Situs inversus totalis is a condition where there is a transposition of all internal organs from their normal anatomical location. This infrequent and rare congenital condition has been described in several species of mammals. Dextorcardia is a series of conditions associated with an abnormal congenital positioning of the heart, and is often associated with situs inversus totalis. Here we report a case of situs inversus totalis and two cases of dextrocardia identified in Sprague Dawley rats during gross necropsy evaluations at both the Health Sciences North Research Institute (HSNRI) in Canada and Finlay Institute of Vaccine Research and Production in Cuba. The intent of this report is to share our findings and aid in the accumulation of data on these rare conditions. Situs inversus totalis (SIT) is a rare congenital malformation characterized by the complete reversal (transposition) of the normal location of the thoracic and abdominal organs [l organs . SIT is l organs , within l organs . The exal organs . SIT doel organs ,6. SIT il organs .Dextrocardia is a broadly encompassing term for any congenital abnormality in the positioning and orientation of the heart . The oveIn addition to humans, there are reports of SIT and dextrocardia in different species of animal, both domestic and wild, including fish , pigs 11, cats , dogs 1,14,15, h® rat cages at the Animal Care Facility at Laurentian University. Interior dimensions: 141 square inch floor space, 7” height , outside dimensions: 17” L × 13.4” W × 7.8” H (maximum), 43.2 × 34.0 × 19.8 cm (maximum), 100% PET plastic and BPA-Free. Rats were provided specialized rodent feed , and Aquavive® acidified rat water was provided in bottles, which were both available ad libitum. The animal room was maintained at a temperature of 21 ± 2 °C and a relative humidity of 55 ± 5%. All protocols were approved by the Animal Care Committee at Laurentian University (Protocol #: 2016-09-02) and the Biosafety Committee at Health Sciences North Research Institute.Studies done at HSNRI involved female SD rats . The animals were housed in Innocage® rat cages at the Animal Care Facility at the Finlay Institute of Vaccine Research. Dimensions and model: 1354 G Eurostandard Type IV, 595 × 380 × 200 mm, floor area 1820 cm, PEI plastic and BPA-Free. Rats were provided specialized rodent feed , and acidified water (2.5–2.7 pH) was provided in bottles (750 mL volume). Both food and water were available ad libitum. The animal room was maintained at a temperature of 22 ± 2 °C and a relative humidity of 60 ± 5%. Blood hematology analysis was performed using an automatic analyzer  and serum chemistry was analyzed using a semi-automatic analyzer . All protocols were approved by the Laboratory Animal Care Committee (Protocol #: 16P03 and 17P01) and the Biosafety Committee at Finlay Institute of Vaccine Research and Production.Studies done at the Finlay Institute involved male rats , Havana, Cuba). The animals were housed in TecniplastThis case describes an observation of SIT discovered during the immunological evaluation of a novel nicotine vaccine developed by Dr. Hoang-Thanh Le and his team at the Health Sciences North Research Institute in Sudbury, Ontario, Canada. Routine pathological investigations were carried out on a group of female SD rats that were approximately 34 weeks of age at the time of euthanasia. SIT was identified incidentally in a single rat during gross necropsy. Macroscopic observations of the thoracic and abdominal cavities showed translocation of organs, including the heart, lungs, liver and spleen . ThroughThese cases describe two separate observations of dextrocardia discovered in male SD rats during preclinical toxicological evaluations at the Finlay Institute of Vaccine Research and Production in Havana, Cuba. Dextrocardia was incidentally identified in two separate rats  during gSIT and dextrocardia in humans is associated with a variety of other medical conditions, including Kartagener’s syndrome ,6,7,14. Here we report a case of SIT and two cases of dextrocardia observed in SD rats. In all three cases, the animals were healthy at the time of discovery, and displayed no evidence or clinical signs of the conditions. Our observation of SIT organ transposition in Case 1  is not tOur incidental discoveries of SIT and dextrocardia left us to determine if these anatomical abnormalities should be considered as exclusion criteria in our vaccine evaluation studies. In surveying the literature, there is no evidence to suggest that immunological and toxicological responses to a vaccine would be modified by the presence of SIT or dextrocardia when not associated with other medical conditions, nor would there be any impact of these anatomical abnormalities on organs at either the macroscopic or microscopic levels. The cases we reported agree with these findings, where other than the organ transpositions, there was no effect observed on any of the diagnostic parameters measured, including organ weights, as well as various serum chemistry and hematological parameters .SIT and dextrocardia are rare congenital abnormalities that have been reported in humans and various other animal species. Typically, the conditions are asymptomatic and often go undiscovered. There is no evidence to support that immunological and toxicological responses to a vaccine or drug would be impacted by these anatomical abnormalities when not associated with other medical conditions. Case reports such as this will aid in the accumulation of data on these rare conditions.


P = 4.58 × 10−9). By looking at differentially expressed genes between CMS patients and controls around these four loci, we suggested AEBP2, CAST, and MCTP2 as candidate CMS causal genes. None of the candidate loci were under strong natural selection, consistent with the observation that CMS affects fitness mainly after the reproductive years. Overall, our results reveal new insights on the genetic architecture of CMS and do not provide evidence that CMS-associated variants are linked to a strong ongoing adaptation to high altitude.Chronic mountain sickness (CMS) is a pathological condition resulting from chronic exposure to high-altitude hypoxia. While its prevalence is high in native Andeans (>10%), little is known about the genetic architecture of this disease. Here, we performed the largest genome-wide association study (GWAS) of CMS  to detect genetic variants associated with CMS. We highlighted four new candidate loci, including the first CMS-associated variant reaching GWAS statistical significance (rs7304081;  Chronic mountain sickness (CMS), or Monge’s disease, is a pathological condition resulting from chronic exposure to hypoxia at high altitude. The syndrome is characterized by an excessive number of red blood cells associated with a high blood hemoglobin concentration ([Hb]), hypoxemia, and, in some cases, pulmonary hypertension. Clinical signs include headache, fatigue, sleep disturbances, dyspnea, digestive complaints, and high risk of thrombotic events. The disease may appear during early adulthood, and turn into a highly prevalent condition mainly in men over 40 years old and in post-menopausal women. The clinical status becomes progressively incapacitating with cardiovascular complications, leading to social exclusion and psychological degradation . CMS shoLittle is known about the genetic architecture of CMS in Andean populations. We expect that this architecture is mainly driven by common genetic variants due to both the strong genetic component of [Hb] at high altitude (heritability greater than 0.8 in both Tibetan and Bolivian populations)  and the EPAS1  is associated with red blood cell abundance and has an allele frequency close to 90%, against 10% in Han Chinese  of this disease by successfully genotyping on extremely dense single nucleotide polymorphism (SNP) chips 166 CMS patients and 146 healthy subjects living at Cerro de Pasco in Peru  where CMS prevalence is around 15% . First, The study population was composed of 387 residents of the city of Cerro de Pasco . They were all high-altitude native of Quechua ancestry, as is most of the population of Cerro de Pasco. Only male individuals with residence longer than 6 months in Cerro de Pasco were recruited in the study. Individuals were not enrolled in the study if they had any other chronic disease or smoking habit (≥5 cigarettes per day). The study was approved by the Institutional Ethics Committee of Universidad Peruana Cayetano Heredia. All participants were enrolled in the study after signing an informed consent.1) were measured by spirometry . All values were within normal limits corrected for age, sex, and height. Blood was drawn from an antecubital vein. Hematocrit was evaluated by microcentrifugation , and pulse O2 saturation was measured by transcutaneous oximetry . 12-lead electrocardiography (EKG) was performed, and usual markers of coronary disease, conduction, or rhythm disorders were looked for. Indirect signs of pulmonary hypertension were detected through right ventricular hypertrophy . Systemic arterial pressure was measured by sphygmomanometry after a 15-min rest in supine position. A quality of life score, adapted and validated to Spanish language, assessed through a form, was completed by the patients under the supervision of a physician . After these steps, 133,649 SNPs remained. This set of SNPs was only used for population structure analyses and admixture analyses (see below).To study the structure of our sample, we used both the Human Genome Diversity (HGDP-CEPH) panel  and the Principal component analysis (PCA) was performed using PLINK. We first pruned the previous dataset to remove sites in linkage disequilibrium (still using PLINK option –indep-pairwise 50 5 0.50) and projected our 312 individuals on the 3,446 individuals of HGDP-CEPH and 1000G panels.K = 2 ancestral population on the 64 Americans individuals of HGDP-CEPH panel and the 503 Europeans of 1000G panel .The genome-wide proportion of European and Native American ancestries of individuals from our dataset was estimated with the software Admixture . To consEPAS1 haplotype has been shown to be associated with high-altitude adaptation in Tibet . We computed the genetic effect that we can detect with a power of 90% and the power to detect a genetic effect of 2.0, 2.5, and 3.0. To take into account the structure and the relatedness of our sample, we performed an association analysis using the standard linear mixed model implemented in GEMMA software (N = 21) and ii) the low level of European admixture in our dataset . Genotypes, age, and the batch effect were modeled as fixed effects, while relatedness was treated as a random effect. We did not use body mass index (BMI), right ventricular hypertrophy (RVH), and oxygen saturation (SaO2) as covariates , as there is no evidence that they are cause or consequence of the disease . Significance of each SNP was determined through a likelihood ratio test. We defined SNPs with a P value < 10−5 as candidate. When multiple candidate SNPs were in the same region, we only reported the top SNP with the smallest P value. We performed conditional analyses to verify that the significance of the surrounding candidate SNPs is due to linkage disequilibrium with the top SNP. Regression coefficients from the linear mixed model  were converted to odds ratio for easier interpretation as proposed by Lloyd-Jones et al.  and GAPDH  as reference. Expression levels were log transformed. We compared the expression between CMS patients and controls using a logistic regression with age as a covariate.Gene expression was measured for candidate genes in a subset of 71 unrelated individuals (30 CMS patients and 41 controls) using RNA extracted from whole blood under hypoxia condition . We manually selected candidate gene around the four candidate regions (based on known function of genes and by giving a higher emphasis to the region containing the genome-wide significant SNP). We also selected 10 additional genes reported in other studies as CMS candidate genes or genes being under adaptation to high altitude. Blood samples were withdrawn in PAXgen tubes and immediately frozen until analyses. After extraction, an RNA quality control was performed on Fragment Analyzer (AATI) with the RNA kit (DNF-471) to check the integrity of the RNA profile and to estimate the RNA concentration. A Reverse Transcription reaction was performed for each sample in 20 µl, according the conditions of the High Fidelity Reverse Transcription kit (Applied Biosystems). qPCRs were performed on the Biomark (Fluidigm) in a microfluidic multiplex 96.96 dynamic array chip according to the Fluidigm Advanced Development Protocol with EvaGreen (PN 100 – 1208 B1). One chip was used to quantify all transcripts. A 14-cycle preamplification reaction was performed for each sample in 10 µl by pooling 48 primer pairs , 3.3 µl cDNA, and 5 µl 2X PreAmp Master Mix (Applied Biosystems) according to Applied Biosystems conditions. For each individual assay, 5 µl 10X Assay Mix containing 9 µM forward primer, 9 µM reverse primer, and 1X Assay Loading Reagent was loaded into one of the Assay Inlets on the chip. The following solution (5 µl) was loaded in sample inlets: 1.25 µl Preamplified sample previously diluted in low TE Buffer, 2.5 µl 2X Taqman Gene Expression Master Mix (Applied Biosystems), 0.25 µl 20X DNA Binding Dye Sample Loading Reagent , 0.25 µl 20X EvaGreen (Biotium), and 0.75 µl low TE Buffer. The Biomark’s default cycling program was used to amplify fragments. All experiments were done in four replicates. CT values were obtained using BioMark Gene Expression Data Analysis version 3.0.2 according to Fluidigm’s recommendations for EvaGreen Gene Expression. Gene expression was normalized using P value was computed as the proportion of SNPs in the sample having a lower or equal index.Selection tests were performed on the subset of 124 unrelated controls. For all SNPs with a MAF above 5% in the whole sample, we first computed, using the selscan program , the iHSP value inside the gene body. We also replicated our analyses by retaining the minimal P value in +/−20 kb and +/−50 kb windows around the genes to include regulatory variants. We next computed association enrichment using two complementary gene set analyses. First, we executed an over-representation analysis that calculates the proportion of genes in the pathway having a P value less than 0.05 . This approach has been widely and successfully used for pathway analysis; see Holmans  pathway and CMS and natural selection, we retained 345 autosomal genes in the nine Gene Ontology (GO) categories retained by Simonson et al. : detecti Holmans  for a ret method , which ht method . P valueotations . We noteIn order to investigate the role of European genomes on our phenotypes, we performed admixture mapping to test if chromosomal segments inherited from European populations were associated with CMS. Inference of chromosomal segments ancestry was performed using RFMix  with twoP values for SNPs that were not present in the 133,649 SNPs used for the analysis.For each individual, and for each of the 133,649 SNPs common to the HGDP-CEPH and 1000G data, we obtained an average number of alleles of European origin (between 0 and 2) deduced from forward-backward RFMix output probabilities. GEMMA was then used to test the association of these probabilities with the different phenotypes, by using the same covariates as in the association analyses. Finally, to merge admixture-mapping results with association results, we interpolated linearly –log10 of the 2 . After quality control, we retained 166 CMS patients and 146 controls . Clinical and physiological data of these patients are presented in P = 0.73; To study the proportion of European genome present in our data, we first projected our 312 individuals with Quechua ancestry on the HGDP-CEPH  and 1000 Genomes  panels (−8 but had a reasonable power (≥60%) to detect very common variants (MAF ≥20%) with odd ratios of 2.5 when considering a discovery threshold of 10−5 (EPAS1 and EGLN1 (Egl-9 Family Hypoxia Inducible Factor 1) genes (P = 4.58 × 10−9). We note the large effect size of this SNP  especially due to its high frequency in the population (57% in CMS and 34% in controls), which explains 13% of the trait variance. We also found three other independent candidate loci with P < 10−5 on chromosomes 5 , 8 , and 15 . Imputation around these loci did not reveal new significant information (We then performed a genome-wide association study on the CMS status Table S11) genes . Neverth2), as these phenotypes are risk factor of CMS ; for rs7832232, the CMS risk allele was associated with RVH risk (P = 0.025); and for rs75810402 and rs7168430, the CMS risk alleles were associated with decreased SpO2 . As a consequence, adjusting for BMI, RVH, and SpO2 decreased the significance of the CMS association with all the candidates (P between 5.48 × 10−7 and 4.68 × 10−3), except for rs7832232. All association results are presented in 2 are still significant after conditioning on each other , right ventricular hypertrophy (RVH), and pulse oxygen saturation  was the closest gene AEBP2 , with lower expression levels in cases than in controls (P = 0.042). We also performed RNA quantification for manually selected candidate genes around other candidate loci and found a significantly different expression in CMS cases and controls for genes MCTP2  and CAST .We performed RNA quantification extracted from whole blood for seven manually selected candidate genes around the genome-wide significant SNP of chromosome 12 (rs7304081) on 71 unrelated individuals of our study (30 cases and 41 controls). The only gene with a significant difference of RNA expression between cases and controls (at the level controls Table S4P < 0.05) in CMS for three CMS candidate genes: SENP1 (sentrin-specific protease 1) (ATM (ATM serine/threonine kinase)  .Finally, we performed expression analyses on 10 additional genes reported in other studies as CMS candidate genes or genes being under adaptation to high altitude  and Andresponse ], suggesEPAS1, the five SNPs of the Denisovan haplotype under selection in Tibetans (th century. Based on a mitochondrial DNA study, O’Fallon and Fehren-Schmitz (Second, we thus postulated that the population history of Andeans might have influenced the selection hallmark of high altitude on genome. Consistent with the absence of selection on Tibetans  were abs-Schmitz  demonstr-Schmitz  reported-Schmitz .In conclusion, CMS may have various determinants. By performing the largest GWAS of CMS, as well as the largest selection scan in native Andeans, we observed no evidence that CMS is a consequence of an ongoing adaptation to altitude where variants under positive selection have not yet reached their optimum frequency. In addition, although the genes of the HIF pathway are under selection in Andeans, these genes are not enriched in the GWAS signal, which does not sustain our first hypothesis that CMS risk variants are under adaptation. Secondly, the influence of genetic admixture with another population, i.e., low interbreeding with Denisovan and recent admixture with lowlander European genomes, may also have a limited impact on adaptation to altitude and CMS outcome. Finally, the physiological pattern of adaptation to hypoxia between Tibetans and Andeans is quite different. In Tibetans, adaptive pathways limit the increase in hemoglobin level, while in Andeans without CMS, a higher [Hb] is a mechanism of adaptation to counteract tissue hypoxia, as it does in all low-altitude natives exposed to high altitude. This mechanism has no major negative impact during youth, and the evolutionary cost, CMS in the elderly, is limited. Finally, without a strong relationship with natural selection in response to altitude-induced hypoxia, CMS may appear as a polygenic trait, involving other risk factors such as ventilatory defects, pulmonary hypertension, or overweight. Thus, a larger sample size will be necessary to replicate the variants we have identified as being associated with CMS, to investigate selection pressure on these variants, and to investigate the role of European admixture.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The study was approved by the Institutional Ethics Committee of Universidad Peruana Cayetano Heredia. All participants were enrolled in the study after signing an informed consent.All the authors read and approved the manuscript.This study was supported by grants from Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051. The labex GR-Ex is funded by the program “Investissements d’avenir” of the French National Research Agency, reference ANR-11-IDEX-0005-02. The study was also supported by a Wellcome Trust grant 107544/Z/15/Z to FCV.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.


Thousands of lower Manhattan residents sustained damage to their homes following the collapse of the Twin Towers on 11 September 2001. Respiratory outcomes have been reported in this population. We sought to describe patterns of home damage and cleaning practices in lower Manhattan and their impacts on respiratory outcomes among World Trade Center Health Registry (WTCHR) respondents. Data were derived from WTCHR Wave 1 (W1) (9/2003–11/2004) and Wave 2 (W2) (11/2006–12/2007) surveys. Outcomes of interest were respiratory symptoms (shortness of breath (SoB), wheezing, persistent chronic cough, upper respiratory symptoms (URS)) first occurring or worsening after 9/11 W1 and still present at W2 and respiratory diseases (asthma and chronic obstructive pulmonary disease (COPD)) first diagnosed after 9/11 W1 and present at W2. We performed descriptive statistics, multivariate logistic regression and geospatial analyses, controlling for demographics and other exposure variables. A total of 6447 residents were included. Mean age on 9/11 was 45.1 years (±15.1 years), 42% were male, 45% had ever smoked cigarettes, and 44% reported some or intense dust cloud exposure on 9/11. The presence of debris was associated with chronic cough (adjusted OR (aOR) = 1.56, CI: 1.12–2.17), and upper respiratory symptoms . A heavy coating of dust was associated with increased shortness of breath , wheezing , and chronic cough . Dusting or sweeping without water was the cleaning behavior associated with the largest number of respiratory outcomes, such as shortness of breath, wheezing, and URS. Lower Manhattan residents who suffered home damage following the 9/11 attacks were more likely to report respiratory symptoms and diseases compared to those who did not report home damage. The terrorist attacks in New York City on 11 September 2001 (9/11) led to the destruction of the World Trade Center (WTC) Twin Towers and six other adjacent buildings. The collapse of the two towers released a massive cloud of dust and debris that damaged surrounding buildings within and around the WTC complex, a 16-acre area that was subsequently called Ground Zero [The contents of settled WTC dust have been extensively analyzed and were characterized as a mixture of cement dust, glass fibers, asbestos, lead, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, organochlorine pesticides, and polychlorinated furans and dioxins ,3,4,5. IApproximately 25,000 lower Manhattan residents were physically and emotionally affected by this disaster . Two monA number of studies demonstrated the deleterious health effects of household exposures in the context of the WTC attacks. Most of them were relatively small case-control studies ,9,10,11.With a large sample of residents in lower Manhattan and a comprehensive set of outcomes, the present study describes patterns of home damage and cleaning practices, as a surrogate for household exposures since measures of exposure in homes is limited. We initially looked at geospatial relationships between Ground Zero location and exposure and health outcomes. We then conducted multivariate logistic regression analyses to find potential associations between those variables among a large sample of WTCHR respondents.https://www1.nyc.gov/site/911health/about/wtc-health-registry.page.The WTCHR monitors the health of people exposed to the 9/11 WTC disaster through periodic health surveys and is housed within the New York City Department of Health and Mental Hygiene (NYCDHMH). The WTCHR is the largest effort in the United States to monitor health after a disaster of this kind and includes data on 68,444 adults, including lower Manhattan residents, rescue and recovery workers, and building occupants and passers-by. More details on the WTCHR can be found elsewhere  and onliFor this report, data were derived from WTCHR Wave 1 (W1) survey (baseline), collected between 9/2003 and 11/2004, and Wave 2 (W2) survey (first follow-up), collected between 11/2006 and 12/2007. We included W1 and W2 participants 18 years and older, whose primary residence on September 11th was located in lower Manhattan, south of Canal Street. Residents who performed rescue and recovery work were excluded from this analysis.The Centers for Disease Control and Prevention (CDC) and NYCDHMH institutional review boards approved the Registry protocols. A Federal Certificate of Confidentiality was obtained, as was verbal informed consent.In the W1 Survey, respondents were asked if they were outdoors after the towers’ collapse and to report their location when they first encountered the dust cloud  . The W2 Wave 2 also included detailed questions about conditions inside the homes after 9/11 as well as cleaning practices and replacing of household items. WTCHR participants were asked about the presence of “a fine coating of dust on surfaces”, “a heavy coating of dust on surfaces (so thick one couldn’t see what was underneath)”, “broken window(s)”, “damage to home or furnishings”, and “debris from the disaster”. In addition, enrollees answered whether or not they had personally “cleaned ventilation ducts”, “cleaned with a damp cloth or wet mop or wet sponge”, “used a vacuum (with or without a high-efficiency particulate air or HEPA filter)”, and “dusted or swept without water.” Moreover, enrollees were inquired whether or not they replaced “carpet or rugs”, “furniture (replaced or re-upholstered)”, “drapes, blinds or curtains”, and “air conditioners” as a result of the 9/11 events.Outcomes of interest were self-reported respiratory symptoms and self-reported physician diagnoses of respiratory diseases, asked at both W1 and W2 surveys. Symptoms included shortness of breath, wheezing, persistent chronic cough, and upper respiratory symptoms, first occurring or worsening after 9/11 and present at W2. Respiratory diseases comprised asthma or reactive airways dysfunction syndrome (RADS) and chronic obstructive pulmonary disease (COPD), first diagnosed after 9/11 and present at W2.Initially, to explore potential geospatial relationships between Ground Zero location and exposures and health outcomes, we mapped exposures and health outcomes of WTCHR resident enrollees in lower Manhattan using ArcGIS version 10.2 . These variables were mapped at the census tract level of geography using 2000 Census tract boundaries. For this part of the analysis, “yes” responses for any of the health outcomes variables were summed per census tracts . Similarly, exposure variables  were also grouped and categorized as “yes” or “no” and mapped by census tract totals. The number of enrollees within each census tract was used as the denominator of the proportions represented on the maps. In addition, point values of dust cloud exposure were modeled using inverse distance weighted (IDW) interpolation to create a surface representation of the dust levels throughout lower Manhattan. IDW uses measured values (point locations) to predict values for unmeasured surrounding areas. We examined demographic characteristics, household exposures resulting from the 9/11 attacks, and cleaning practices among the study population using descriptive statistics . We also calculated the prevalence (%) of self-reported respiratory symptoms and respiratory diseases. The relationship between cleaning practices and conditions inside the homes after 9/11 was assessed using multivariate logistic regression analyses adjusted for age, race, education, income, sex. Analyses were also controlled for “priority group”, a stratification of registrants into higher and lower priority groups for the purposes of targeting and outreach, which to a degree reflects a resident’s distance from the WTC site. Priority group 1 included residents as of 9/11 at addresses located south of Chamber Street; Group 2 included residents located on or north of Chamber but south of Canal Street; a third resident group, Group 0, was defined upon recognition of the inclusion of respondents living on or north of Canal in zip codes overlapping the Canal boundary or in other NYC boroughs. Also, the analyses were controlled for any exposure outside, to the dust cloud.a priori selection. The predictors or covariates included five home exposures . The covariates also included four replacement behaviors . Lastly, each model contained terms for eight confounders, as listed in Each of the cleaning practices  was modeled using logistic regression. Based on medical considerations and plausibility of real relationships being present, the list of all variables was narrowed to 17 potential explanatory variables for cleaning practices using ‘purposive’ or The cleaning practices themselves, other than the cleaning behavior specified outcome, are also modeled as predictors. These other cleaning practices were conditioned on the presence of dust in the home. For example, “cleaned ventilation ducts” was modelled including the other three cleaning practices as predictors . The covariate cleaning practices were considered only if reported in conjunction with the presence of either fine or heavy dust . We fitted the four logistic models with all 17 variables included. Adjusted odds ratios (aOR), and their 95% confidence intervals (95% CI), for each cleaning method are provided in a priori selection). Each of the six health outcomes was modeled using logistic regression with covariates as follows: (a)Five demographic variables ;(b)Resident priority group, indicating degree of registrant recruitment by area ;(c)Dust cloud exposure category ;(d)Ever having smoked (at least 100 cigarettes in a lifetime);(e)Five variables of exposures in the home ;(f)Four variables containing data on behavior regarding replacement of various major items in the home ; and(g)Four variables for the presence of cleaning practices, which are conditioned on the presence of dust in the home.Each health outcome variable was modelled in a similar way. Based on medical considerations and likelihood of real relationships being present, the list of all covariates was narrowed to 21 explanatory variables for health outcomes . Smoking status was queried as “Have (you) smoked at least 100 cigarettes in (your) entire life?” Adjusted odds ratios (aOR), and their 95% confidence intervals (95% CI), for each health outcome were computed for all variables in the model including demographic, exposure, and cleaning practices. All analyses were performed using SAS version 9.3 . A total of 6447 lower Manhattan residents were included in this study. Using a damp cloth, sponge, or mop was also associated with reporting a fine coating of dust . In contrast, those who used water for cleaning were less likely to report broken windows. Lower Manhattan residents who reported using a vacuum to clean, as well as those who reported cleaning ventilation ducts, were less likely to report a fine or a heavy coating of dust, but more likely to report damage to home or furnishings, Prevalence of respiratory outcomes was as follows: shortness of breath (16.1 %), wheezing (10.7%), chronic cough (6.8%), upper respiratory symptoms (60.8%), asthma or reactive airways dysfunction syndrome (RADS) (7.9%), and chronic obstructive pulmonary disease (COPD) (5.4%) . A totalThis is the largest study to evaluate respiratory outcomes among lower Manhattan residents who reported household exposures as a result of the 9/11 terrorist attacks. Our findings are consistent with increased respiratory symptoms and diseases, which are associated with several levels of home damage and different cleaning practices, and corroborate other studies in similar populations ,11,13 orThe explosion and collapse of buildings and subsequent fires at Ground Zero produced a large plume of dust and smoke that released particles and gases into the air. Characterization of both outdoor and indoor dust samples identified asbestos, glass fibers, lead, and polycyclic aromatic hydrocarbons (PAHs), among numerous other contaminants ,4,5. In As a result of the presence of dust, debris, and other damage, lower Manhattan residents personally cleaned their homes using a variety of methods. Lin et al. reported that 74.3% of residents in the affected area personally cleaned their homes . The majUpper respiratory symptoms were the most common health outcomes reported by lower Manhattan residents in the WTCHR. This finding is consistent with other WTC studies among residents  and rescOur results demonstrate an association between respiratory health outcomes and numerous types of home damage and cleaning practices. In particular, the presence of a heavy coating of dust was associated with shortness of breath, wheezing and cough and the presence of debris with chronic cough and upper respiratory symptoms. Lin et al. reported that upper respiratory symptoms were associated with both dust and physical damage to home, with statistically significant cumulative incidence ratios (CIRs) ranging from 1.27 to 1.71. In addition, the association between lower respiratory symptoms and exposures yielded CIRs between 1.31 and 1.44 . In our A strength of this study is the ability to demonstrate persistent health outcomes in a large sample of lower Manhattan residents exposed in their homes to airborne contaminants resulting from the 9/11 terrorist attacks. Our analyses controlled for potential confounders, such as the intensity of exposure to the cloud of dust and debris, smoking, and priority group or area of recruitment, which may serve as a surrogate for location in lower Manhattan. reduced the apparent effect of the reported home damage on respiratory health outcomes.However, this study is subject to several limitations. Self-selection bias may affect our results. Lower Manhattan residents who developed respiratory symptoms and diseases after 9/11/2001 may have been more likely to enroll in the WTC registry than asymptomatic persons. Medical records were not obtained which might have been used to verify reported conditions. In addition, smoking was defined as ever/never, without taking into account pack-years. Also, there was essentially no ability to validate the reported exposures quantitatively, because environmental sampling was not conducted. Nonetheless, it is unlikely that differences between respondents and non-respondents and misclassification of disease status would have an influence on the intensity of home damage reporting and selection of cleaning practices. Recall bias is also a limitation since questionnaires for W2 were applied over 5 years after the event of interest. In an attempt to account for this aspect, we verified responses on W1 and W2 for consistency. In addition, information on time to return home and use of respiratory protection while cleaning was not available. It is possible that severely damaged home inhabitants took longer to return to their homes or did so only after renovations, which potentially reduced dust exposures. Similarly, residents may have used different degrees of respiratory protection while cleaning surfaces. These factors would have This analysis demonstrates that lower Manhattan residents who suffered home damage following the 9/11 attacks are more likely to report respiratory symptoms and diseases in the WTCHR. It also highlights the specific kinds of damage or other specific exposure events, such as cleaning practices, which are statistically related to such increased symptoms and diseases. These health outcomes persisted for at least 5–6 years after the event, which may have translated into lower quality of life.


The preweaning phase is the period for the rapid growth and development of dairy calves. During this period, dairy calves receive their nutrients through milk. Feeding hygienic milk is of great benefit to optimum growth rate and health status of dairy calves. Previous studies focused on the effects of hygienic milk by acidification on dairy calves’ health and growth. Reconstituted milk, as the common source of milk, is being used in dairy calves feeding. However, no previous studies reported the effects of feeding acidified reconstituted milk on dairy calves’ health and growth. Our study will provide the evidence that the acidification of reconstituted milk had positive effects on growth performance and health status of preweaning dairy calves.p < 0.01). Calves fed AFM had more feed intake than those fed ARM and RM (p < 0.01). Feed efficiency was significantly lower for calves offered ARM than those offered RM and AFM (p < 0.01). A lower withers height growth was found for calves fed RM than those fed ARM and AFM (p <0.05). Diarrhea rate and white blood cell (WBC) and lymphocytes (LYM) counts were greater for calves fed RM than those fed ARM and AFM (p < 0.05). These findings suggested that ARM and AFM had positive effects on growth performance and health status of the preweaning dairy calves.The present experiment was carried out to assess the effects of reconstituted milk (RM), acidified reconstituted milk (ARM), and acidified fresh milk (AFM) on growth performance, diarrhea rate, and hematological parameters of preweaning dairy calves. For this purpose, a total of 27 Holstein female calves (one month of age) with initial body weight of (67.46 ± 4.08) kg were divided into three groups in such a way that each group contained nine calves. Calves were housed individually, and starter was offered ad libitum to each calf. The dietary treatments were RM, ARM, and AFM. The highest milk intake was observed in calves receiving AFM as compared to other treatments ( The preweaning phase is the period for the rapid growth and development of female dairy calves . ProvidiOn large scale dairy farms, pre-weaning dairy calves are usually reared by milk or milk replacer ,4,5. DelReconstituted milk is resulted from the addition of water to dried or condensed milk powder in the amount necessary to reestablish the specified water:solids ratio. Reconstituted milk is also being used on the dairy farms for preweaning dairy calves . However−1 to 10−4) using distilled water, and a volume of 1 mL of each dilution was inoculated onto the surface of plate count agar (PCA), violet red bile agar (VRBA), and MRS agar for the cultivation of total bacteria, lactobacillus, and Escherichia coli, respectively. Total bacteria and Escherichia coli were incubated at 37 °C for 48 and 24 h. Lactobacillus was incubated anaerobically at 37 °C for 72 h. After incubation, the numbers of total bacteria, lactobacillus and Escherichia coli colony-forming units (cfu) were calculated. Bacterial numbers of different types of milk are shown in The animal care and experimental procedures were conducted by the Animal Welfare and Ethics Committee of Heilongjiang Bayi Agriculture University. Animal care and handling were followed the guidelines by the regulations for the Administration of Affairs Concerning Experimental Animals . Twenty-seven Holstein female calves procured from large dairy herd of same age with initial body weight of (67.46 ± 4.08) kg were selected and randomly divided into three groups in such a way that each group contained nine calves. Three types of milk were applied to different treatment groups—(1) reconstituted milk (RM), which was made by mixing milk powder with warm water (35–40 °C) in a weight ratio of 1:7; (2) acidified reconstituted milk (ARM), which was obtained by adding 30 mL formic acid (food grade) of 8.5% concentration in 1L reconstituted milk at temperature 5–10 °C; and (3) acidified milk made by using fresh milk (AFM), which was obtained by adding 30 mL formic acid (food grade) of 8.5% concentration in 1L fresh milk at temperature 5–10 °C. The fresh milk was procured from general and healthy herd and not pasteurized before feeding. Milk samples of 50 mL were collected every 10 days to detect the compositions. A milk analyzer  was used to determine the composition of milk, and milk composition are shown in Prior to onset of trial, all the calves were offered the same basal ration from day 1 to 21. Experimental treatments were applied from day 22 onward. Calves were given a nine-day adaptation period before the start of data collection. No abnormal behaviors were observed during the adaptation period, and the initial data were collected on day 30. Calves were housed individually in calf hutches, and 6 L milk were offered twice daily at 5:00 and 16:30. All calves had free-choice access to clean, fresh water and starter throughout the experiment. The chemical compositions of the starter diets are shown in Calves were weighed before the morning feeding for three consecutive days at the start and end of the experiment to calculate the initial body weight, final body weight, and average daily gain (ADG). Milk and starter consumption were recorded daily. Feed efficiency was determined from total feed consumption and ADG.In addition, body measurements of each calf including body length, withers height, heart girth, and shin circumference were taken at the beginning and end of the trial according to the study made by Li et al. (2014) .Fecal consistency was scored everyday on a scale of 1 to 4, where 1 = normal-firm but not hard, 2 = soft-does not hold form, 3 = runny-spreads easily, and 4 = devoid of solid matter. Fecal score of 3 or 4 were considered as diarrhea. The diarrhea rate was calculated according to the procedure described in the recent study  and formBlood samples (about 10 mL) were taken from the jugular vein in the morning, approximately 3h post feeding on the first and final day of the experiment. Blood was anticoagulated with EDTA for hematological analysis within three hours after collection. Hematological parameters values including white blood cell (WBC), lymphocytes (LYM), monocytes (MO), red blood cell (RBC), hemoglobin (Hb), and hematocrit (HCT) were detected by using automatic hematology analyzer .ij is the observed variable, μ is the overall mean, Di is the fixed effect of diet treatment, Cj is the random effect of calves, and εij denotes the residual error. Significance was declared for p < 0.05, and trends were reported at 0.05 < p < 0.10. When a significant effect of treatment was detected (p < 0.05), differences between the means were tested using Bonferroni multiple comparison test.All the data were analyzed by PROC MIXED of SAS version 9.4 , the statistical model wasp < 0.01). Calves fed AFM had increased total feed intake compared to those fed ARM and RM (p < 0.01). The value of feed efficiency was significantly lower for calves offered ARM than those offered RM and AFM (p < 0.01), which suggested that the feed efficiency was better for calves fed ARM than RM and AFM.The results of growth performance of dairy calves are shown in p < 0.05). Compared with calves fed ARM and AFM, a lower withers height growth could be found for calves fed RM (p < 0.05). Similarly, final withers height was lower for calves fed RM as compare to ARM and AFM (p < 0.05).Body measurements data of different treatments are presented in p < 0.01).Fecal score and diarrhea rate of dairy calves were presented in p < 0.05).Hematological parameters of different treatments are presented in Preweaning growth of female dairy calves is considered an important factor of milk production in their later stage of life. Feed intake, ADG, and feed efficiency are important indicators for growth performance of female dairy calves. Feed intake affects the level of nutrient intake, thus affecting ADG and final BW. ADG and final BW play an important role in assessing the feed efficiency and overall health status of dairy female calves . Akins rBody measurements usually reflect body growth and development, which can be used to estimate liveweight . Calves Escherichia coli [Escherichia coli  and 2.67–4.77 (109/L) of weaning Hanwoo and Holstein calves [9/L) and LYM was 4.83 (109/L) for calves fed RM, which were much higher than that of the research mentioned above. However, mean value of WBC and LYM for calves offered ARM and AFM were very close to the values of the research we have reviewed. Therefore, it can be concluded that the calves offered RM were susceptibility to infection, which may result in detrimental effects on calf health.Hematological parameters such as WBC, LYM, and RBC counts and Hb concentration are considered as important clinical indicators that are widely used to reflect health and disease status . Diseasen calves ,32,33. IResults from this research indicated that calves fed acidified reconstituted milk or acidified fresh milk had greater average daily gain and withers height growth, lower diarrhea rate, and white blood cell and lymphocytes counts. We concluded that calves offered reconstituted milk were susceptible to infection, which may have a negative effect on calf health. Acidification of reconstituted milk as well as fresh milk had positive effects on growth performance and health status of the preweaning calves.


The importance of soil microbial flora in agro-ecosystems is well known, but there is limited understanding of the effects of long-term fertilization on soil microbial community succession in different farming management practices. Here, we report the responses of soil microbial community structure, abundance and activity to chemical (CF) and organic fertilization (OF) treatments in a sandy agricultural system of wheat-maize rotation over a 17-year period. Illumina MiSeq sequencing showed that the microbial community diversity and richness showed no significant changes in bacteria but decreased in fungi under both CF and OF treatments. The dominant species showing significant differences between fertilization regimes were Actinobacteria, Acidobacteria and Ascomycota at the phylum level, as well as some unclassified genera of other phyla at the genus level. As expected, soil organic matter content, nutrient element concentrations and bacterial abundance were enhanced by both types of fertilization, especially in OF, but fungal abundance was inhibited by OF. Redundancy analysis revealed that soil enzyme activities were closely related to both bacterial and fungal communities, and the soil nutrient, texture and pH value together determined the community structures. Bacterial abundance might be the primary driver of crop yield, and soil enzyme activities may reflect crop yield. Our results suggest a relatively permanent response of soil microbial communities to the long-term fertilization regimes in a reclaimed sandy agro-ecosystem from a mobile dune, and indicate that the appropriate dosage of chemical fertilizers is beneficial to sandy soil sustainability. A growing global demand for agricultural crops is one of the main challenges in the 21st century. The pursuit of high productivity, long-term sustainability and optimal resource use efficiency without negative effects in the restricted land available for agricultural cultivation has led to the emergence of a variety of management practices . BecauseBecause microorganisms play a leading role in soil development and preservation , there iThe effects of farming management practices on changes in the soil microbial community  have been extensively studied in different soils, but the results have differed. For example, most studies of long-term chemical fertilization have revealed that a significant increase in soil microbial biomass  and longTo provide a comprehensive understanding of whether sandy fields will be healthy and sustainable after application of integrated farming management, changes in soil microbial community structure and activity and their relationships with soil characteristics and crop yield were analyzed in a 17-year fertilization treatment experiment. Illumina MiSeq sequencing was used to analyze the prokaryotic 16S rDNA and fungal 18S rDNA in different fertilization regimes , as well as in uncultivated mobile sand as a control. Soil physicochemical properties, bacterial and fungal abundance, and the activity of microbial intracellular oxidizing enzymes and extracellular hydrolases involved in nitrogen and phosphorus cycles in the soil were also determined. We hypothesized that (i) the composition, abundance and activity of the soil microbiome are driven by different fertilization regimes in the primary successional process; (ii) soil nutrient content is the main determinant for bacterial and fungal structure, whereas organic fertilizers  are more helpful for the development of microbial community; and (iii) shifts in the structure and abundance of soil microorganisms affect the agro-ecosystem crop yield.The study site was in the southeast fringe of the Tengger Desert, at the Shaptou Desert Research and Experiment Station, Chinese Academy of Sciences . This site is in a semiarid area with a mean annual precipitation of less than 200 mm. The local climate is that of a typical temperate continental desert and the area has a mean annual temperature of 9.6 °C and an average wind velocity of 3.5 m/s. The selected farm field was established in 2000 on mobile sand consisting of 1.0% silt, 20.01% clay and 78.99% sand from truncated sand dunes.2, and three replicates with areas of 10 × 10 m2 were randomly set in one field. The sources of chemical fertilizers were urea for N, single super-phosphate for phosphorus (P) and muriate of potash for potassium (K), and soils fertilized with CF received an annual dose of 150:45:60 kg ha−1 year−1 of N/P/K. The fertilizer application norm and recommended dosage were based on the data of the Second Soil Fertility Census in China  were used in a fertilization experiment with wheat and maize rotation every year, and no planted sand was used as a control (CK). The size of each experimental field was 666.6 m2 individually using a sterile sampler. The five obtained cores consisted of a mixed sample. Triplicate composite samples for each treatment were collected and preserved in an ice box, then taken to the laboratory and immediately sieved (by 1 mm) to remove stones and plant roots. For microbial DNA extraction, samples were stored at −80 °C. For soil bulk density, samples were dried at 105 °C. To measure the soil other physiochemical properties and soil enzyme activity, samples were dried in natural air.Soil samples were collected from different treatment fields in October 2017, three months after harvest of the wheat crop. To decrease spatial heterogeneity, we collected five soil cores (20 cm in depth and 5 cm in diameter) from four vertices and a diagonal point crossing of a square of 10 × 10 mSoil bulk density (SD) was estimated using a metallic cutting ring of known internal volume. Water holding capacity (WHC) was determined using the Wilcox method . Silt an2O2 with KMnO4, respectively. One unit of enzyme activity was defined as the amount of enzyme required to free 1 µg/µmol of substrate per hour per gram of soil under the experimental conditions.The soil enzymes urease (UE) and alkaline phosphatase (AKP), which are involved in N and P cycling, respectively, were analyzed using well-known assays described by Plant biomass and yield  were analyzed by the weighing method. Stem length was measured using a ruler, and leaf area index was determined with a CI-202 Portable Laser Leaf Area Meter . All soil biogeochemical properties and crop agronomic characteristics were carried out in triplicate.SRP148524 (BioSample accession: SAMN09229018 –SAMN09229029) for bacteria and SRP148525 (BioSample accession: SAMN09229048 –SAMN09229059) for fungi. After the raw reads were demultiplexed and filtered using QIIME (version 1.17), as described in http://drive5.com/uparse/).An E.Z.N.A Soil DNA Kit  was used to extract the total DNA from 1 g of each of the soil samples described above according to the manufacturer’s instructions. The obtained DNA was then examined on 1.0% agarose gels, and the concentration was determined with a NanoDrop 2000c spectrophotometer , and the purified DNA was then used for PCR. The universal primers for the 16S rRNA gene for prokaryotic microbes and the 18S rRNA gene for fungi were used in the PCR. The primer information, as well as detailed procedures describing the amplicon and Illumina MiSeq sequencing, are described in qPCR was conducted to determine the absolute 16S and ITS rRNA gene abundance using the primer set 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) for bacteria and ITS1F (5′- CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) for fungi, to quantify the total microbial abundance. The detailed procedures of qPCR are described in Alpha biodiversity indices analysis was used to assess the richness and diversity of soil microbial diversity. Hierarchical clustering analysis and principal co-ordinate analysis (PCoA) were conducted to reflect the overall differences in community compositions. The major soil characteristics contributing to the microbial community structure were evaluated with redundancy analysis (RDA), which was also used to identify the attributes of microbial diversity and abundance in relation to the crop yield. We excluded OTUs with fewer than 5% occurrences in RDA analysis, and RDA ordinations were performed using CANOCO version 4.5 . NonparaThe soil biogeochemical properties and crop agronomic characteristics differed markedly among fertilization strategies. Both CF and OF clearly improved the soil texture and increased the levels of soil nutrients such as OM, AN and AP, and the effects of OF on OM content were significant . HoweverTo analyze the taxonomic compositions of the microbial community, a total of 627,173 16S rDNA and 437,477 18S fungal effective sequences with mean lengths of 273 bp and 401 bp, respectively, were identified by Illumina MiSeq sequencing in 12 soil samples (three replicates in four treatments). The sequences were clustered into 3,307 and 243 OTUs for prokaryotic microbes  and fungp < 0.05), and were higher in OF than in CF (p < 0.001) and decreased in OF relative to C  than no fertilization (an in CF . Howeverive to C . Both thp < 0.05), and only two phyla (Ascomycota and an unclassified phylum) were significantly different in fungi. Notably, three unclassified genera of Crenarchaeota, Actinobacteria and Gemmatimonadaceae among prokaryotic microbes  and fungal community . Among the soil factors, soil pH, soil nutrient content  and soil texture  were strongly related to the prokaryotic microbial community structure, and soil enzyme activities  also highly contributed to axis 1  was 91.3% and 89.4%, respectively, with the first axis explaining 80.2% and 73.5% of the prokaryotic microbial and fungal changes, respectively, and the corresponding second axis explaining 7.6% and 11.9% . Accordio axis 1 . Thus, so axis 1 .p < 0.01), and changes in crop agronomic characteristics were significantly correlated with bacterial abundance and fungal diversity (p < 0.05). The bacterial abundance was the major determinant for almost all crop yield indices except the underground biomass. The Shannon index of the fungal community was significantly and negatively correlated with above ground biomass and seed yield (p < 0.05), whereas soil enzyme activity was significantly and positively correlated with seed yield, stem length, leaf area index and above ground biomass.The relationships between crop agronomic characteristics and microbial community characteristics  were analyzed by RDA . The firTransformation from mobile sand to arable land in arid areas is good not only for economic returns but also for the future sustainable development of land in China. Therefore, reasonable management strategies without negative effects on soil quality and environmental systems are the main objectives of land development in agroecosystems. This study provides an overview of the responses of soil microbial communities to long-term fertilization from a traditional farming practice, and it could be useful in the development of methods of sustainable land management for newly developed land.Our first hypothesis that the composition, abundance and activity of the soil microbiome are driven by different fertilization regimes was verified by investigation of shifts in prokaryotic microbial and fungal community composition, abundance and soil enzyme activities between CF and OF. In the present study, long-term chemical and organic fertilization had significantly different effects on prokaryotic microbial and fungal community structures. There were no substantial changes in the prokaryotic microbial community diversity and richness; however, there were clear shifts in the proportion of prokaryotic microbial community compositions in relation to fertilization treatments. Fungal communities were much more sensitive to fertilization, showing a significant decrease in community diversity in response to organic fertilizers. These different responses in bacterial and fungal community structures have been reported in most previous studies of long-term fertilization treatments  and evenp < 0.05) between treatments, Acidobacteria and Actinobacteria showed the most significant changes (p < 0.001). Actinobacteria, the predominant phylum in all soils investigated in this study, play a major role in agricultural soil quality promotion via organic matter decomposition  ions by oxidation of ammonium (Soil pH is an important factor that drives prokaryotic microbial community composition . The apptivation . Howevertivation . Variatitivation . These cIn recent years, some studies have shown that soil nutrient elements such as OC and TN are more closely correlated to the bacterial community structure than pH, whereas no correlations were observed between the fungal community and these elements . ExtensiThe third hypothesis, in which shifts in the structure and abundance of soil microorganisms affect the crop yield of the agro-ecosystem, was verified by our observation that bacterial abundance and fungal diversity indexes were closely related to crop yield. Many studies have attempted to link crop productivity to below-ground microorganisms and have addressed the potentially important role of bacterial taxa in soil OM accumulation, increases in soil enzyme activity, plant growth promotion and disease suppression in different fertilization regimes ; howeverThe living soil system, which provides several ecosystem services including nutrient cycling, water regulation and controlling pests and diseases , is of pMany studies have shown that organic fertilization stimulates the release of plant available nutrients in soil and enhances soil biological activities, thereby establishing a sustainable agricultural ecosystem . HoweverSuccession of microbial community characteristics and soil biogeochemical properties from an arid sandy system showed that both long-term chemical and organic fertilization enhanced the soil nutrient content available to plants, changed the microbial community structure, promoted soil enzyme activity, and increased crop plant yield. These results were compatible with our first hypothesis. However, contrary to our second hypothesis, no significant differences were observed in prokaryotic microbial community diversity in relation to fertilization regimes, and FYM inhibited the development of the fungal community. In contrast, chemical fertilization was more beneficial to bacterial propagation and crop yield, and bacterial abundance might be the main determinant of crop yield. Thus, changes in soil microbial activities and crop yield under long-term fertilization practices may be achieved mainly by bacteria rather than fungi. This field experiment indicates a relatively permanent response of soil bacterial and fungal communities in a sandy agro-ecosystem to long-term fertilization, and provides a comprehensive understanding of the effects of fertilization regimes on sustainable soil development. Overall, the results obtained from a sandy agricultural system managed by chemical and organic fertilization over a 17-year period indicated that it is possible to perform reasonable chemical fertilization management while maintaining soil health during farming practices.10.7717/peerj.6497/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6497/supp-2Figure S1Click here for additional data file.10.7717/peerj.6497/supp-3Table S1Details of crop agronomic characteristics under different fertilization treatments.Click here for additional data file.10.7717/peerj.6497/supp-4Table S2The OTU numbers of bacteria in different samples by using a threshold of 0.97.Click here for additional data file.10.7717/peerj.6497/supp-5Table S3The OTU numbers of bacteria in different samples by using a threshold of 0.97.Click here for additional data file.


Fungi are important decomposers of woody biomass in natural habitats. Investigation of the mechanisms employed by decay fungi in their attempt to degrade wood is important for both the basic scientific understanding of ecology and carbon cycling in nature and for applied uses of woody materials. For wooden building materials, long service life and carbon storage are essential, but decay fungi are responsible for massive losses of wood in service. Thus, the optimization of durable wood products for the future is of major importance. In this study, we have investigated the fungal genetic response to furfurylated wood, a commercial environmentally benign wood modification approach that improves the service life of wood in outdoor applications. Our results show that there is a delayed wood decay by the fungus as a response to furfurylated wood, and new knowledge about the mechanisms behind the delay is provided. Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point . Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment.The aim of this study was to investigate differential expression profiles of the brown rot fungus IMPORTANCE Fungi are important decomposers of woody biomass in natural habitats. Investigation of the mechanisms employed by decay fungi in their attempt to degrade wood is important for both the basic scientific understanding of ecology and carbon cycling in nature and for applied uses of woody materials. For wooden building materials, long service life and carbon storage are essential, but decay fungi are responsible for massive losses of wood in service. Thus, the optimization of durable wood products for the future is of major importance. In this study, we have investigated the fungal genetic response to furfurylated wood, a commercial environmentally benign wood modification approach that improves the service life of wood in outdoor applications. Our results show that there is a delayed wood decay by the fungus as a response to furfurylated wood, and new knowledge about the mechanisms behind the delay is provided. Wood as a building material has a number of attractive properties, including carbon sequestration during its service life and its aesthetic aspects as a natural material. One of the main challenges with using wood as a building material is its susceptibility to attack by wood-degrading microorganisms. Traditional wood preservatives with a biocidal mode of action  cause concerns due to their perceived negative environmental impacts. In Europe, the Construction Products Regulation and the Biocidal Products Regulation have a major impact on the wood industry, and the number of active ingredients allowed for wood preservatives is decreasing . This, tThe modern wood modification approach, in contrast to more traditional wood preservatives, is to modify the wood matrix so that it can no longer act as a suitable substrate for wood-degrading organisms. Different wood modification approaches were described in detail by Hill . CurrentThe commercial wood modification process furfurylation uses furfuryl alcohol, which is manufactured industrially by catalytic reduction of furfural obtained from agricultural waste such as sugarcane bagasse or corn cobs . It invoIndependently of how the furfurylation modifies the wood, the process results in improved resistance to fungal deterioration –4, 6, 7.–Rhodonia placenta  is the extracellular reductant of Fe3+, and DMHQ has been demonstrated to play a bioactive role when wood is attacked by members of three divergent fungal lineages, Boletales, Gloeophyllales, and Polyporales  placenta is a commonly used brown rot decay fungus in laboratory wood decay tests; strain FPRL 280 is included the European standard CEN-EN 113 (R. placenta strain MAD 698-R and its monokaryotic strain MAD698-R-SB12 have been sequenced by the Department of Energy-Joint Genome Institute (JGI) (22––R. placenta (and Phanerochaete chrysosporium) was influenced by wood substrate composition (three Populus trichocarpa genotypes) and the duration of incubation. For early stages of modified wood, a possible shift toward increased expression of genes related to oxidative metabolism and concomitant reduction of several gene products related to the breakdown of holocellulose in furfurylated wood compared to unmodified wood have been suggested  , 22. Thete (JGI) , 22–24 a(JGI) 22––31, inclJGI) 22––, 30. TheJGI) 22––. The expJGI) 22–– demonstruggested .Rhodonia placenta harvested at several time points when grown on Pinus radiata (radiata pine) with three different levels of furfurylation. For comparison, we also investigated the gene expression during decay of unmodified radiata pine. Radiata pine is native to central and southern coastal California but is also widely planted throughout the Southern Hemisphere. Radiata pine was selected in this study because it is the most frequently used wood species by the largest commercial producer of furfurylated and acetylated wood (wood modifications). They use radiata pine because of the low heartwood proportion and because it is easy to modify .In this study, we investigated the differential expression profiles of the brown rot fungus The overall aim is to understand the mechanisms utilized by brown rot decay fungi in their attempt to degrade modified and unmodified wood. This is important for further optimization of future modified wood products and for an expanded understanding of the fungal decay process in general.In the following experiments, the radiata pine wood was modified at three different levels. The three modification levels had mean weight percent gain (WPG) levels of 3.8% ± 0.7%, 24.0% ± 3.5%, and 36.6% ± 5.0%. For simplicity, the treatments are named WPG4, WPG24, and WPG37, respectively. The experiment on the unmodified radiata pine is named “unmodified wood.”R. placenta strain FPRL 280 on the unmodified wood blocks, they had a mean mass plus treatment loss of 28.8% ± 4.0%, while the modified WPG37 only had a mean mass plus treatment loss of 13.5% ± 3.3% after 21 weeks . We therefore produced Illumina HiSeq paired-end data to be used for a transcriptome assembly. The resulting assembly had 56,520 contigs and a total of 114,539 transcripts when including all isoforms (Table S1). The transcriptome covered 99.3% of the conserved BUSCO fungal genes (Table S2). In the transcriptome, 9,355 contigs had an annotation from the UniProt database in BLASTX, and 18,917 contigs were given a PFAM annotation. In all further analyses, the sequence data were mapped to this annotated transcriptome assembly. The mapping success of sequence data to this transcriptome was more than 90% for all libraries.Even if the genome and transcriptome of the American The gene expression data demonstrate consistent results for each treatment with a gradient clustering of replicate samples according to treatment, with few outliers . There is some biological variation between replicates as expected from natural variation in wood and the variation in modification of the wood blocks. The unmodified wood and WPG37 experiments display a wider range of biological variation than do WPG24 and WPG4.To obtain an overall impression of the entire data set, we ran a multifactorial differential expression (DE) analysis, factoring in time and treatment. Thus, all harvest time points were included for all treatments, and we extracted contrasts describing the different modified treatments versus the unmodified wood experiment, i.e., WPG4 versus unmodified wood, WPG24 versus unmodified wood, and WPG37 versus unmodified wood. . FunctioAs an alternative analysis method to the multifactorial DE analyses mentioned above, we also clustered the transcripts with similar expression profiles. The read counts were grouped into 10 clusters (K) for each treatment, and samples from unmodified wood, WPG4, WPG24, and WPG37 were analyzed separately . For theFor most of the clusters, an observed pattern of higher expression in one or a few of the harvest points was found . For theR. placenta genes were differentially expressed between weeks 1 and 2, while only 10 genes were differentially expressed between weeks 2 and 3 (Table S5).Pairwise comparisons of different harvest points within treatments revealed large gene expression differences between the first and later harvest points for all treatments. In the unmodified wood experiment, 2,450 The furfurylated wood modification demonstrated a temporal pattern of DE genes similar to that of the unmodified wood experiment (Table S6). For WPG4, there were 103 DE genes between week 3 and week 6 and 481 DE genes between week 3 and week 9. No DE genes were observed between week 6 and week 9. For WPG24, large numbers of DE genes were observed between week 3 and the later time points, but again, there were very few in between the later harvest points. For WPG37, few genes were differentially expressed (between harvest points) in general. However, also here, more DE genes were found between week 3 and the later harvest points and fewer among the later harvest points.Functional enrichment analyses of the pairwise analyses conformed to the expected two-step decay in this system (Table S7). These results demonstrated that for the unmodified wood experiment, there was an increased expression of functions related to hydrolase and catalytic activity from week 1 to the later harvest points, especially pronounced in weeks 2 and 3 (Table S7). This was also found, but to a lesser extent, in WPG4 and WPG24. In WPG4, most of this response was found between week 3 and week 9, while for WPG24, the response mainly started in the comparison between week 3 and week 12 to week 18. For WPG37, this was not pronounced, and no hydrolase activity was enriched. In the first harvest points of the unmodified wood and WPG4 and WPG24 treatments, we found less enrichment of upregulated functions, with the exception of some upregulation of GO terms related to protein binding and metal ion binding. However, in WPG37, there was an enrichment of several terms related to iron binding, heme binding, oxidoreductase activity, and cytochrome P450s in the upregulated gene set of week 3 compared to week 18. Significant enrichment of a GO term of oxidation-reduction process was found in later stages in WPG4 and WPG24.As week 1 in the unmodified wood was considered the first initial step of decay, with only 0.8% mass loss, we compared unmodified wood week 1 to all other time points and treatments (Table S8). As with the time series analyses, a signal of downregulation of protein degradation was found in all the WPG37 harvest points compared to unmodified wood. This was seen as enrichment of protein kinases, proteasome, F-box domain, and endopeptidase activity in unmodified wood. This pattern was also found in the early harvest points of WPG24. In contrast, week 1 of the unmodified wood showed upregulation of wood decay-related functions as sugar transporters, GHs, and dehydrogenases.The gene expression of annotated carbohydrate-active enzymes (CAZymes) that are involved in wood decay according to Floudas et al.  was plotn = 4 for both data sets), i.e., the same samples are not used for both analyses. Wood samples for each experiment were selected to keep the variation small within experiments. This caused some variation between the experiments. The main effect believed to be reflected in the results is a slightly faster decay for qRT-PCR WPG37 samples than for the RNA-seq WPG37 samples. It is also worth to keep in mind that qRT-PCR included an additional harvest point (week 21).Large RNA sequencing (RNA-seq) data sets tend to contain a lot of biological variation, as seen in our principal-component analysis (PCA) plot, which will affect downstream differential expression analyses . Quantitative reverse transcription-PCR (qRT-PCR) serves as an RNA-seq control but also provides deeper/more-detailed insight into the expression of individual genes. Here, we have used qRT-PCR primers for key genes involved in wood decay and applied a traditional qRT-PCR approach , and no statistically significant trends were found. In contrast, the RNA-seq data showed a strong induction at early-intermediate stages in the different experiments. The comparison with the lowly expressed qRT-PCR data should be interpreted with caution, but the general trends were similar .R. placenta decay included three glucose-methanol-choline (GMC) oxidoreductases  are widely distributed among wood decay fungi , oxidizeBenzoquinone reductases (CAZy family AA6) are intracellular enzymes involved in oxidative depolymerization of wood cell wall polymers by the synthesis of hydroquinone compounds that can act as mediators for laccases or reductants of iron in brown rot systems , 37. In Figure S6 illustrates the selected genes involved in hemicellulose hydrolysis. One endomannanase in CAZy family GH5 Man5a;  was founFor the two endoxylanases in CAZy family GH10 .Beta-xylosidase in CAZy family GH3 βXyl,  hydrolyzO- or de-N-acylation of substituted saccharides. The selected carbohydrate esterase 16a  are important enzymes in lignocellulose depolymerization that cause oxidative chain breaks in crystalline and amorphous polysaccharides. We selected a cellulose-active AA9, lytic polysaccharide monooxygenase LPMO; . For qRTExpansins are hypothesized to increase enzyme accessibility by loosening plant-cell wall interactions, although no catalytic mechanism is known –40. The –Rhodonia (Postia) placenta, using RNA-seq and qRT-PCR, in unmodified and furfurylated radiata pine wood.We have investigated if wood modification by furfuryl alcohol causes altered decomposition response in From our four treatments , we found that WPG4 closely followed the trend on unmodified wood, while WPG24 seemed to be an accelerated version of the decay pattern of WPG37. For simplicity, we mainly focus on unmodified wood decay processes and the comparison of unmodified wood decay versus WPG37 decay in the Discussion.In our unmodified wood decay experiments, we observed wood decay from incipient stages with mean mass loss of 0.8% in week 1, to severely decayed wood with a mean mass loss of 29% at week 5. Our results support the previously suggested two-step decay mechanism of brown rot fungi , 18, 41,This early response was followed by a polysaccharide degradation stage, with upregulation of transcripts encoding hemicellulases, e.g., endoxylanases (Xyl10b and Xyl10a), beta-xylosidase (βXyl), and endomannanase (Man5a), in weeks 2 and 3. In our study, there was an abrupt decline in transcripts encoding many core hemicellulases in week 4 during the later stages of decay . It has Some transcripts encoding putative cellulases are also accumulated in week 2 and were further upregulated in week 3. This was particularly obvious from the qRT-PCR expression pattern of the GH3 beta-glucosidase (βGlu) and the LPMO . Some cellulase transcripts are also highly expressed in the more advanced stages of decay, e.g., both the endoglucanase GH5 (Cel5b) and the glucoside hydrolase GH12 (Cel12a) are most highly expressed in week 5. This high expression of cellulases at a late decay stage was also observed by Zhang and Schilling . Thus, wR. placenta and other decay fungi is influenced by the wood substrate. For example, Vanden Wymelenberg et al. (Populus grandidentata) and pine (Pinus strobus) as the substrates. Furthermore, for this fungal species, gene expression profiles were influenced by wood substrate composition between wood genotypes of a single species (Populus trichocarpa) and the duration of incubation  as well as for the modified WPG37. This confirms the finding in Alfredsen et al.  where R.ss clear . This isR. placenta grown on wood modified with both furfurylation and acetylation shows similar or decreased levels of core wood-hydrolyzing enzymes  pathway, protein degradation, and the Ras pathway. All of these functions are related to carbon starvation and are highly enriched in the late decay of the unmodified wood. Thus, we hypothesize that Paxillus involutus  pathway is a conserved pathway in all eukaryotic organisms and is important to various cellular processes, such as recycling of intracellular protein (where unnecessary proteins are degraded to amino acids that can be reused to produce new proteins) and programmed cell death. Recently, the pathway was shown to be activated by carbon starvation in the ectomycorrhizal basidiomycete species nvolutus . In P. iThe other functions observed to be upregulated in unmodified wood compared to WPG37 were several domains related to Ras proteins. These proteins are involved in cell proliferation and growth of the mycelia. Carbon starvation supports an upregulation of transcripts encoding these proteins. In starving mycelia, it has been shown that the diameter of the hypha is reduced while it grows to cover a larger area , 52. RasFomitopsis pinicola [Serpula himantioides [In all our experiments, we investigated one strain, FPRL 280. Thaler et al.  suggestepinicola  and Serpntioides ). This hR. placenta hyphal front. This wafer approach works well to separate different initial decay stages and provides information about spatial separation along the decay gradient. For more advanced stages of decay, a different approach is needed. In the current study, we selected small and homogeneous (earlywood) samples in order to enable colonization on a uniform solid wood substrate for decay as fast as possible. Still, some variation in gene expression is expected, as new colonization pathways will be found close to areas with previously colonized wood. This effect is expected to increase with increasing WPG level, since areas that are long colonized will cease to provide nutrients able to sustain survival, while the others that are more recently invaded will provide nutritious substrates. In our experiments, the wood modification treatment modifies the composition of the wood, and the fungus is therefore forced to respond differently from when it encounters unmodified wood. Hence, comparison at similar mass losses between treatments was not the goal of this study, but rather, it was the shift of gene expression over time between and within the different treatments.Furthermore, for decay testing, the sample size, sample geometry, and wood anatomy influence the colonization rate. Sampling time and extent of decay are crucial when examining gene expression or secretome of fungal colonization of wood. The studies by Zhang et al.  and ZhanR. placenta colonizing furfurylated wood follows the same modality of gene expression as for unmodified radiata pine wood but with a delayed and elongated response (increasing delay with increasing furfurylation level).The novelty of this study compared to previous work on transcriptome studies of fungal decay of modified wood (including furfurylation) is the combination of (i) following the entire transcriptome instead of just a few selected genes as in previous studies of modified wood and (ii) including decay stages from initial decay to advanced stages of decay. Hence, this is a significantly more comprehensive data set than used in previous studies. The major findings based on this approach were (i) the identification of a decay threshold for the highest treatment level (WPG37) and (ii) that Increased knowledge about brown rot decay mechanisms is important for an expanded understanding of the fungal decay process in general since ecosystem carbon flows are closely linked to wood-degrading fungi. From an industry perspective, the findings in this study show that successful inhibition of the initial oxidative decay is a clue to success for future wood protection systems.In the present study, the entire gene expression pattern of a decay fungus is followed in untreated and modified wood from initial to advanced stages of decay. From these observations, we have demonstrated how the furfurylated modification delays the fungus gene expression while growing on this substrate. All treatments (modified and unmodified) expressed the expected staggered decay mechanism, i.e., enzymes implicated in oxidative decay were expressed first, and then hydrolytic enzymes were expressed later. Thus, we show that the fungus follows the same modality of gene expression on the modified wood. The major difference is that the responses are delayed and elongated compared to unmodified wood. However, from the downregulation of CAZymes in the latest harvest points in the WPG37, the fungus seems to be finalizing the decay also in the furfurylated wood at a mass loss of only 14%, compared to 29% in the unmodified wood. This suggests that the fungus never gets access to the remaining carbohydrates from the modified wood even at low mass loss. The lower levels of modification in the WPG4 and WPG24 treatments show intermediate expression differences and higher mass loss. The exact mode of action of the modification is still uncertain. However, the similar decay mechanisms and no obvious signs of gene expression related to detoxification of the furfuryl alcohol may indicate that the furfurylation functions as both a physical barrier and a factor that creates a less-hydrated environment for the fungus. These hypotheses should be investigated to further improve environmentally friendly modification processes. Further studies could include studies of chemical, mechanical, and structural properties of the decaying wood cell wall at a nanoscale using novel microscopy techniques, for example, combining atomic force microscopy and infrared spectroscopy.Pinus radiata D. Don) earlywood according to Beck et al.  were prepared from radiata pine  Niemelä, K.H. Larss. & Schigel (syn. Postia placenta) strain FPRL 280 (= BAM113). The strain was obtained from Bundesanstalt für Materialforschung und –prüfung, 4.1 Division Biodeterioration and Reference Organisms (BAM), as a dikaryotic strain. The fungus was first grown on 4% (wt/vol) Difco malt agar medium (VWR), and plugs from actively growing mycelia were transferred to a liquid culture containing 4% (wt/vol) Difco malt (VWR). After 2 weeks, the liquid culture was homogenized with a tissue homogenizer .The brown rot fungus in this experiment was A modified E10-16 soil/block test  was usedSamples were incubated at 22°C and 70% rH and harvested every third week in experiments with modified wood and every week for the unmodified wood material. Fungal mycelium was manually removed from the wood surface with delicate task wipes  in order to remove most of the fungal mass for the wood mass loss measures. Eight samples from each treatment and each harvesting point were dried at 103°C for 18 h in order to provide data for mass loss . The remaining samples were wrapped individually in aluminum foil and put directly into a container with liquid nitrogen. The samples were then stored at −80°C.For RNA-seq analyses, samples from the following harvesting points were used: unmodified wood, weeks 1 to 5; WPG4, weeks 3, 6, and 9; and WPG24, weeks 3, 6, 9, 12, 15, and 18. For qRT-PCR, samples from the following harvesting points were used: unmodified wood, weeks 1, 3, and 5; WPG4, weeks 3, 6, and 9; WPG24, weeks 3, 9, 15, and 21; and WPG37, weeks 3, 9, 15, and 21. The harvest interval was shorter for the experiments on unmodified wood, as this wood was known to decay more rapidly. Furthermore, for WPG4, the experiment was ended after 9 weeks because of severe mass loss at this stage.Wood powder from frozen samples was obtained by cutting the plugs into smaller pieces with garden shears wiped with 70% alcohol and, thereafter, Molecular BioProducts RNase AWAY surface decontaminant . The wood samples were immediately cooled again in Eppendorf tubes in liquid nitrogen. Fine wood powder was produced in a 300 mill . The wood samples, the 100-mg stainless steel beads , and the containers were chilled with liquid nitrogen before grinding at maximum speed for 1.5 min. They were then cooled in liquid nitrogen again before a second round of grinding.For Illumina sequencing, the MasterPure plant RNA purification kit  was used, according to the manufacturer’s instructions, for total RNA extraction with a 100-mg wood sample. The mean WPGs for the four replicate samples were WPG4, 3.6% ± 0.5%; WPG24, 24.8% ± 1.2%; and WPG37, 39.7% ± 2.2%.16) primer in RNase-free water . The solution was incubated for two cycles in a PCR machine  at 65°C for 5 min and 4°C for 2 min. The PCR machine was paused and the master mix added. The next three cycles included 37°C for 30 min, 95°C for 5 min, and 4°C for an indefinite time. In addition to the test samples, two samples without RNA were added as controls and used for each primer pair. After the cDNA synthesis, 50 μl RNase-free water (Qiagen) was added to the samples and mixed well.For qRT-PCR, the MasterPure complete DNA and RNA purification kit  was used, according to the manufacturer’s instructions, with a 90-mg wood sample . The mean WPGs for the four samples were WPG4, 4.1% ± 0.4%; WPG24, 20.9% ± 0.9%; and WPG37, 33.7% ± 3.8%. A NanoDrop 2000 spectrophotometer  was used to quantify RNA in each sample. To convert RNA to cDNA, the TaqMan reverse transcription reagent kit (Thermo Scientific) was used according to the manufacturer’s instructions. The total reaction volume was 50 μl. Three hundred nanograms of RNA was reacted with oligo(dThttps://www.sequencing.uio.no/). All samples were prepared with the strand-specific TruSeq RNA-seq library preparation method. In order to produce a high-quality de novo transcriptome, we sequenced the strain grown on a 4% (wt/vol) Difco malt agar medium (VWR) in addition to 10 selected samples spanning the entire experimental setup on one lane on the HiSeq 2500 platform, producing 125-bp paired-end sequences. These libraries generated ∼395 million read pairs, which after quality control and trimming were reduced to ∼250 million read pairs.All Illumina libraries and sequencing were performed by the Norwegian Sequencing Centre  from the Ensembl Fungi FTP server. The genome-based attempt using Bowtie 2, TopHat 2, and Cufflinks resulted in poor map back (∼50 to 70%) despite doing parameter sweeps (data not shown). We also attempted to use the genome-guided option of the de novo transcriptome assembler Trinity that yielded a more fragmented transcriptome compared to the clean de novo version (data not shown).We initially attempted to use the de novo strategy with the HiSeq trimmed reads and Trinity version 2.2.0  on our local computing cluster. The Trinity bfly process failed for 110 elements which were rerun with more memory. The finished transcriptome contained 56,520 contigs, with a total of 114,539 transcripts, including isoforms (Table S1).The final transcriptome was generated using a We also evaluated the assembly using BUSCO version 2.0 using the recommended parameters and the corresponding fungal database created on 26 January 2017 with 85 species and 290 BUSCO genes . Of the R. placenta genome, we manually annotated our assembly using the predicted proteins reported at the JGI Fungi Portal (https://genome.jgi.doe.gov/mycocosm/home) using TBLASTN from BLAST+  and set a full model consisting of treatment, time, condition, and mass loss to explore the data in VariancePartition. We also made heatmaps using pheatmap version1.0.8 and logMultifactorial differential expression analysis was performed in both edgeR and DESeq2 (the latter to enable use of various plot functions). In DESeq, we opted for a ∼treatment+time formula. Running a more complex design with interaction (:) was not possible, as DESeq2 cannot handle partial models (time points for the different treatments do not overlap completely). Furthermore, we opted for the likelihood ratio test (LRT), which examines two models for the counts, the full model and a reduced model. This then determines if the increased likelihood of the data using the extra term(s) in the full model is more than expected.In edgeR, we opted for an ∼0 + treatment+time formula without generating an intercept term. When estimating overall dispersion, we used the robust=TRUE option to better handle outliers in the data. For the multifactorial test itself, we chose to replace the standard glmFit with glmQLFit, which uses a quasi-likelihood F-test on the likelihood ratio statistics instead of the chi-square approximation. In this way, we should obtain a more conservative control of the type I error rate, as it takes into account the uncertainty in estimating dispersion for each gene, especially when the number of replicates is small.To enable an exploration of specific contrasts between given conditions, we also ran an ∼0 + condition formula in edgeR using the same setup as described above.We evaluated each treatment versus the unmodified wood over time from the treatment+time multifactorial analysis. For the pairwise contrasts between harvest times within treatments, we used the ∼condition multifactorial analysis.The overall exploration of the data revealed that the WPG24 treatment series has a few outliers. RNA-seq data are highly variable, and the individual variation between replicates can be large. We found that 2 replicates in the WPG24 3-week condition were relatively deviating from the other two replicates. We reran the above-described analysis without the most extreme outlier  and observed a large change in the number of reported significant differentially expressed genes. However, seeing as these differences did not necessarily improve the results, we decided to keep all samples in the final data set.As an alternative to the pairwise differential expression analyses, the read count data were clustered based on similarity in expression patterns using the MBCluster.Seq package in R. The EM clustering algorithm was used.Functional enrichment analysis was used to characterize the functions of the differential expressed genes of the various treatments and clusters. The functional enrichment of PFAM domains and GO terms in the DE gene sets was calculated using a Fisher’s exact test with Benjamini-Hochberg correction for multiple testing.https://github.com/TransDecoder/TransDecoder/wiki). All protein sequences were annotated in dbCAN2 (The transcripts were translated using TransDecoder (n dbCAN2 . The stan dbCAN2 .mT) of 60°C and to yield a 150-bp product. qRT-PCR was performed using ViiA 7 by Life Technologies . Primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/), using the genome of Postia placenta MAD 698-R as the template (https://genome.jgi.doe.gov/Pospl1/Pospl1.home.html). Target genes were selected based on existing transcriptomic data of genes known to be upregulated during decay by R. placenta MAD 698-R (The qRT-PCR-specific primers used to determine the transcript levels of selected genes were designed with a target melting temperature (AD 698-R . The genPostia placenta MAD 698-R v1.0 genome from the Joint Genome Institute (https://genome.jgi.doe.gov/pages/search-for-genes.jsf?organism=Pospl1). Threshold cycle (TC) values obtained here were used to quantify gene expression.Two constitutive housekeeping genes, β-tubulin  and α-tubulin , were used as a baseline for gene expression. The target genes (Tg) and the endogenous controls in this study are listed in TC values was the QuantStudio real-time PCR system . The TC values of βt, αt, and Tg were used to quantify gene expression according to the following equation: 4. As an internal control, the expression levels of βt and αt were compared using the same equation, showing a stable expression, with αt being expressed at approximately 80% relative to βt. Only data for βt were included in this paper.The software used to export the All statistics were performed in JMP . The significance of differences in expression levels of each gene was calculated with Tukey’s honestly significant difference (HSD) test. A probability of ≤0.05 was the statistical type I error level.PRJEB32334. The transcriptome assembly and all lists of differentially expressed genes were deposited in Dryad at https://doi.org/10.5061/dryad.vc6s461.RNA-seq data generated for this study were deposited at the European Nucleotide Archive (ENA) under accession number