formation. Fruiting bodies were harvested, and the yield was determined based on fresh weight (g). Harvested fruiting bodies were kept at −20 °C.α-glucan content were measured for each individual fruiting body according to McCleary and Draga [β-glucan assay kit. In brief, the principle of the assay is to solubilize and hydrolyze the total glucan of the sample using sulfuric acid, exo-1, 3-β glucanase and β-glucosidase enzymes. α-Glucan and sucrose were dissolved using sodium hydroxide. Glucose was measured with amyloglucosidase and invertase using GOPOD reagent. β-Glucan was determined by subtracting α-glucan from the total glucan measurements. Total glucan, α-glucan and β-glucan content are expressed as a percentage of the freeze-dried fruiting body . Analyses were done as duplicates; two samples from each pulverized fruiting body were measured separately.The fruiting bodies were freeze-dried  and kept at −20 °C. Fruiting bodies including both cap and stipe were cut into pieces and then milled with a hammer mill with a 1 mm sieve . Total glucan and nd Draga  and usinG. lucidum strain, wood substrate and cold treatment on successful fruiting (0/1), yield (g of the fresh fruiting body) and total glucan, α-glucan and β-glucan content  were evaluated by regression modeling. To avoid overfitting, only the two-way interactions of strain, wood and treatment were considered during modeling.The effects of p and y are, respectively, the probability of successful fruiting and the fruiting body yield (g of the fresh fruiting body) of the substrate bag , which produced fruiting bodies; S is the strain of G. lucidum ; W is the wood substrate ; Tr is cold treatment ; and e is the random error term with a mean of 0 and constant variance. Square root transformation was needed to meet assumptions for linear regression .A logistic regression model was fitted for the probability of successful fruiting (Equation (1)) and a linear regression model was fitted for the yield of fruiting bodies, if any (Equation (2)):Glucan is the content of total, α or β-glucan  of each fruiting body.The content of glucan was modeled by fitting a linear mixed model (Equation (3)):u), the predicted means were computed by averaging the random effects over subjects . Using a given cold treatment, all pairwise comparisons of strain and wood substrates were performed to find out which strains and wood substrates have a statistically significant higher yield and glucan content.The models were fitted and the marginal means were estimated for all level combinations of the factors  using the MIXED procedure in IBM SPSS Statistics 25 . If the model involved random effects (


Without salt substitution, this chronic diarrheal disorder causes severe dehydration and electrolyte disturbances. Homozygous variants in the nearby gene SLC26A4 disrupt anion exchange in the inner ear and the thyroid, causing Pendred syndrome , which is the most frequent form of syndromic deafness.Congenital chloride diarrhea  is a rare autosomal recessive disorder caused by pathogenic variations in the SLC26A3 and SLC26A4 genes, causing a rare combination of both CLD and PDS in two siblings. Although the clinical pictures were typical, the combined loss of these anion transporters might modulate the risk of renal injury associated with CLD.We report an unusual co-occurrence of two rare homozygous mutations in both the SLC26A3 and SLC26A4 genes cause CLD and PDS in siblings, shedding light on co-occurrence of rare recessive traits in the progeny of consanguineous couples.Familial presentation of two rare autosomal recessive disorders with loss of function of different SLC26 anion transporters is described. Independent homozygous variants in the  OAE was negative, and tympanometry showed normal curves. The hearing loss was suspected because of poor language acquisition and because the younger sister had just been diagnosed with the same condition. The hearing loss was progressive in the right ear but remained stable in the left. The boy received bilateral hearing aids and is now being considered for cochlear implants. Currently at the age of 12, he is attending a special school for hard of hearing/deaf children and communicates with spoken language as well as with some sign language.Pregnancy ultrasound at gestational week 25 showed a distended bowel but no polyhydramnios. A baby girl was born at 35 weeks with a birth weight of 2600 g. Her abdomen was slightly distended and the meconium watery. After admission to the neonatal intensive care unit, oral feeding was started, but the baby developed hypokalemia, hypochloremia, hyponatremia, and metabolic alkalosis, similar to her older brother. High fecal chloride confirmed the diagnosis of CLD. Six days after the birth, salt replacement therapy was started according to the recommendations [The girl failed the otoacoustic emission test (OAE) at 7 days and again at 12 days. At the age of 6 weeks, brainstem response audiometry revealed a bilateral moderate to severe sensory hearing impairment . Bilateral hearing aids were fitted. The hearing loss was progressive, and at the age of 5 years, she received a cochlear implantation (CI) on the right ear. The pre-operative radiological examinations (CT and MRI) revealed minor malformations of the cochleas, and the semicircular canals were normal. The internal auditory canals were wide and the vestibular aqueducts were enlarged (EVA). At the time of the operation, the so-called “gusher” (increased perilymphatic pressure) appeared as a characteristic of Pendred syndrome. CI in the right ear and a hearing aid on the left resulted in significantly improved language acquisition and speech. The girl currently attends a special preschool for hard of hearing/deaf children and learns both Swedish spoken language and sign language.SLC26A3 exons were amplified using intronic primers as described [Genetic testing for CLD was performed at our research laboratory  when the boy was 1 year old. escribed . The Ara2, 200 μM of each dNTPs, 0.8 μM of each primer, 0.03 U/μL of Hot Star Taq Plus DNA polymerase , and 10 ng of genomic DNA. Cycling conditions were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s, and by the final extension of 72 °C for 10 min. The PCR products were studied on 2% agarose gels. ExoSAP-IT PCR clean-up was performed according to manufacturer’s instructions (Thermo Fisher Scientific) and PCR products were further studied by direct sequencing into both directions utilizing the BigDye Terminator v.3.1 Cycle Sequencing Kit and the ABI3730xl DNA Analyzer (Thermo Fisher Scientific).The suspected deletion around the non-coding exon 1 was further mapped by multiple PCR and sequencing reactions on both the patient sample and the parental samples. All PCR reactions were run in a volume of 25 μL involving 1 x PCR buffer, 1.5 mM MgClSLC26A3 gene. We identified regions in which PCR was unsuccessful in the patient sample but successful in the parental samples. This approach allowed localization of the approximate borders of the deletion, which was further characterized using specific primers (5′-CCGTGTTCAAGGACAATGTG-3′ in intron 1 and 5′-TGAGCCAGTCAGTGCAAAAC-3′ in the promoter). The deletion involved 8596 nucleotides from 4.3 kilobases upstream of exon 1 to 4.2 kilobases downstream of it. This homozygous deletion found in the patient sample, and present in a heterozygous form in the parental samples, results in a deletion of a large segment including the intestinal promoter for transcription and the non-coding exon 1 of the SLC26A3 gene [SLC26A3 was done because the clinical diagnosis of CLD – both the clinical picture and high fecal chloride – were consistent with the disease.We designed primers to amplify flanking sequences around 16 single nucleotide polymorphisms (SNPs) from 54 kilobases upstream rs10274491) to 5 kb downstream (rs4730264) from the exon 1 of the 274491 toSLC26A4 change c.384delT (p.Phe128Leufs#17). The parents were heterozygous carriers for this pathogenic variant. This deletion is previously unreported and not found in genetic databases but lies at the location where one pathogenic variant c.382_384delTTTinsAA (p.Phe128Lysfs) has been reported in a single individual [When hearing impairment was clinically diagnosed, both the boy and the girl had additional genetic testing  and were found to be homozygous for the dividual .SLC26A3 and SLC26A4 genes, including their promoters, is shown in Fig. SLC26A3 deletion found in the patient does not cover any enhancer elements of the gene. Thereby, the co-occurrence of these diseases is apparently not affected by any enhancers, further supporting the causative role of the two rare and independent mutations for CLD and PDS in these patients.The region covering SLC26 gene family. As for CLD, founder populations in Finland, Poland and the Arab Middle East account for the majority of the affected individuals [SLC26A4 gene and not covering any enhancer elements of the genes (Fig. SLC26A4 nonsense change is most likely independently causative for PDS in these patients. These cases highlight the importance of consanguineous family history in diagnostics of rare childhood disorders.The described co-occurrence of two rare recessive pathogenic variants independent of each other is highly unusual and previously unreported for the members of the ividuals . The affividuals . As thisnes Fig. , the rarSLC26A3 disease mutations have revealed consistent loss of function [The clinical picture of CLD in these patients was typical as such, and the age at the diagnosis of the older child in the family was close to the mean of 2.7 months reported in the literature [function , differefunction . This isfunction . NotablyBoth the boy and the girl showed a typical pattern of hearing loss attributed to Pendred syndrome. This hearing loss is often progressive and, in many cases, profound in young adulthood . The radSLC26A3 is mostly expressed by the apical intestinal epithelium and the male reproductive tract [SLC26A4 shows expression in the inner ear and the thyrocyte [SLC26A4 alone is compensated with other transporters in the nephron, and neither Pendred patients nor SLC26A4-deficient mice develop overt acid-base disturbances [SLC26A4 in the kidneys is regulated by systemic chloride load and acid-base status [SLC26 anion transporters show a highly differential tissue distribution . SLC26A3ve tract , while Shyrocyte . Althoughyrocyte . While lurbances , SLC26A4e status . Furthere status . TherefoSLC26A3 and SLC26A4. The clinical picture in the siblings highlights the challenges in diagnostics of rare disorders in children of consanguineous couples.This study describes an unusual presentation of two unique recessive diseases caused by homozygous changes in two nearby genes,


We examined the immediate and long-term impacts of military aircraft noise exposure on noise-induced hearing loss (NIHL) in fighter pilots and ground staff. We recruited 40 pilots, 40 ground staff, and 136 age-matched controls; all participants underwent hearing tests, including conventional pure-tone audiometry (PTA) (0.25–8.0 kHz), extended high-frequency (EHF) audiometry (9.0–18.0 kHz), and distortion-product otoacoustic emission (DPOAE) as a recent reference. A subsequent hearing test immediately after flight-mission noise exposure was requested. The results revealed higher recent hearing thresholds in pilots and ground staff than in controls. Threshold shifts at many octave band frequencies were also significantly elevated in ground staff. The grouped frequency threshold was significantly elevated in the 4–8 kHz high-frequency range. After a single flight-mission noise exposure, both ground staff and pilots showed decreased signal-to-noise ratios for DPOAE (1–8 kHz), whereas only ground staff showed significantly elevated left-ear hearing thresholds at 3, 11.2, and 12.5 kHz by conventional and EHF PTA. Fighter pilots and ground staff serve in hazardous noise-exposed environments that cause hearing damage and subsequent NIHL, but ground staff may be more vulnerable. A comprehensive hearing conservation program should be implemented to protect high-risk service members, and especially ground staff, from high-intensity noise exposure.  The adverse impact of noise from military operations on hearing among military service members has long been considered an important global health issue. The resulting hearing impairment can also further interfere with the effective execution of military operations. Hearing loss is one of the most common occupational health hazards, and tinnitus and hearing impairment are the most common disabilities among veterans of the United States military forces ,2. Noise can be classified into different types, including continuous, variable, intermittent, or impulsive, depending on how the noise changes over time. Continuous noise may cause greater cochlear damage and a permanent hearing threshold shift compared to intermittent noise . ImpulseSeveral clinical auditory functional tests are used currently, but pure-tone audiometry (PTA) has long been a useful and widely used tool for assessing auditory function for hearing screening in both the clinical and on-site workplace settings. However, otoacoustic emissions (OAEs) have subsequently been introduced for research and clinical applications ,6. The OAnother hearing test, extended high-frequency (EHF) audiometry, is now drawing much interest for early detection of sensorineural hearing loss. EHF audiometry is defined as a threshold measurement above the frequency of 8 kHz and is especially useful for detecting damage at the basal turn of the hair cells. Several studies have suggested that EHF audiometry is particular useful for assessing ototoxic drug-induced and noise-induced hearing loss, as well as age-related hearing loss (presbycusis) associated with sub-clinical hearing difficulties ,16,17,18The aim of the present study was to use these audiometric examination tools to survey the hearing status of young military personnel serving at an air force base as a way to establish the best diagnostic tool for NIHL detection. A secondary aim was to identify the immediate noise impact of a single flight mission on the hearing of fighter pilots and air force ground staff. This cross-sectional study evaluated the hearing condition of military personnel before and after a one-time noise exposure during flight missions. We recruited military members who served at a military air base, enrolling ground staff, fighter aircraft pilots, and age-matched control subjects whose duties did not involve fighter aircraft operation. Participants with a history of ear trauma, any previous ear surgery, or otitis media were excluded. All participants completed a questionnaire regarding their daily work, hearing condition, and whether they routinely wore a hearing protection device (HPD) in or over the ears while exposed to hazardous noise levels exceeding 85 dB within the workplace. Before performing the hearing tests, all participants underwent otoscopic examinations by an otologist to ensure the patency of the external auditory canal and to exclude any otological diseases. The recent hearing conditions of personnel in each group, reflecting the long term impact of military noise on hearing, were determined by conventional PTA, EHF audiometry, and DPOAE tests. Pilots and ground staff who were exposed to a single flight task-related noise were requested to undergo another hearing test battery as above within 2–4 h after the exposure. The Tri-Service General Hospital Institutional Review Board approved the research project with ethical approval code: TSGHIRB 2-105-05-107.Participants were tested in a sound-attenuating test booth established in a van equipped with a mobile audiologic unit that conformed to ANSI/ASA S3.1-1999 (R2018) specifications for audiometric test rooms. We obtained conventional PTA of all subjects at octave frequencies from 0.25 to 8 kHz for each ear using a Grason-Stadler GSI 61 clinical audiometer  with TDH-50P headphones . Frequencies over 8 kHz are termed extended high-frequencies (EHFs). Pure-tone hearing thresholds in the EHF range  were determined using a GSI 61 clinical audiometer with Sennheiser HDA 200 circumaural earphones . A descending⁄ascending method in 5 dB steps revealed hearing threshold levels. Responses were considered reliable if the difference between the test and retest thresholds was no more than 5 dB. If thresholds exceeded the equipment’s maximum output level, a “Χ” symbol was indicated. We calculated the arithmetic mean of the grouped-frequencies threshold as pure-tone averages (PTAs) of three-frequency , four-frequency , high-frequency , overall-frequency , and extended high-frequency  PTAs. Participants were tested in a sound-attenuating test booth, as described previously for obtaining the PTA. The distortion product otoacoustic emissions (DPOAEs) were recorded for both ears of all participants using the Bio-Logic Scout Sport System . The DPOAE test consisted of presenting two primary tones at frequencies f1 and f2 and levels L1 and L2. The frequency ratio f2/f1 was fixed at 1.22. The stimulus sound pressure levels were set at L1 = 65 dB and L2 = 55 dB. These parameters are expected to elicit optimal DPOAE magnitudes ,20. InseThe noise level was measured and recorded from within the hardened aircraft shelter, where the fighter aircraft is protected and the ground staff can perform necessary maintenance and prepare the aircraft for flight. For acoustic measurements, the equipment included an acoustic fixture  and a blast probe microphone (GRAS 67SB) operated with power support modules . This equipment was placed 7 m from the aircraft, as this setting simulated the real working distance between the ground staff and aircraft and met ANSI S12.42 standards. Computers (Lenovo Ideapad 700 15ISK) equipped with SpectraPlus-DT and SpectraPlus-DT9837A software  were used for data collection and analysis. All equipment was calibrated using the GRAS Type 42AP Intelligent Pistonphone. Application of fast Fourier transform gave a noise spectrum and spectrogram that showed the distribution of noise intensities as a function of frequency over time.p-value less than 0.05 was considered statistically significant.The statistical differences among the groups were analyzed using the Kruskal-Wallis nonparametric ANOVA. Results for the rate of HPD wearing were compared with the chi-squared test or Fisher’s exact test. Results of audiograms before and after flight mission noise were compared with a paired t-test. An independent t-test compared the audiograms between the pilots and ground staff. All statistical analyses were conducted using SPSS software, Version 22.0 , and a n = 196) of the participants, reflecting the significant gender difference not only in this study (p < 0.002) but also in military air bases in general. Although control subjects were recruited with the criteria of no history of working in a high A-weighted equivalent SPL places (> 85 dB), the habit of HPD wearing on the air base was also investigated. No significant statistical difference was detected among the three groups in terms of HPD wear or between the ground staff and fighter pilot groups  and HFPTA (p = 0.025 for right ear and p = 0.029 for left ear) that involved a high frequency greater than 4 kHz showed significantly higher threshold averages in the ground staff than in the fighter pilots , and 9, 10, 11.2, 12.5, 14, and 16 kHz for the left ear , and at 9, 10, 11.2, 14 and 16kHz in the left ear of the ground staff group compared to the pilot group . These data also suggest a greater vulnerability to noise injury among the ground staff at the air base than among the pilots.Comparison of the total number of “X” symbols in both groups revealed that the number was larger for the ground staff than for the pilot groups. However, the number of “X” symbols at 14, 16, and 18 kHz showed no significant difference between the groups using Fisher’s exact test for comparison . The ovep = 0.016), 11.2 , and 12.5 kHz in the left ears  of the ground staff group  and 6 kHz  in the right ear and at 3 kHz  and 6 kHz  in the left ear ; therefore, it could contribute a further acoustic gain and present a relatively realistic recapitulation of the noise impact on an individual’s hearing system. During a one-hour recording and measurement by the blast probe microphone, the measured equivalent SPL was 85.3 dB. The sound levels that exceeded 90% (LA90), 50% (LA50), and 10% (LA10) of the measurement period were 93.2, 123.4, and 124.1 dB, respectively. Conversely, the use of the acoustic test fixture for recording gave a measured equivalent SPL of 115.3 dB, and the LA90, LA50, and LA10 of the measurement period were 123.0, 156.6, and 157.4 dB, respectively. These data indicated that the hardened aircraft shelter is a hazardous and noisy work environment and that the ground staff require HPD appropriate to their practical needs.One of the main goals of this research was to examine the prevalence and severity of hearing loss in military personnel serving on an air force base. Surprisingly, the mean pure-tone averages from 0.25–8 kHz in each group were within normal limits (<20 dB), not to mention the characteristic noise notch at 4 kHz demonstrated in the audiogram. A focus on the overall trend of the age distribution of recruited participants revealed that 77.5% of the ground staff and 90% of the pilots were approximately 35 years old, whereas 92.5% of the ground staff and 97.5% of the pilots were under 40 years old. In other words, the vast majority of the participants in this study might have had a relatively shorter duration of noise exposure than had occurred in the other studies that recruited members who had over 20 years of military service ,22. The However, comparison of the pure-tone audiograms from the two aircraft groups and the control group still revealed a clear threshold elevation in the pilots and a statistically significant threshold shift (STS) in the ground staff group. Interestingly, both the ground staff and pilot groups showed increases in their hearing thresholds starting at 3 kHz, suggesting that aircraft noise exposure had affected the personnel’s hearing even though the exposure duration was shorter than 20 years. The SNR of DPOAEs of the three groups also revealed a similar trend by showing relatively low SNR values for the ground staff, followed by the pilot values. The EHF audiometry metric may provide more robust evidence to complement the observations obtained with conventional PTA. The higher rate of measurements that were out of the maximum output level of the equipment shown in the recent EHF audiometry of the ground staff, and particularly for the left ear, was consistent with the results obtained using conventional PTA and DPOAEs. The conventional PTA and DPOAEs are the most common audiometry methods used for screening and monitoring personnel under the regulations of occupational hearing conservation programs. Both hearing tests are also applied to investigate hearing in military service members who participate in gunfire practice or artillery exercises involving high-intensity impulse noise. Recently, EHF audiometry was reported to provide more information regarding the hearing status covering the high-frequency range from 9 kHz to 20 kHz; therefore, EHF may be able to detect deficits that might be overlooked in noise-exposed populations assessed by conventional PTA ,25. The Only limited reports have described the different impacts of aircraft noise on pilots and ground staff. The previous literature mentions a higher-than-limit noise exposure and the vulnerability of ground staff working in civilian airports ,30, but Conventional PTA and EHF audiometry revealed statistically significant differences in STS in the ground staff across a wide range of frequencies. By contrast, the recent DPOAEs records showed only one significant decrease in the SNR level at frequency of 4 kHz in the right ears of the ground staff group. Much of the literature has documented that DPOAE amplitudes are poorly correlated with PTA thresholds ,37. InteThe investigation of the immediate impact of noise on the hearing system after only one military flight task was another goal of this study. In agreement with the early findings in the recent PTA values, only the ground staff showed significant temporary thresholds shifts and only at some band frequencies  in the left ear after flight noise exposure. The pilots showed negligible changes in the PTA values. By contrast, the DOPAE measurements were more sensitive for detection of subclinical impairments than was behavioral tonal audiometry. Again, the significantly reduced SNR level shown by the ground staff indicated that aviation-related personnel may exhibit different vulnerabilities to aircraft noise. The reduction in the SNR may result from louder noise exposure experienced by the ground staff than by the pilots, or the ground staff may be more vulnerable to noise impacts and show greater changes in the SNR than the pilots do after noise exposure. Unlike civilian or enterprise factories that follow noise control regulations and receive annual noise survey/auditing, military camp sites usually restrict noise assessment to specific equipment, weapons, exercises, and training. Calculation of the eight-hour time-weighted average sound level (8-hour TWA), by averaging different exposure levels during an exposure period for each person, would provide more fundamental information that could clarify the difference in hearing status among personnel experiencing different noise impacts. However, the real situation for conducting investigations on the military air base may not allow us to survey every possible environment that has extra high-level noise. That is a limitation of our current study. We chose the hardened aircraft shelter as a realistic example of a high-level noise environment because the ground staff spend most of their working hours per day there and because both the ground staff and the fighter pilot would encounter substantial noise during each single flight mission.Both the fighter pilot and the ground staff are exposed to different types of noise with different intensities, even when they serve in the same workplace—the air base. The recognition of this difference prompted the writing of this article, as we wanted to point out that personnel situated in different noise-exposed environments should have different regulations regarding hearing protection.To our knowledge, this is the first prospective study that has proposed the possibility of a hearing insult among the pilots and the ground staff during a single flight mission. The results of immediate monitoring of the changes in hearing acuity after a one-off high-intensity noise exposure and the recent hearing status after a long-term noise exposure point to an urgent crisis in the hearing health of military personnel. However, these results also highlight the need for a concerted effort to improve the auditing practice based on current hearing conservation programs that have varying levels of success. Both EHF audiometry and the DPOAE test provide additional information for predicting military aircraft noise that results in NIHL. EHF is more sensitive in detecting potentially lasting NIHL, whereas DPOAEs are more able to reveal the immediate noise impact on hearing. These findings suggest that EHF audiometry is a suitable adjunct to conventional PTA during regular hearing monitoring, while DPOAE is helpful for instantaneous evaluation of noised-induced cochlear insult. A significant hearing threshold shift clearly occurred in both the pilots and the ground staff; therefore, a more comprehensive hearing conservation regulation should be implemented at military air bases to prevent at-risk service members, and particularly aircraft ground staff, from high-intensity noise exposure.