PDGFRB) gene rearrangements define a unique subgroup of myeloid and lymphoid neoplasms frequently associated with eosinophilia and characterized by high sensitivity to tyrosine kinase inhibition. To date, various PDGFRB/5q32 rearrangements, involving at least 40 fusion partners, have been reported. However, information on genomic and clinical features accompanying rearrangements of PDGFRB is still scarce. Here, we characterized a series of 14 cases with a myeloid neoplasm using cytogenetic, single nucleotide polymorphism array, and next-generation sequencing. We identified nine PDGFRB translocation partners, including the KAZN gene at 1p36.21 as a novel partner in a previously undescribed t chromosome change. In all cases, the PDGFRB recombination was the sole cytogenetic abnormality underlying the phenotype. Acquired somatic variants were mainly found in clinically aggressive diseases and involved epigenetic genes , transcription factors (RUNX1 and CEBPA), and signaling modulators (HRAS). By using both cytogenetic and nested PCR monitoring to evaluate response to imatinib, we found that, in non-AML cases, a low dosage (100–200 mg) is sufficient to induce and maintain longstanding hematological, cytogenetic, and molecular remissions.Platelet-derived growth factor receptor B (The online version contains supplementary material available at 10.1007/s00277-021-04712-8. PDGFRB gene, at 5q32, encodes for the ß chain of the cell surface receptor for platelet-derived growth factor (PDGFRß), a class III receptor tyrosine kinase (RTK) that activates signaling pathways involved in cell growth and differentiation [. PDGFRB is a frequent target of chromosomal translocations in a subgroup of hematological malignancies recognized in the 2017 World Health Organization (WHO) as a stand-alone category under “Myeloproliferative neoplasms with eosinophilia and gene rearrangement” [PDGFRB is rearranged in lymphoid malignancies, including cases of both B- and T-cell acute lymphoblastic leukemia/lymphoma (ALL) [. In addition, in the same patient, a PDGFRB rearrangement may underlie the occurrence of two different malignancies of both myeloid and lymphoid lineages, which may be diagnosed concomitantly or sequentially [PDGFRB have been identified. Among them, ETV6 is the most frequently involved gene as a consequence of the t [PDGFRB gene always participates in the fusion with its 3′ end tyrosine kinase domain, resulting in the constitutive activation and in the deregulation of downstream signaling cascades, including Ras/mitogen-activated protein kinase, phosphatidylinositol 3′-kinase, and phospholipase-5γ pathways [PDGFRB rearrangement is still scarce mainly affecting ASXL1, TET2, STAG2, DNMT3A, NRAS, ZRSR2, BCOR, and STAT5B [The ntiation . PDGFRB ngement” . Less frma (ALL) –6. In adentially . To dateq32;p13) , while aq32;p13) . Whatevepathways . Informad STAT5B , 11. Mald STAT5B , 9. Althd STAT5B , 12.PDGFRB-positive cases recruited in our center during the last 23 years. In addition, we evaluated the response to imatinib by cytogenetic and molecular monitoring.We carried out an in-depth characterization of genomic events accompanying the 5q32 rearrangement in a series of 14 PDGFRB were recruited from the files of the Laboratory of Cytogenetics at the Department of Medicine and Surgery-Hematology section of the University of Perugia  or home-brew FISH assays with bac probes  and even more specific cosmid clones: cosmid 4–1, for the 5′, and 9–4, for the 3′ of the gene [KAZN breakpoint was characterized using a series of locus-specific probes mapping at 1p36–1p35 regions (Supplementary Table Fluorescence in situ hybridization (FISH) was used to investigate the gene . The 1p3http://dgv.tcag.ca/dgv/app/home).Copy number variations (CNVs), gains, losses, and copy neutral loss of heterozygosity (cnLOH) were studied by SNP array in 9 cases with available material at diagnosis  using a Cytoscan HD Array Kit  following manufacturer’s instructions. Data were analyzed by Chromosome Analysis Suite (ChAS 4.1) software with hg19 build as reference. Filters were set at 400 kb, 50 markers for CNVs, and 10 Mb, 50 markers for cnLOH. Polymorphic copy number variants were excluded from the analysis by comparing with the Database of Genomic Variants , according to the manufacturer’s protocol. One microgram was retrotranscribed using 50 U of SuperscriptII (Invitrogen). Nested PCR was performed to identify the fusion transcripts in 13/14 cases  and for monitoring the minimal residual disease in 11 cases  with a monitoring range of 1–12 months  were defined as germline. All other variants were defined as somatic.Mutational analysis of thirty genes mutated in myeloid malignancies and included in the Myeloid Solution SOPHiA GENETICS  was performed following the manufacturer’s instructions on 13 cases at diagnosis , in 8 cases  also at cytogenetic and/or molecular remission, and in one case (no. 14) also after HSCT. The resulting captured libraries were further processed on a Miseq® sequencing platform . FASTQ sequencing files were uploaded to SOPHiA DDM® platform version 4 and, following adapter trimming and quality filtering, reads were aligned to the human reference genome (hg19 assembly) through the artificial SOPHiA™ intelligence. Variant calling of the resulted alignments was then performed using an in-house somatic variant caller, which takes into account the background noise level at each region. Only exonic and splice sites variants with MAF < 0.01 were taken into consideration and classified according to ACMG criteria . Mutatio9/L); eosinophilia (≥ 0.5 × 109/L) or hyper-eosinophilia (≥ 1.5 × 109/L) was demonstrated in 12 of them (range: 1.07–14.4 × 109/L) (Table 9/L). Anemia was seen in seven cases and thrombocytopenia in three. Hepatomegaly and/or splenomegaly was present in eleven cases (Table Fourteen cases were collected. The median age at diagnosis was 38 years (range: 2–75) with a male/female ratio of 2.5 Table . Eleven  ≥ 0.5 × 09/L or h ≥ 0.5 × 09/L or h ≥ 0.5 × 09/L or h ≥ 0.5 × 09/L or hPDGFRB involvement and identified the translocation partner in all cases. Seven partners were already reported [ETV6 (n = 4), ERC1 (n = 2), and CCDC88C (n = 2) were recurrent partners. Moreover, in case no. 14, we identified KAZN at 1p36.21 as a novel partner gene of PDGFRB. Metaphase FISH identified the 1p breakpoint between RP11-1079F4, flanking the 5′, and RP11-317H5, covering the 3′ of the gene. Additional experiments, showing the splitting of WI2-0883F15 and WI2-0968K05 fosmids, narrowed the breakpoint between exons 4 and 5 of KAZN (KAZN (NM_201628.3) and exon 12 of PDGFRB (NM_002609.3)  Fig. 1CySNPa revealed a total of 13 events  in nine investigated cases. However, they were mainly concentrated in case no. 13 showing nine imbalances , TET2, DNMT3A, and CEBPA (n = 1 each) were found in 5 out of the 10 cases with chronic presentation . Longitudinal studies showed disappearance of HRAS and TET2 variants at remission in case nos. 4 and 8, respectively, whereas DNMT3A mutation load increased at cytogenetic remission in case no. 11 (Table RUNX1 was affected by multiple variants (n = 9), while one mutation at ASXL1 was found in case no. 14   and one case as myeloid sarcoma (no. 12).PDGFRB::ETV6 and the absence of somatic mutations in bone marrow cells. Hematological, cytogenetic, and molecular response were achieved after 2 cycles of chemotherapy [A 66-year-old man was diagnosed with myeloid sarcoma in another center based on a lymph node biopsy. At the same time, we found t/otherapy . RemissiPDGFRB translocation, we found five somatic variants at the RUNX1 gene -positive AML that underwent chemotherapy and autologous transplant. At relapse, in addition to the -positiveRUNX1 and one at ASXL1   received a cytoreductive therapy before imatinib while four cases  received imatinib as front-line therapy .In four cases with complete monitoring , the median time lapse between cytogenetic and molecular remission was 24 months (range: 14–32). The earliest cytogenetic remission was obtained in case no. 3 after 1 month of treatment, although the disease persisted at molecular level up to month 25. In case nos. 1 and 4, remission was simultaneously documented by FISH and RT-PCR after 24 and 9 months of treatment, respectively. The median overall survival after molecular remission in nine cases was 42 months (range: 6–197), with ongoing follow-up in seven cases.PDGFRB in myeloid malignancies are rare events. Here, we characterized a series of 14 cases with a myeloid neoplasm at diagnosis and a rearrangement of PDGFRB, providing data from in-depth genomic characterization by SNPa and NGS analysis. At cytogenetics, the 5q32/PDGFRB rearrangement involved multiple partners identified by banding in all cases but one with a cryptic change. Notably, in one case, complex chromosome rearrangements corresponded to a PDGFRB::ETV6 fusion underlying a four-break translocation. These results confirm the clinical relevance of FISH studies for PDGFRB gene rearrangements in patients with myeloid neoplasms, especially when associated with eosinophilia, even in the absence of karyotypic abnormalities. Notably, the 5q32/PDGFRB rearrangement was the sole cytogenetic abnormality in our cohort and SNPa confirmed a low burden of co-occurring abnormalities in all cases with chronic disease, strongly supporting the driver role of PDGFRß tyrosine kinase activation to address the clinical phenotype.Chromosomal rearrangements involving KAZN gene at 1p36 as a novel partner of PDGFRB in one case with a t. The KAZN gene is involved in intercellular adhesion, cell differentiation, signal transduction, cytoskeletal organization, and apoptosis [PTBP2, TMEM51, and MTOR have been found as fusion partners of KAZN by RNA sequencing studies in solid tumors [KAZN have never been described in hematological malignancies. Recurrent KAZN variants have not been specifically associated with human tumors .TET2, ASXL1, and STAG2 genes [TET2, DNMT3A, HRAS, and CEBPA. Notably, five and four variants affected the RUNX1 gene in our AML case no. 13 and no. 14, respectively. These findings suggest that instability at RUNX1 is a nonrandom event accompanying an acute clinical phenotype in PDGFRB + malignancies. This hypothesis is supported by the work of Stengel et al. [RUNX1 gene in a large series of mutated de novo AML cases. With respect to the nature of RUNX1 mutations in our acute cases, they are all predicted to be pathogenic. Six of them fell in the Runt-Homology DNA binding Domain (RHD), one fell in the Trans-Activation Domain (TAD), and two non-sense mutations truncated the protein between RHD and TAD.In a literature review, we found only 8 cases in chronic phase with information on the mutational background that showed variants at G2 genes , 7, 10. l et al. , who rarHRAS and TET2 gene variants disappeared concomitantly to cytogenetic and molecular remission of the PDGFRB abnormality. These cases, in addition to one case with somatic mutation at the BCOR gene that disappeared at remission of the PDGFRB rearrangement [PDGFRB + clonal population sensitive to imatinib. Instead, a peculiar dynamics of somatic gene variants, as compared to the PDGFRB rearrangement, was seen in our case no. 14, in which one of the RUNX1 variants  decreased concomitantly to cytogenetic remission, whereas both the ASXL1 and the c.497G > A RUNX1 variants appeared as the leukemic fil rouge over the whole disease course in this case, from diagnosis till post-transplant relapse, when PDGFRB was absent. Altogether, these results raise the question whether AML in the last case was a second malignancy instead of an acute phase originated by linear clonal evolution [PDGFRB-positive cells. Single-cell analysis will be helpful to better understand the origin of AML in PDGFRB-positive cases. Finally, the increased clonal size of DNMT3A in another responder (case no. 11) with cytogenetic disappearance of the PDGFRB rearrangement is consistent with clonal hematopoiesis, similar to that observed in other myeloid malignancies [Longitudinal studies helped us to characterize the behavior of somatic mutations over the disease course. In particular, both angement , suggestvolution  of the Pgnancies .PDGFRB rearrangement is deeply influenced by the clinical presentation [Our study confirmed that prognosis in cases with entation , as our PDGFRB-related disorders presenting in chronic phase is still not available. The present series underlines the low dosage of 100–200 mg as a successful approach to inducing and maintaining cytogenetic and molecular remission. Moreover, molecular analysis is essential to carefully monitoring response to therapy, to evaluate the amount of residual disease, and to address changes of imatinib dosage over the disease course.A consensus on the posology of the TKI inhibitor in Supplementary file1 (XLS 35 KB)Supplementary file2 (XLS 34 KB)Supplementary file3 (XLS 37 KB)Below is the link to the electronic supplementary material.


Depression among older adults is an important public health concern associated with increased risk of suicide and decreased physical, cognitive, and social functioning. This study identified trajectories of depressive symptoms and investigated predictive variables of group-based trajectory modeling among Korean community-dwelling older adults.Participants comprised 2016 community-dwelling Korean adults over 65 years. Data from the years 2006–2016 of the Korean Longitudinal Study of Aging, a nationally representative panel survey that has been conducted every two years since 2006, were used. We employed a group-based trajectory modeling analysis to identify depressive symptom trajectories. Multinomial logistic regression analysis was conducted to identify predictors of each class of depressive symptoms.Five depressive symptom trajectory groups were identified: Group 1, “None” (28.9%); Group 2, “Slowly worsening” (24.3%); Group 3, “Rapidly worsening” (17.5%); Group 4 “Improving” (12.4%); and Group 5, “Persistently severe” (16.9%). Older adults followed five distinct depressive symptom trajectories over 10 years. Mini-Mental State Examination scores, number of chronic diseases, educational level, and social activity were predictors associated with increasing depressive symptoms.This study showed that many older adults living in the community have depressive symptoms. To prevent and treat depression and aid successful mental health aging among older adults, the development of interventions should be tailored to target specific needs for each symptom trajectory. It is necessary to develop community-based interventions and strategies to identify and prevent depressive symptom trajectories among older adults. Depression among older adults is an important public health concern associated with increased risk of suicide and decreased physical, cognitive, and social functioning . DepressDepressive symptoms experienced in older age have serious implications for health, functioning, and emotional distress. They are influenced by an individual’s subjective mood at the time of assessment and may vary according to one’s life course . DepressMulti-component collaborative intervention, through a collaboration of specialists, professional mental health care providers, and social networking, has the greatest impact on reducing depression . StudiesThe Korea Labor Institute collected data for the Korean Longitudinal Study of Aging (KLoSA) . The KLoIn the baseline data from 2006, 10,254 individuals were surveyed, but we excluded 6,090 participants who were below 65 years. Attrition data can be heavily influenced by unnoticed outcomes of dropout cases, leading to potential misclassification of these cases into the GBTM . In thisr = 0.88, p < 0.001) with verified validity [Depressive symptoms were measured using the Korean version of the Center for Epidemiologic Studies Depression Scale (CES-D10), which was developed in Boston . The CESvalidity . At the validity ; for thiDemonstrated risk factors in previous studies were selected as predictors of the trajectory of depressive symptoms. Demographic and socioeconomic factors included age, gender, existence of spouse, educational level, religion, current employment, and total household income. Educational level was divided into four categories . Religion, existence of spouse, and current employment were measured as dichotomous variables (yes or no), and total household income and age were treated as continuous variables. For religion, respondents were instructed to indicate if they believed in any religion. A “yes” response indicated that the participant belonged to a religion while a “no” response indicated no affiliation with any religion.Two tools were used to assess older people’s functional limitation: Korean activities of daily living (K-ADL) and Korean instrumental activities of daily living (K-IADL). The K-ADL assessed seven activities: bathing, dressing, eating, grooming, walking, transfers, and toileting . For eac2). Cognitive function was measured using the Korean-Mini-Mental Status Examination (K-MMSE); total scores ranged from 0 to 30, with higher scores indicating better cognitive function [Physical health was assessed as the total number of physician‐diagnosed chronic diseases , with the total count ranging from 0 to 8. Body Mass Index (BMI) was calculated as body weight (kg) divided by squared height  and contact with children . Responses were measured on a 9-point scale . For analysis, the frequencies of social activity and contact with children were divided into five categories .GBTM according to CES-D10 scores was used to identify trajectories of depressive symptoms during the follow-up period. GBTM was developed to identify different homogeneous subgroups with similar growth trajectories within longitudinal data of heterogeneous groups , 42. BasThe final number of trajectories for this study was identified based on the smallest Bayesian information criterion (BIC), sample size adjusted BIC (SSABIC) , which in = 143) and was labeled as “No depressive symptoms” (Class 1). The second trajectory group (Class 2) comprised 21.8% (n = 440) of the sample and was labeled as “Persistent minimal depressive symptoms.” The Classes 1 and 2 showed no clinical depressive symptoms at baseline and consistently showed no clinical depressive symptoms after 10 years. The third and largest trajectory group comprised 24.3% of the sample (n = 491) and was labeled as the “Slowly worsening depressive symptoms” (Class 3). This trajectory group showed no depressive symptoms at baseline; the depressive symptoms increased slowly and worsened to a clinical level after six years. The fourth trajectory group (Class 4) comprised 17.5% (n = 353) of the sample and was labeled as “Rapidly worsening depressive symptoms.” This trajectory group showed no depressive symptoms at baseline, but depressive symptoms started increasing after 6 years till 10 years. The fifth trajectory group (Class 5) comprised 12.4% (n = 249) of the sample and was labeled as “Improving depressive symptoms.” This trajectory group showed depressive symptoms at baseline, but no depressive symptoms were present after 10 years. The final trajectory group (Class 6) comprised 16.9% (n = 340) of the sample and was labeled as “Persistently severe depressive symptoms.” This trajectory group showed depressive symptoms at baseline and the symptoms were maintained over time  or higher , 46. We The six classes of GBTM were reclassified into five trajectory groups according to the clinical criteria for depressive symptoms. Clinically, the cutoff point of depressive symptoms is CES-D10 ≥ 4 . Class 1Table The five trajectory groups differed significantly in age, gender, education level, existence of spouse, employment, K-ADL, K-IADL, K-MMSE, number of chronic diseases, exercise patterns, frequency of social activity, and CES-D10. The highest average baseline age (mean = 71.74), females (75.3%), and those with elementary or lower education (85.3%) were found in the “Persistently severe” trajectory group. In contrast, the lowest average baseline age (mean = 70.19), females (47.2%), and those with elementary or lower education (58.8%) were found in the “None” trajectory group. The highest and lowest values of existence of a spouse were found in the “None” (77.7%) and “Persistently severe” trajectory groups (57.1%), respectively. The average scores for K-ADL at 0.35 (SD = 1.28) and K-IADL at 1.00 (SD = 2.29) were highest in the “Persistently severe” trajectory group. The K-MMSE score was the highest in the “None” trajectory group at 25.03 (SD = 4.39). For the frequency of regular social activities, the highest score for meeting almost daily was found in the “Improving” trajectory group (15.3%), while the lowest was found in the “Rapidly worsening” trajectory group (5.1%).p = 0.001). The "Rapidly worsening " trajectory group was associated with decreased K-MMSE scores . Furthermore, compared with participants engaging in social activities almost daily, the frequency of regular social activities was associated with increased depressive symptoms: 2–3 times a week , 1–4 times a month , 1–6 times annually , and none . Compared to the "Persistently severe " group, the “Improving trajectory” group was more likely to have standard K-MMSE scores  and fewer chronic diseases . Moreover, compared with those who engaged in social activities almost daily, the frequency of regular social activities decreased the relative risk significantly: 2–3 times a week , 1–4 times a month , 1–6 times annually , and none .Table p =  < 0.001). The relative risk of being on the “Rapidly worsening” trajectory increased with age , and decreased with education level . Having a current job  was associated with a higher relative likelihood of being on the “Improving” trajectory.The relative risk of being on the “Slowly worsening” trajectory was associated with education level  were identified by K-MMSE scores, the number of chronic diseases, and frequency of social activity. In previous studies, lower levels of education were associated with depressive disorders , whereasUnemployment status was associated with a higher relative risk of being “persistently severe” than “improving” in the trajectory group. In Europe and the USA, unemployed older adults had a higher incidence rate for depression . Older aIn the present study, there was a relationship between depressive symptoms and cognitive function; participants with depressive symptoms were more likely to show a decrease in cognitive function scores (K-MMSE), while reduced depressive symptoms were associated with an increase in cognitive function scores. One study found that participants with depressive symptoms were at high risk for dementia . WhetherPrevious studies have suggested that chronic diseases are associated with an increased risk of depressive symptoms . Older aSuccessful healthy aging is defined as reaching older adulthood with the absence of major disease, depression, or disability; high cognitive and physical function; active social engagement; and satisfaction with life . IndividThere were several limitations to this study. First, our study targeted older adults living in the community and did not include those who were admitted to nursing homes and hospitals. Second, it was not possible to fully assess whether participants had been diagnosed with depression and treated with medication. Third, participants were asked to state if they engaged in exercise at least once a week; however, this may not have effectively indicated the level of exercise needed for the prevention of diseases among older adult populations. Finally, the study could not account for substantial classification uncertainty. Therefore, there may be bias in estimating the associations between subgroup membership and secondary variables such as education, MMSE, and ADL. Despite these limitations, our study is significant, as it used a representative sample for longitudinal follow-up. In addition, by identifying risk factors affecting CES-D10 scores, the results can be utilized to prevent depression among older adults in the future.Given the rapidly aging populations globally, it is likely that the number of older adults with depressive symptoms will continue to increase. Our findings showed that community-dwelling Korean older adults followed five distinct depressive symptom trajectories over 10 years. Our findings indicated that low educational level, no current employment, decreased K-MMSE scores, more chronic diseases, and low frequency of regular social activities were related to an increased risk of depressive symptoms among community-dwelling Korean older adults. Health care providers should periodically screen older adults for depressive symptoms, and interventions should be developed to address individual risk factors. Depression management and prevention programs by local health managers should be applied through screening in clinics and hospitals for older adults belonging to trajectories that may be headed toward increased depression. Health care providers should consider our findings to prevent depressive symptoms among older adults. Furthermore, given that the suicide rate among older adults in South Korea is the highest among OECD countries, we recommend that policy makers and other relevant stakeholders establish a national monitoring system for depression among older adults.


Deep-sea organisms must have proteins that function under high hydrostatic pressure to survive. Adaptations used in proteins from “pressure-loving” piezophiles may include greater compressibility or greater stability against pressure-induced destabilization. However, while greater compressibility can be accomplished by greater void volume, larger cavities in a protein have been associated with greater destabilization and even unfolding as pressure is increased. Here, computer simulations of dihydrofolate reductase from a moderate piezophile and a hyperpiezophile were performed to understand the balance between adaptations for greater compressibility and those against pressure destabilization and unfolding. The results indicate that while compressibility appears to be important for deep-sea microbes, adaptation for the greatest depths may be to prevent water penetration into the interior.Moritella profunda (Mp) isolated at ~2.9 km in depth and from a hyperpiezophile Moritella yayanosii (My) isolated at ~11 km in depth were compared using molecular dynamics simulations. Although previous simulations indicate that MpDHFR is more compressible than a mesophile DHFR, here the average properties and a quasiharmonic analysis indicate that MpDHFR and MyDHFR have similar compressibilities. A cavity analysis also indicates that the three unique mutations in MyDHFR are near cavities, although the cavities are generally similar in size in both. However, while a cleft overlaps an internal cavity, thus forming a pathway from the surface to the interior in MpDHFR, the unique residue Tyr103 found in MyDHFR forms a hydrogen bond with Leu78, and the sidechain separates the cleft from the cavity. Thus, while Moritella DHFR may generally be well suited to high-pressure environments because of their greater compressibility, adaptation for greater depths may be to prevent water entry into the interior cavities.Proteins from “pressure-loving” piezophiles appear to adapt by greater compressibility via larger total cavity volume. However, larger cavities in proteins have been associated with lower unfolding pressures. Here, dihydrofolate reductase (DHFR) from a moderate piezophile  TG and growth pressure PG) of the microbe [The discovery of life thriving under extreme conditions of temperature, pressure, and composition has led to intriguing questions about the limits at which life can survive. Mechanisms used by “extremophiles” to adapt their biological macromolecules to these extremes could assist in our understanding of these limits of life. Studying the sequence-structure-function relationship of proteins from extremophiles compared to proteins from organisms living under ambient conditions, “mesophiles,” is useful in understanding adaptations used to maintain functional enzymes under all conditions . So far, microbe  and that microbe  is mainl microbe ,5, and “ microbe ,7, studiFor proteins, the effects of pressure are compression and denaturation . Below 4Numerous studies of pressure unfolding of proteins have shown that larger cavity size within a protein apparently leads to lower pressure stability  because Shewanella benthica (Sb) with that from the mesophile Shewanella oneidensis (So) find a larger total cavity volume in SbIPMDH, although this was attributed to more numerous small cavities rather than larger cavities [However, adaptations for smaller cavity volumes in piezophile proteins have not been found. In fact, comparisons of crystal structures of 3-isopropyl malate dehydrogenase (IPMDH) from the obligate piezophile cavities . Furthercavities  and singcavities  find thaTG = 37 °C, PG = 1 bar) Escherichia coli (Ec) with that from the moderate psychropiezophile  [Moritella DHFR so far, it is not in other piezophile DHFR, indicating it may allow but is not necessary for high-pressure activity [Pu) at 25 °C is 2.7 kbar for apo-EcDHFR but only 0.7 kbar for apo-MpDHFR, indicating MpDHFR is actually more sensitive to pressure denaturation than EcDHFR [Dihydrofolate reductase (DHFR) is an ~160 residue, ~20 kDa enzyme that is a prime target for comparative studies of extremophile proteins because it is ubiquitous. DHFR catalyzes the hydride transfer and protonation of dihydrofolate (DHF) from the coenzyme NADPH to form tetrahydrofolate (THF), an essential precursor in the purine biosynthesis pathway ,20. Conf 220 bar ) Moritelnda (Mp) ,27,28, anda (Mp) ,30,31. Aactivity . In addiactivity . In addin EcDHFR . Since mn EcDHFR .δ hydrogen bond under pressure leads to the monotonic pressure deactivation in EcDHFR by overcorrelating collective motions while strengthening of the weak Thr113 O…Glu27 Oε hydrogen bond to a strength similar to EcDHFR at 1 bar leads the pressure activation in MpDHFR and D27E EcDHFR.Molecular dynamics (MD) simulation can provide an important molecular perspective to experimental studies. Our previous MD simulation studies of EcDHFR and MpDHFR showed that MpDHFR had higher overall atomic fluctuations than EcDHFR, and pressure appeared to increase fluctuations ,36. The σ02, an intrinsic isobaric expansivity αP, and an intrinsic isothermal compressibility κT. At 279 K, 1 bar, the QHA indicates MpDHFR (κT = 76 × 10−3 / kbar) was more compressible compared to EcDHFR (κT = 67 × 10−3/kbar) [In addition, since the compressibility of a protein is difficult to measure, a quasiharmonic analysis (QHA) is a method based on computer simulations that allows another assessment of compressibility. A QHA probes the local energy landscape through a series of short simulations at a variety of temperatures and pressures around a reference set of conditions . The eff−3/kbar) , consistMoritella [Moritella DHFR may have been adapted for the cold by having fewer interactions so that they are more flexible, but that this adaptation may also be fortuitously favorable for high pressures by making them more compressible. However, weaker interactions also would lead to lower stability under either or both higher temperatures or higher pressures. DHFR from Moritella yayanosii (My), which has an optimum TG = 10 °C and PG = 800 bar [Pu = ~700 bar [Finally, our previous sequence comparison and molecular dynamics studies of DHFR from oritella  indicate 800 bar  but is f 800 bar  in contr~700 bar . Notably~700 bar ,30 of MyM. profunda and hyperpiezophile M. yayanosii were performed to explore adaptations for high pressure, focusing on sequence differences between these homologous enzymes. Note that a consensus sequence numbering of aligned Moritella DHFR sequences with E. coli DHFR is used in this text , a presumed analog of the Michaelis complex, were obtained from the PDB [+ and folate were modified to the Michaelis complex cofactor NADPH and substrate DHF, respectively, using Ligand Reader and Modeler [M. profunda and M. yayanosii DHFR at T = 279 K and P = 1, 800 bar were performed using the molecular mechanics package OpenMM [NPT ensemble using a leapfrog Verlet integrator with a time step of 0.001 ps, Andersen thermostat [NVT ensemble using a velocity Verlet integrator with a timestep of 0.001 ps and a Nosé-Hoover thermostat [Methods have been described previously , so they is used . Residue the PDB . The fir the PDB  in PDB R Modeler  in CHARM Modeler ,47, wate Modeler , the lig Modeler , and cof Modeler . MD simue OpenMM . Each sye OpenMM . Heatingermostat  and Montermostat . Afterwaermostat ,56,57,58rHA2〉 were calculated within 10 ns blocks with respect to the average structure within each block and then averaged over all blocks. The mean-squared fluctuations of Cα atoms, 〈ΔrCα2〉, were calculated from the entire 50 ns production run with respect to the average structure over the entire production run.Average properties were calculated from coordinates written at 1 ps intervals except as noted. Averages and standard deviations were calculated by block averaging over 10 ns blocks. The mean-squared fluctuations of the protein-heavy atoms 〈ΔHydrogen bonds were defined as having a distance between the donor hydrogen atom and acceptor atom smaller than 2.40 Å  and an a3 were used. Volumes were calculated from structures at every 5 ns from the 50 ns simulation. The average volume and root mean square (RMS) fluctuations in volumes for each cavity or cleft were then obtained from volumes over the ten structures, where volumes less than the 1.0 Å3 cutoff were given a volume of 0 Å3. Since many cavities transitioned between cavities and solvent-accessible clefts, they were termed as “cavity” or “cleft” based on the larger population.Cavities and clefts were calculated using defaults, except, as noted, using McVol . A searcP-T grid of short, 1 ns simulations in the NPT ensemble (with coordinates saved every 0.1 ps) starting from the end of the 5 ns NVT equilibration at P = 1 bar, T = 279 K. This P–T grid was comprised of all combinations of P = 1, 2500, 5000, 7500, and 10,000 bar and T = 40, 80, 120, 160, 200, 240, 280, 320 K.A quasiharmonic analysis ,40 from σ2, were calculated for each of the 1 ns P−T grid simulations. σ2 were calculated by averaging fluctuations with respect to the average structure of each 10 ps interval and then performing a second average over the 100 × 10 ps intervals in the 1 ns simulation. The fits to the quasiharmonic equations were performed in gnuplot. The reference state is P0 = 1 bar and T0 = 279 K. First, σ2, from the MD simulation data at all pressures and T ≥ 200 K were fit using Equation (1) to σ02, Pα,0, and Tκ,0.The average fluctuations, σ2 from the MD simulation data for all pressures and T < 200 K were fit using Equations (2) and (3) to find values for Tg,0 and c.Next, Moritella DHFRs in the 50 ns MD simulation are similar and do not change much with pressure  O hydrogen bond within helix F in MpDHFR is replaced by the Tyr103 (105) Oη…Leu78 (80) O hydrogen bond between helix F and E in MyDHFR, especially at 800 bar  in MyDHFR than MpDHFR. Interestingly, the thermal property αP,0 is nearly identical between the two Moritella DHFRs, while the compressibility κT,0 is slightly greater for MyDHFR than MpDHFR, and barriers between wells Tg,0 are somewhat lower for MyDHFR than MpDHFR  of MyDHFR, which is at the end of the FG loop, and cleft 5 is embedded within the Met20 and FG loops, adjacent to the unique residue 119 (121) of MyDHFR. Average volumes of cavities 3, 4, and 5 are similar for both proteins and appear to be largely pressure-independent. While cavities 3 and 4 are not observed in the crystal and starting structures , this maied here . MpDHFR σ02) and lower barriers between wells  as well as slightly larger gR. However, the difference in the intrinsic isothermal compressibility between the two of 5 × 10−3/kbar is much less than between EcDHFR and MpDHFR of 9 × 10−3/kbar. Note that the previous work comparing compressibilities [Pressure leads to a decreased total volume of the system, which can occur by decreasing the total cavity volume or increasing the hydration of cavities. QHA indicates that the potential energy landscape of MyDHFR may be slightly more compressible than MpDHFR due to slightly shallower potential energy wells  that fortuitously also makes them function better at high pressure. However, while greater flexibility/compressibility is generally accomplished by fewer or weaker interactions, there may be a limit to how much the flexibility/compressibility can be increased while maintaining the three-dimensional fold so that these two DHFRs may have reached that limit. Moreover, since MpDHFR begins to show deactivation at ~500 bar and unfolding at ~700 bar, the higher pressures that MyDHFR experiences are more likely to result in water penetration. Thus, the evolutionary driver may be preventing water penetration rather than increasing compressibility. Since cleft 2 and cavity 2 can form a pathway from the surface to the interior in MpDHFR, the unique Tyr103 (105) in MyDHFR, which forms a hydrogen bond that prevents the merging of cleft 2 and cavity 2, may be an adaptation to block water from reaching the interior by this pathway.


Aspergilli section Nidulantes series Versicolores are recurrent in bioaerosols and are mainly responsible for allergies and asthma aggravation. Phylogenetic studies recently identified 12 new species within this series. This study is the first to identify species of Aspergillus series Versicolores in French bioaerosols and to characterize them macroscopically, microscopically and molecularly. Bioaerosols were collected in a cancer treatment center, in contaminated homes and in agricultural environments. A total of 93 isolates were cultured on selective media, observed by optical microscopy and identified by benA amplification before sequencing. The field data (temperature and relative humidity) were statistically tested to explore the ecology of these species. Eight species were identified from bioaerosols: Aspergillus creber and A. jensenii, which represent more than 80% of the isolates, and A. protuberus, A. puulaauensis, A. sydowii, A. tabacinus, A. amoenus and A. fructus. Aspergilli series Versicolores are distributed differently depending on the sampling site and climatic determinants. Aspergillus protuberus was found in bioaerosols collected under significantly lower relative humidity (p = 3.899 × 10−4). Characterization and repartition of these isolates belonging to the Versicolores series constitute an important step to better assess exposure to fungal bioaerosols.Air quality can be altered by fungal contaminants suspended in the air, forming bioaerosols.  Over the last decades, air quality has become a major public health issue, especially in industrialized countries. It is estimated that the population of these countries (including France) spends up to 90% of its time indoors ,2, whichAspergillus belonging to the series Versicolores section Nidulantes are found in hypersaline water [Aspergillus versicolor was considered until a few years ago as the most recurrent species of the Versicolores series present in bioaerosols. Recent phylogenetic studies [Versicolores series. Among these new species, Aspergillus creber is considered as the most frequent species and was misidentified as Aspergillus versicolor [Versicolores series in different matrices seem to confirm this hypothesis [Versicolores series do not all produce the same metabolites and are not as frequent in human pathology as each other [Molds of the genus ne water , in soilne water , in varine water  and in ine water . These sne water , aggravane water  and be ine water . They arne water  or pulmone water . More rene water . These ane water , a mycotne water . The spe studies ,27 have rsicolor . The fewpothesis ,30. We ach other .Aspergillus series Versicolores collected from French bioaerosols in order to evaluate their diversity and contribute to fungal exposure to Aspergillus series Versicolores.In this study, we characterize and describe 93 environmental isolates of n = 24) [n = 65) [n = 4) [Serpula lacrymans-damaged homes [Bioaerosols were collected indoors from a cancer treatment center  (n = 24)  and in c[n = 65) ,33, and  [n = 4) ,35. Duried homes .Aspergillus isolates belonging to the Versicolores series (n = 93) were stored on slant agar at −4 °C and in a cryoprotective agent composed of sterile water  and 10% glycerol  at −80 °C before characterization.Samples were cultured on Malt Extract Agar medium with 0.02% chloramphenicol  (MEA+). Plates were incubated at 25 °C and checked daily. Each fungal colony was isolated and purified on the same medium. All DNA extraction was performed using a modified protocol of the Nucleospin™ Plant II kit . Fungal colony was introduced in a 2 mL microtube with glass beads. The microtube underwent two incubation cycles of 15 min at 80 °C and then 15 min at −80 °C. It was placed into a Qiagen Tissue Lyser with 400 μL of lysis buffer PL1  for 15 min at 20 Hz, and incubated with 10 µL of RNAse  and 20 μL of proteinase K at 10 mg/mL  at 65 °C for 15 min. Then, 400 μL of chloroform  was added to the mixture. The aqueous phase was recovered and extracted using the precipitation kit and washing buffer according to the protocol described by the supplier.DNA was purified using the NucleoSpin gDNA Clean-up kit  following the instructions of the manufacturer. For each isolate, quantification and quality of purified DNA were realized using a NanoDrop 2000 spectrophotometer  .benA) using Bt2a/Bt2b (5′-GGTAACCAAATCGGTGCTGCTTTC-3′/5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) primers [Aspergillus species of the Versicolores series  [Molecular characterization was performed by amplification of the beta-tubulin gene , Czapek medium (CZ), Czapek Yeast Autolysate Agar (CYA25) and Czapek Yeast Extract Agar with 20% sucrose (CY20S) at 25 °C and on CYA at 37 °C (CYA37). Conidia from 7- to 14-day-old cultures were mixed in soft agar ). Each medium was inoculated in three points equidistant from the center with 2 µL of this spore suspension. After 7 days of culture, the diameters, colors (given as hex color codes), textures and reliefs of the colonies were noted. After 14 days of culture, the presence or absence of exudate and diffusible pigment was noted [Macroscopic characterization was performed by culture of the as noted .Microscopic characterization was performed with an optical microscope Olympus CX31  after 10 days of culture on MEA, or on CY20S in case of low sporulation on MEA. For each isolate, the appearance and size of vesicles, the size of metulae, phialides and conidia, the texture of conidia, the color of fungal structures and the presence or absence of Hülle cells were specified ,38.p < 0.05 were considered statistically significant. All statistical analyses were realized using XLSTAT .Descriptive statistics were calculated to provide information on isolates, and environmental data were subjected to the Mann–Whitney test. Only results at Aspergilli series Versicolores were ranked fifth in bioaerosols from the cancer treatment center, first in Serpula lacrymans- and mold-damaged homes and third in agricultural environments, with concentrations ranging from 2.48 CFU/m3 to 3.44 × 105 CFU/m3 (mean: 9.30 × 103 CFU/m3). Aspergilli series Versicolores were associated with 109, 100 and 28 other fungal species in bioaerosols collected from the cancer treatment center, Serpula lacrymans- and mold-damaged homes, and in agricultural environments, respectively. The most represented genera in all these environments were Aspergillus , Penicillium  and Cladosporium  [Among all the mold species found in the different bioaerosols, spermum) ,32,33,34n = 93), we were able to molecularly identify 8 species of the Versicolores series: Aspergillus creber (43%) ranked first, followed by A. jensenii (40%), A. protuberus (7%), A. puulaauensis (4%), A. sydowii (2%), A. tabacinus (2%), A. amoenus (1%) and A. fructus (1%). Bioaerosols collected in the cancer treatment center (n = 24) contained more A. jensenii (58%), followed by A. protuberus (25%), A. creber (13%) and A. puulaauensis (4%). A. protuberus was only found in bioaerosols collected in the cancer treatment center. Bioaerosols collected in Serpula lacrymans- and mold-damaged homes (n = 65) were mainly composed of A. creber (57%) and A. jensenii (34%). A. puulaauensis (5%), A. sydowii (3%) and A. amoenus (2%) were also present. Among the four isolates obtained from bioaerosols collected in agricultural environments, we identified two isolates of A. tabacinus, one isolate of A. jensenii and one isolate of A. fructus. Aspergillus tabacinus and A. fructus were only found in bioaerosols collected in agricultural environments. A. jensenii was the only species found in all environments. We also observed a similar distribution of Versicolores species within the contaminated homes independently in the sampling campaign with the highest species richness.As shown in Aspergillus amoenus, A. fructus and A. tabacinus were found in bioaerosols collected at low temperatures, while A. protuberus and A. sydowii were found in bioaerosols collected at higher temperatures. Aspergillus fructus, A. protuberus and A. tabacinus were also found in bioaerosols collected with low relative humidity, while A. amoenus, A. creber and A. sydowii were found in bioaerosols collected with high relative humidity. It should be noted that only Aspergillus protuberus was found in bioaerosols collected in the cancer treatment center with a significantly lower relative humidity than in bioaerosols not containing Aspergillus protuberus (p = 3.899 × 10−4).The temperature measured in these different environments varied between 9.4 and 26.1 °C, and the relative humidity between 23.8% and 74.3% . AspergiAspergillus species in the series Versicolores , mycelium was white to buff, reverse uncolored to yellow brown (#c1a457), texture was velutinous to granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies flat with an entire margin, conidia were pale greyish green to greyish green (from #d9cd97 to #a8a975), mycelium were white to buff, reverse yellow brown (from #c89847 to #c59951), and texture was velutinous to granular; when present, exudate was uncolored or brownish orange to brownish red (from #63512a to #4b210f), and when present, soluble pigment was orange (#ba8d2e). On CY20S, circular and umbonate colonies radially sulcate with an entire margin, conidia grey to greyish green (from #a8a687 to #9ea067), mycelium was white to buff, reverse brownish yellow to orange (from #d1b236 to #c29833), texture was velutinous and there was no exudate or soluble pigment. On CYA25, circular to irregular and flat colonies radially sulcate with an entire margin, conidia were dull green to greyish green (from #a6b27a to #989467), mycelium was white to buff, reverse brownish yellow to orange (from #d1b236 to #c29833), texture was velutinous to granular and exudate was uncolored to brownish orange (#be985d); when present, soluble pigment was brownish orange (#c59145). On CYA37, there were dense, convex and crateriform colonies with an entire margin, no conidiation with mycelium white to pale brown (#cdb489), reverse brown (#a27931), texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus amoenus isolates. Conidia were 3–4 µm in diameter, mostly globose to sub-globose, with finely or distinctly roughened walls. No globose Hülle cells or chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform vesicles 12–15 µm in diameter, biseriate, metulae of 4–6 µm covering all the vesicles and phialides of 5–6 µm. No diminutive conidial head was observed for n = 40 isolates , CZ (8–) 10–16 (–17), CY20S (18–) 19–26 (–28), CYA25 15–22 (–25) and CYA37 0–6 (–8).On MEA, there circular and flat colonies with an entire margin, conidia yellow to dark green (from #e3bc25 to #52582a), mycelium was white to buff, reverse uncolored to orange (#eea620), texture was velutinous to granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies with an entire margin, conidia were pale yellow or dull green to greyish green (#ffdc61 or #beb790 to #77723c), mycelium was white to buff, reverse orange to brownish red (from #be7329 to #602909), texture was velutinous to granular; when present, exudate was yellow, brownish red, brown or black  and soluble pigment was orange pink to dark pink (from #dfa06b to #b63346). On CY20S, circular and flat colonies sometimes radially sulcate with an entire margin, conidia were beige to greyish green (from #e0bb74 to #6b6023), mycelium was white or buff to brownish orange (#905a21), reverse brownish orange to brownish red (from #cc9700 to #501a00), texture was velutinous; when present, rare exudate was yellow to brownish orange (#eccc53 to #894d20) and soluble pigment was dark red (#a53000). On CYA25, circular and flat colonies often radially sulcate with an entire margin, conidia were yellow beige or dull green to greyish green (#f2cb67 or #646945 to #76774e), mycelium was white to buff, reverse brownish orange (#d57a18), texture was velutinous to granular; when present, exudate was brownish with shades from yellow to red or black (#a3884a to #501c01 or #170d03), and when present, soluble pigment was dull orange to dark pink (#e8a052 to #8e1c24). On CYA37, when present, colonies were circular and flat with an entire margin, white to dull orange (#db9441), reverse brownish yellow to brown (#ca9329 to #6c4b0a), texture was velutinous and there was no exudate; when present, soluble pigment was brownish orange (#a46016).Microscopic characteristicsAspergillus creber isolates. Conidia were of 2.5–4.5 µm in diameter, mostly globose to sub-globose, with finely or most of the time distinctly roughened walls. Hülle cells (12–14 µm) were found, but no chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform vesicles of (3–) 5–13 (–15) µm in diameter, biseriate, metulae of (3–) 4–6 (–7) µm covering two thirds to all the vesicles and phialides of 4–6 (–8) µm. Diminutive conidial heads were observed for n = 1 isolate , mycelium was white to buff, reverse uncolored, texture was granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies with an entire margin, conidia were greenish (#ac9f37) with inconstant shades of dull pink (#d1aa98), mycelium was white to buff, reverse brownish red (#5c2e04), texture was velutinous to granular, exudate was greenish yellow or brownish orange to brownish red (#bdaa30 or #a77d00 to #6a2e00) and there was no soluble pigment. On CY20S, circular and umbonate colonies sometimes radially sulcate with an entire margin, conidia were greenish (#a89950), mycelium was white or buff, reverse dull orange (#d3b072), texture was velutinous to granular and there was no exudate or soluble pigment. On CYA25, circular and flat colonies often radially sulcate with an entire margin, conidia were greyish green (#78681d), mycelium was white to buff, reverse brownish orange (#a35f00), texture was velutinous to granular; when present, exudate was brown (#553400), and there was no soluble pigment. On CYA37, when present, colonies were white and wrinkled and flat with an entire margin, reverse brownish yellow (#b59e73), texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus fructus isolates. Conidia of 3–4 µm in diameter were mostly globose or globose to sub-globose, with smooth or finely roughened walls. No Hülle cells or chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform or spathulate vesicles of 12–13 µm in diameter, biseriate, metulae of 4–6 µm covering two thirds to all of the vesicles and phialides of 5–6 µm. No diminutive conidial heads were observed for n = 37 isolates  3–13 (-18), CZ (6–) 8–14 (–16), CY20S (11–) 16–25, CYA25 (8–) 12–18 (–20) and CYA37 0–5 (–8).On MEA, there were circular and flat colonies with an entire margin, white to yellow (#eec64e), mycelium was white, reverse uncolored to orange (#f6b54f), texture was granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies with an entire margin, conidia were greenish (#a5aa79), mycelium was white, reverse brownish pink to brownish red (#d39f72 to #68260f), texture was granular; when present, exudate was orange to brownish orange (#fa6a00 to #b9611a), and when present, soluble pigment was beige to brownish orange (#e1b589 to #c78107). On CY20S, circular and flat colonies radially sulcate with an entire margin, conidia were pale yellow to dull green (#f2dd9e to #a2a462), mycelium was white, reverse orange red (#9c3d17), texture was velutinous and there was no exudate; when present, soluble pigment was orange red to brown (#b54400 to #341000). On CYA25, there were circular and flat colonies with an entire margin, conidia were greyish green (#a7ac75) with non-constant pale orange shades (#d9bd81), mycelium was white, reverse dark orange to brown (#bf6327 to #6f4215) and texture was velutinous; when present, exudate was orange pink to brown (#fbbe82 to #644533), and when present, soluble pigment was orange (#c66927). On CYA37, when present, there were circular and convex white colonies with an entire margin, reverse brownish yellow (#e0ad5a), texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus jensenii isolates. Conidia of (2–) 3–4 (–5) µm in diameter were mostly globose or globose to sub-globose, with mostly roughened or finely roughened walls. Hülle cells (12–16 µm) and chlamydospores were observed.Conidial heads radiate uncolored to yellow orange vesicles expanding into pyriform or spathulate vesicles of (3–) 7–14 µm in diameter, biseriate, metulae of 4–6 (–7) µm covering two thirds to all of the vesicles and phialides of 4–7 (–8) µm. Diminutive conidial heads were observed for n = 6 isolates .On MEA, there were circular and flat colonies with an entire margin, conidia yellow to greyish green (#e3bc63 to #a0904d), mycelium was white to buff, reverse uncolored to orange (#d5a553), texture was granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies with an entire margin, conidia were greyish green or buff (#ac9c56 or #b08d61), mycelium was white, reverse brownish orange (#6b350f), and texture was velutinous; when present, exudate was brown with orange or red shades (#7d4e16 to #531b00), and when present, soluble pigment was pink (#e09a7a) or brownish orange (#a56719). On CY20S, circular and flat or crateriform colonies radially sulcate with an entire margin, conidia were yellowish to greyish green (#ddce61 to #988c62), mycelium was white, reverse brownish orange (#b8671c), texture was velutinous and there was no exudate; when present, soluble pigment was orange to brownish red (#621400). On CYA25, circular and flat colonies often radially sulcate with an entire or undulate margin, conidia were brown to greyish green (from #ae9166 to #aaa581), mycelium was white to buff, reverse brownish orange to brownish red (from #8d531a to #4d2108), and texture was velutinous to granular; when present, exudate was brown (#3e1800), and when present, soluble pigment was orange pink to brownish orange (from #f5b67e to #ac4b00). On CYA37, when present, colonies were dense and convex white to beige (#d3b37c) with an entire margin, reverse brownish yellow (#c5a43b), texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus protuberus isolates. Conidia of 2.5–3.5 µm in diameter were mostly globose to sub-globose, with smooth or finely roughened walls. Hülle cells (14 µm) were found but no chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform or spathulate vesicles of 11–16 µm in diameter, biseriate, metulae of 3–5 µm covering all of the vesicles and phialides of 4–7 µm. No diminutive conidial heads were observed for n = 4 isolates , mycelium was white, reverse uncolored to orange (#a85907), texture was granular and there was no exudate or soluble pigment. On CZ, there were circular and flat colonies with an entire margin, conidia were greyish green (#b6a35b) with orange shades (#daa74e), mycelium was white, reverse brown (#5f412c), texture was granular, exudate was brownish orange (#cc5c00) and soluble pigment was beige to orange (#d78e69 to #d17b19). On CY20S, circular and flat or colonies radially sulcate with an entire margin, conidia were greyish green (#9e8d52), mycelium was white, reverse brownish orange (#8d4d1f), texture was velutinous and there was no exudate; when present, soluble pigment was orange (#db7314). On CYA25, circular and flat colonies radially sulcate with an entire margin, conidia were greyish green to dark green (from #72815c to #354532), mycelium was white, reverse brownish orange (#7f4b1a), and texture was velutinous; when present, exudate was brownish orange to black (#a64601 to #0f0b00), and when present, soluble pigment was orange pink to pale orange (#e3935d). On CYA37, when present, there were dense and convex white colonies with an entire margin, reverse uncolored, texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus puulaauensis isolates. Globose green conidia were of 3–4.5 µm in diameter, with roughened walls. No Hülle cells or chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform vesicles of 9–15 µm in diameter, biseriate, metulae of 4–6 µm covering all of the vesicles and phialides of 5–6 µm. No diminutive conidial heads were observed for n = 2 isolates  14–21, CZ 12–22, CY20S (14–) 21–34, CYA25 16–26 (–31) and CYA37 0–18.On MEA, there were circular and flat colonies with an entire margin, conidia were dark blue (#353a40), mycelium was white, reverse uncolored, texture was velutinous to granular and there was no exudate; when present, soluble pigment was orange (#c5792a). On CZ, there were circular and flat colonies with an entire margin, conidia were blue green (#404a45), mycelium was white, reverse brown (#463b2a), and texture was granular; when present, exudate was yellowish to brownish red (#cdac3f to #732f00), and when present, soluble pigment was dull yellow to dull orange (#e5c069 to #c08c35). On CY20S, circular and flat or colonies sometimes radially sulcate with an entire margin, conidia were greyish blue (#5f6a65), mycelium was white, reverse brownish orange (#975f04), and texture velutinous; when present, exudate was black (#12110c), and when present, soluble pigment was pale orange (#d9ad4e). On CYA25, there were circular and flat colonies with an entire margin, conidia were greyish blue (#81827a), mycelium was white, reverse brown (#7e5631), and texture was velutinous; when present, exudate was brownish orange to brownish red (#c0761b to #762f00), and when present, soluble pigment was brownish yellow (#c18919). On CYA37, colonies were dense and convex or wrinkled and crateriform with an entire margin, conidia were grey to dark grey (#aca38a to #554e33), sometimes with shades of brown (#8d6d4a), reverse brownish black (#1b1612), and texture velutinous; when present, exudate was brownish orange (#93520e), and there was no soluble pigment.Microscopic characteristicsAspergillus sydowii isolates. Globose conidia were of 3–4 (–5) µm in diameter, with roughened to distinctly roughened walls. Chlamydospores were found but no Hülle cells were observed.Conidial heads radiate brown to greyish blue vesicles expanding into globose to spathulate vesicles of 6–12 (–14) µm in diameter, biseriate, metulae of 4–7 µm covering two thirds to all of the vesicles and phialides of 5–7 (–8) µm. No diminutive conidial heads were observed for n = 2 isolates , mycelium was white, reverse orange (#ce7c11), texture was granular and there was no exudate; when present, soluble pigment was uncolored. On CZ, there were circular and flat colonies with an entire margin, conidia were dark beige to greyish green (#b89656 to #8a7429), mycelium was white to buff, reverse brownish orange (#975409), and texture was velutinous to granular; when present, exudate was yellowish to dark brown (from #d8c96e to #221600), and when present, soluble pigment was pale red orange (#b25128). On CY20S, circular and flat colonies often radially sulcate with an entire margin, conidia were greyish green (#ab934d) with non-constant shades of orange (#dea419), mycelium was white, reverse orange (#e36a15), texture was velutinous and there was no exudate or soluble pigment. On CYA25, circular and flat colonies radially sulcate with an entire margin, conidia were dark beige to greyish green (#e9bc73 to #7d6b14), mycelium was white, reverse brownish orange (#8f3d00), and texture was velutinous; when present, exudate was yellowish to dark brown (from #cfac52 to #0f0b02), and when present, soluble pigment was dull orange (#b2621d). On CYA37, when present, colonies were circular and flat with an entire undulate margin, white, reverse brown (#583306), texture was velutinous and there was no exudate or soluble pigment.Microscopic characteristicsAspergillus tabacinus isolates. Globose conidia were of 2.5–3.5 µm in diameter, with distinctly roughened walls. Hülle cells were found but no chlamydospores were observed.Conidial heads radiate uncolored to greyish green vesicles expanding into pyriform to spathulate vesicles of 9–14 µm in diameter, biseriate, metulae of 4–6 µm covering all of the vesicles and phialides of 5–7 µm. No diminutive conidial heads were observed for Aspergillus series Versicolores. This study constitutes the first report on the distribution of these species in French bioaerosols. Molecular identification by benA gene amplification revealed 8 different species among the isolates found in our bioaerosols, which is one of the highest species richness reported to date. Indeed, other studies conducted on isolates collected from indoor environments (air and dust samples) identified by CaM gene amplification show that the species richness within the Versicolores series ranges from 3  to 11 species  [Aspergillus creber and A. jensenii were the most frequently isolated species from French bioaerosols (83% of isolates), which is consistent with studies on Aspergillus species belonging to the Versicolores series that always find these species, and especially with Jakšić et al.’s study, where these two species represent 62% of the isolates [Aspergillus cvjetkovicii, A. griseoaurantiacus, A. pepii, A. tennesseensis or A. venenatus, which have been isolated from indoor air bioaerosols of the USA and Croatia [A. protuberus and A. puulaauensis are not as frequent but remain quite recurrent in indoor air and dust, as indicated by Micheluz et al. and Jakšić et al. [Aspergillus protuberus was only found in bioaerosols collected in the cancer treatment center with significantly lower relative humidity in comparison to that observed for isolates of other species (p = 3.899 × 10-4). Nevertheless, no other study on the Versicolores series provides information on the climatic conditions during sampling, which does not allow to support if low relative humidity can be considered as a factor facilitating the growth of A. protuberus. Measurement of climatic conditions (temperature and relative humidity) during sampling should be systematically performed to provide additional data to explore the fungal ecology of this species. Moreover, all this information could be useful to determine if Aspergillus protuberus is an indicator of healthy homes, as opposed to A. creber, which is prevalent in damp indoor environments. Aspergillus sydowii was only isolated from two bioaerosols collected in contaminated homes, which confirms its presence at a rather low frequency in air compared to food matrices or in human pathology [Aspergillus amoenus was isolated by Jurjevic et al. [Berberis sp. fruit) but also from indoor dust in Japan [Aspergillus tabacinus has been isolated from bioaerosols, whereas it is more frequently isolated from tobacco, brined meat or plants (maize) [Aspergillus fructus which had never been identified in bioaerosols, even though it has already been recovered from dust of water-damaged and control homes in Croatia. However, this species seems more commonly identified from fruits [Aspergillus versicolor stricto sensu was not found in our bioaerosols, which confirms the observations of Jurjevic et al. [Ninety-three fungal isolates isolated from bioaerosols collected in a cancer treatment center, in contaminated homes and in agricultural environments were identified macroscopically and microscopically as belonging to sydowii) ,39. Aspeisolates ,40. Howe Croatia ,29,40 bu Croatia ,30. A. pathology ,30,31. Ac et al.  from dif (maize) . We alsom fruits . Aspergic et al.  and KobaAspergillus creber, which were not described by Jurjevic et al. [We also reported the first observation of globose Hülle cells and the presence of diminutive conidial heads for c et al. .Versicolores series. Nevertheless, the most recurrent species Aspergillus creber and A. jensenii have distinct macroscopic and microscopic aspects: A. creber has a grey-green appearance, while colonies, hyphae, conidial heads and conidia of A. jensenii are yellow-colored.We observed, as previously described ,38, an iVersicolores and a better assessment of the exposure to bioaerosols containing these molds and their effects on health.Further studies will be conducted to explore the metabolic and toxicological profiles of these different species. An analytical approach using HPLC-MS/MS will allow the identification and quantification of target metabolites, while cytotoxicity assays on lung cells will provide supplementary information on the toxicity of the isolates. All these data will allow a better knowledge of the species belonging to the series


Although less frequent than in adults, taste loss also occurs in childhood. “Taste Strips” are frequently used for diagnosing taste dysfunction; however, normative values are lacking for children. In this study, we will create normative values for the “Taste Strips” in children.This cross-sectional study included 609 children aged 6–15 years. “Taste Strips” were used to determine sweet, sour, salty, and bitter taste scores by a non-forced procedure. The 10th percentile was used to distinguish normal taste function from a reduced sense of taste. Multivariable generalized linear models (GLM) were estimated to study the effect of age (group), sex, and 6‐n‐propylthiouracil (PROP) status on taste function.Taste function changed with age, allowing for a distinction of three age groups: (I) 6–7 years, (II) 8–9 years, and (III) 10–15 years. Normative values were created for the age groups and boys and girls separately. Additionally, GLM showed a significant effect of (1) age (group) on sweet, salty, bitter, and total taste scores; (2) sex on sweet, sour, and total taste scores; and (3) PROP status on total taste scores.This study provided normative values for the “Taste Strips” in children, highlighting age- and sex-related differences.Taste dysfunction can be harmful and impacts quality of life, a topic that became increasingly important since the COVID-19 pandemic.Although taste dysfunction is thought to be rare in childhood, the detrimental impact of such dysfunction might be large, as children’s eating habits are strongly influenced by input from the chemical senses.Measuring taste function may elucidate the relationship between taste dysfunction and disease, fostering the development of more appropriate supportive strategies. However, adequate tools are lacking for children.Normative values of the “Taste Strips” are now available for children, which bolster the clinical utility of this test. Medical conditions such as cancer, diabetes mellitus, kidney disease, and obesity are only a few examples of etiologies associated with chemosensory dysfunction in children, consequently affecting food choice, dietary intake, and overall health status.6 Measuring taste function may elucidate the exact relation between taste dysfunction and disease, fostering the development of more appropriate supportive strategies  in treating disease.7 Therefore, it is not a matter of debate that clinical assessment of taste function in children is needed.Little is known about taste dysfunction in children. In general, it is thought to be rare as chemosensory problems are typically associated with aging.8 Indeed, most patients complaining about taste dysfunction actually suffer from smell dysfunction.9 The “Taste Strips” test is a frequently employed and well-validated clinical taste test.10 Advantages of this test are its long shelf-life, easy administration, commercial availability, and short time of investigation. In the context of research, the “Taste Strips” have already frequently been used in children.13 However, normative values are hitherto restricted to individuals aged 15–87 years.14 Especially within a clinical setting, if taste function is expected to be compromised as a result of disease burden and/or treatment, one needs to be able to determine a child’s taste function and interpret these scores by using population specific normative values.In contrast to smell tests, few taste tests are available in a clinical setting. In part, this is due to the low prevalence of taste disorders (relative to smell disorders).16 Secondly, although most studies found no clear sex-related differences in taste function in children, there is some indication that girls outperform boys.20 Thirdly, the ability to taste bitter compounds such as 6-n-propylthiouracil (PROP), which can be partly explained by genetic variations in TAS2R38 polymorphisms, correlates with an increased intensity perception to taste stimuli and other orosensory sensations.22 However, it remains unclear whether PROP status is associated with taste scores as measured by “Taste Strips” in children.Several factors can modulate taste function in children that should be taken into account and needs further investigation concerning the “Taste Strips.” First of all, taste function seems to improve as a child grows older, although results differ per tastant. For example, sucrose intensity has been reported to increase from childhood to adulthood, but sensitivity to bitter compounds was found to be similar between children and adults.Thus, the present study aimed to (1) provide normative values for the “Taste Strips” from a large sample of healthy children, (2) study the effect of age, sex, and PROP status on taste scores in children, and (3) identify to what extent scores on the “Taste Strips” test are associated with self-reported taste function in children.This study was performed at the NEMO Science Museum Amsterdam, the Netherlands. All consecutive visitors to the museum were asked to participate. Participants were eligible for participation if they were between 5 and 17.99 years of age, able to understand Dutch or English, and reported to be healthy. Exclusion criteria for participation were: having a cold, smoking, being pregnant or a self-reported allergy to quinine. Parents provided written informed consent.Taste function was assessed by using “Taste Strips” . “Taste Strips” are filter-paper strips impregnated with a taste solution and determine sweet, sour, salty, and bitter taste scores. Four concentrations of each taste quality were used: sweet , sour , salty , and bitter . To each child, 16 impregnated strips and 2 blank strips were presented in the same pseudo-randomized order starting with the lowest concentration.Children were asked not to eat and drink except water 1 h before the test. Before the test began, taste qualities were explained by presenting photographs . “Taste Strips” were placed on the middle of the tongue, approximately 1.5 cm from the tip. Participants were then asked to close the mouth and indicate whether the perceived taste was sweet, sour, salty, bitter, or tasteless. Scores for each taste quality range from 0 to 4 and the total taste score was derived by summing the scores of each taste quality (range 0–16). A higher score represents a better taste function.23 Participants who answered “no” or reported that the strip “tastes like paper” were classified as “non-tasters.” Children who indicated that the strip tasted “bitter,” “sour,” “bad,” or “spicy” were classified as “tasters.” In addition, participants who immediately removed the strip because of its “foul” taste or showed other signs of taste rejection were classified as “tasters” as well.24PROP taster status was determined by a filter paper strip impregnated with PROP . Participants were instructed to place the strip on the dorsal surface of the tongue for approximately 30 s and were then asked whether they tasted anything (yes/no).Participants were asked to self-assess their taste function by rating their taste perception  and by estimating their taste function relative to their peers on a 5-point Likert scale (1 “much worse than others” to 5 “much better than others”).10 Non-parametric tests were used to assess differences between sex, age or self-reported measures in relation to taste scores. Bonferroni post hoc procedure to adjust for multiple testing was used when required. To study the association between taste scores  and the independent variables age (group), sex, and PROP status, multivariable generalized linear models (GLM) were estimated since the dependent variables were not normally distributed. To study the association between subgroups and outcome, interaction terms were created and included in the model if significant. An alpha level of 0.05 was used. Data analysis was performed with IBM SPSS Statistics (version 25.0).Descriptive statistics are presented as median with interquartile range (IQR) and the number of participants (n) with percentage (%). The 10th percentile was used to distinguish normal taste function from a reduced sense of taste .n = 23), as well as the small number of 16-year-old (n = 5) and 17-year-old participants (n = 3). Thus, 609 participants aged 6–15 years were included for final analysis.In total, 645 children aged 5–17 years participated. Figure Participants’ mean age was 9.3 ± 2.3 years; 365 of the participants were girls (60%). Overall, 541 participants (89%) were living in the Netherlands, 56 (9%) were living in other European countries, and 12 (2%) came from non-European countries.Median scores (IQR) for the individual taste qualities were as follows: sweet 4.0 (3.0–4.0), sour 3.0 (2.0–3.0), salty 4.0 (2.5–4.0), and bitter 4.0 (3.0–4.0). Median total taste score (IQR) was 13.0 (11.0–14.0). In total, 373 children (61%) were PROP tasters.p = 0.001) and positive correlation  between age and total taste score was observed, showing major age- and sex-related differences between 6 and 9 years of age. Based on these results, three arbitrary age groups were distinguished: group I, 6–7 years; group II, 8–9 years; group III, 10–15 years. A significant difference in sweet (p < 0.001), sour (p = 0.044), salty (p < 0.001), bitter (p < 0.001), and total taste score (p < 0.001) was found between the three age groups . For sour taste, no significant differences were found between age groups when adjusted for multiple testing.A sex difference , “better than others” by 125 participants (21%), “similar to others” by 447 participants (73%), “worse than others” by 16 participants (3%), and “much worse than others” by 3 participants (<1%).This is the first study that provides normative data for the “Taste Strips” from a large sample of children, divided into three age groups  and separated for sex. The present study also shows that taste scores (1) increased with age for sweet, salty, bitter, and total taste; (2) was higher in girls compared to boys for sweet, sour, and total taste scores; (3) was higher for PROP “tasters” compared to “non-tasters” regarding total taste scores; and (4) did not correlate with self-report.20 In addition, they compared 8–9-year-old children’s taste function with adults, showing no significant differences in taste function between 8–9-year-old girls and adults, but 8–9-year-old boys showed a poorer ability to detect sweet, sour, and salty taste than adult males and a poorer ability to detect all four taste qualities than adult females. A similar trend can be found in the current study, when comparing children’s taste scores with those previously found among adults.10 Our results corroborates prior findings, suggesting that taste is functionally mature around an age of 10 years and that taste function matures a bit faster in girls.25Age and sex differences in taste function among children were also found by James and colleagues, who showed higher detection thresholds for sucrose and sodium chloride in 8–9-year-old boys compared to 8–9-year-old girls when using freshly prepared taste solutions.26 They found that detection thresholds and recognition thresholds for sweet, sour, salty, bitter, and umami did not correlate with PROP bitterness, but also not with fungiform papillae density. In general, only detection and recognition thresholds were related, which highlights the complexity of identifying taste function. Thus, the current study found PROP status to be associated with taste function, but not to the extent that it can be seen as a measure of overall taste function in children.PROP status was associated with taste function in children, with higher total taste scores in “tasters’ relative to “non-tasters.” Although significant, this difference was moderate (0.6 points) and was not found for individual taste qualities. This is in line with a study that studied five common ways of measuring taste function  in women.27 For that reason, self-reported taste abilities, which can provide meaningful information to the clinician, should be always accompanied by a more objective test such as the “Taste Strips.”Self-reported taste function was not correlated with total taste scores. This indicates that children are not aware of their own taste function (or indeed dysfunction). One might argue that the absence of a correlation between self-reported taste function and performance on the “Taste Strips” test indicates that the “Taste Strips” are simply not valid, at least not in children. However, this also appears to be the case in adults as self-report of taste dysfunction has been shown inaccurate when using focused questions.10 This allows the participant to indicate a strip as “tasteless,” instead of being forced to choose between sweet, salty, sour, or bitter. Forced choice testing has the advantage of limiting response bias and malingering.29 The major disadvantage of a forced-choice method, however, is the inability to determine whether a “hit” or “miss” reflects an individual’s taste function or random guessing. As this information might be useful for the clinician if taste sensitivity is expected to be lost or changed, a non-forced choice method was chosen. Secondly, the 10th percentile was used to separate normogeusia from hypogeusia, according to the original protocol and originating from research into adults’ smell function.30 As we do not know how many children suffer from taste dysfunction, though presumably <10%, using the 10th percentile could lead to false-positive results and overdiagnosis. Using two standard deviations from the mean might be an alternative and more conservative approach.31 However, if this remains unclear, we have chosen to apply the commonly used 10th percentile as a cut-off value for children’s taste function, additionally considering their self-report and clinical symptoms.Two methodological choices should be noted. First of all, a non-forced choice paradigm was used, according to the original protocol for the “Taste Strips.”11Strengths of our study were the large sample size and wide age-range of included participants. In addition to Dutch children, also children from other European countries were included which increases the generalizability of our findings. However, some limitations should be noted. As mainly European children were included, it is uncertain whether these normative values extend to children outside of Europe. Furthermore, body weight or body mass index was not reported or measured, which could be a confounding factor as obesity is associated with a lower taste sensitivity in children.In conclusion, normative values for the “Taste Strips” were obtained for children aged 6–15 years, highlighting age and sex differences. These results bolster the clinical utility of the “Taste Strips” among children in a clinical setting, beyond its easy and quick administration.