In Situ Remediation of TNT Red Water Contaminated Soil: Field Demonstration

In China, about 1.5 × 105 m3 soil was contaminated by TNT red water, which contained mainly dinitrotoluene sulfonates (DNTS). These nitroaromatic explosives are toxic and exhibit human mutagenic and carcinogenic potential. Biotechnology capable of decontaminating these contaminated sites and applicable to a larger scale field application was in urgent need. A two-year pilot study was conducted in the TNT red water contaminated sites (1000 m3). The sites were treated by integrated treatment technologies of “Desorption-Biostimulation and Bioaugmentation-Phytoremediation.” Soil samples were taken every six months to determine the concentration of nitro-aromatic compounds. Acute toxicity and Fluorescein diacetate (FDA) hydrolytic activity tests were conducted to evaluate the remediation effect. After 2 years of remediation, the total nitroaromatic compounds were effectively removed, with the average removal efficiency of 99.88% and 90.47% in sites planted with alfalfa and reed, respectively. The toxicity was significantly reduced. The average luminescence inhibition ratio reduced from 92.64% to 3.37% and 29.16%, respectively. The soil microbial activity was significantly improved, with the highest FDA hydrolase activity in the surface layer. The remediation costs for the treatment of 1 cubic meter of contaminated soil were estimated to approximately $126 US. The integrated technologies used have huge potential for decontaminating munitions contaminated sites.</p

Data_Sheet_1_Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.docx

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.</p

Functional convergence of <i>gliP</i> and <i>aspf1</i> in <i>Aspergillus fumigatus</i> pathogenicity

<p>Gliotoxin contributes to the virulence of the fungus <i>Aspergillus fumigatus</i> in non-neutropenic mice that are immunosuppressed with corticosteroids. To investigate how the absence of gliotoxin affects both the fungus and the host, we used a nanoString nCounter to analyze their transcriptional responses during pulmonary infection of a non-neutropenic host with a gliotoxin-deficient Δ<i>gliP</i> mutant. We found that the Δ<i>gliP</i> mutation led to increased expression of <i>aspf1</i>, which specifies a secreted ribotoxin. Prior studies have shown that <i>aspf1</i>, like <i>gliP</i>, is not required for virulence in a neutropenic infection model, but its role in a non-neutropenic infection model has not been fully investigated. To investigate the functional significance of this up-regulation of <i>aspf1</i>, a Δ<i>aspf1</i> single mutant and a Δ<i>aspf1</i> Δ<i>gliP</i> double mutant were constructed. Both Δ<i>aspf1</i> and Δ<i>gliP</i> single mutants had reduced lethality in non-neutropenic mice, and a Δ<i>aspf1</i> Δ<i>gliP</i> double mutant had a greater reduction in lethality than either single mutant. Analysis of mice infected with these mutants indicated that the presence of <i>gliP</i> is associated with massive apoptosis of leukocytes at the foci of infection and inhibition of chemokine production. Also, the combination of <i>gliP</i> and <i>aspf1</i> is associated with suppression of CXCL1 chemokine expression. Thus, <i>aspf1</i> contributes to <i>A. fumigatus</i> pathogenicity in non-neutropenic mice and its up-regulation in the Δ<i>gliP</i> mutant may partially compensate for the absence of gliotoxin.</p> <p><b>Abbreviations</b>:PAS: periodic acid-Schiff; PBS: phosphate buffered saline; ROS: reactive oxygen species; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling</p

Activation and Alliance of Regulatory Pathways in <i>C. albicans</i> during Mammalian Infection

<div><p>Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive <i>Candida albicans</i> infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either <i>ZAP1</i> or the Zap1-dependent zinc transporter gene <i>ZRT2</i> restores pathogenicity to a <i>sut1</i> mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host–pathogen interaction occurs.</p></div

Comparison of <i>C</i>. <i>albicans</i> proliferation-defective transcription factor mutants.

<p>(A). Heat map representation of gene expression ratios for 148 environmentally responsive genes at 24 hr postinfection (<i>rim101Δ/Δ</i>, <i>rob1Δ/Δ</i>, <i>rpn4Δ/Δ</i>, <i>sut1Δ/Δ</i>, or <i>zap1Δ/Δ</i>, each relative to the wild type) or 48 hr postinfection (<i>efg1Δ/Δ</i>, relative to the wild type). Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>. (B). Yield of <i>C</i>. <i>albicans</i> RNA relative to total kidney RNA for each mutant and complemented strain at 24 hr postinfection. Asterisks mark significant differences (<i>p</i>-value < 0.05) between mutants and respective complemented strains. (C). Histopathology images of kidney sections. All samples were at 24 hr postinfection. Arrows indicate fungal cells. The scale bar corresponds to 50 microns. For panel A, the specific strains used and the dose of viable cells per mouse, as determined by plating the inocula, were: CW 696 (wild type) 8.4 × 10⁵; CW730 (<i>rob1</i>) 10.2 × 10⁵; DAY25 (<i>rim101</i>) 11.1 × 10⁵; CW1018 (<i>efg1</i>) 9 × 10⁵; CW792 (<i>rpn4</i>) 7.9 × 10⁵; CW756 (<i>zap1</i>) 8.8 × 10⁵; CW704 (<i>sut1</i>) 8.6 × 10⁵. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Expression of <i>C</i>. <i>albicans</i> environmentally responsive genes during invasive infection.

<p>Changes in expression levels during mouse kidney invasion for 248 <i>C</i>. <i>albicans</i> genes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s001" target="_blank">S1 Data</a>) are presented in a heat map format. Mean values of biological triplicates are shown for up-regulation (yellow) and down-regulation (blue) of genes at 12, 24, and 48 hr postinfection relative to mean inoculum levels (0 hr). Color saturation represents the extent of the expression change, with full saturation at 10-fold up- or down-regulation. (All heat maps in this article follow the same color scale.) Portions of the heat map are expanded to illustrate representative early up-regulated genes (top), late genes (middle), and early down-regulated genes (bottom). In these portions, individual samples are presented separately to illustrate reproducibility. We define early expression changes as significant differences between the inoculum and 12 hr sample. We define late expression changes as significant differences between the 12 and 48 hr samples. Significance refers to changes of ≥2-fold and a <i>p</i>-value < 0.05. The data for each sample were normalized to RNA levels from control gene <i>TDH3</i> before mean values were calculated. Our assignment criteria allow some dynamically regulated genes to fall into both the early and late expression classes. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Mouse immune response gene expression during <i>Candida</i> infection.

<p>(A). RNA levels for 46 mouse immune response genes were assayed at 12, 24, and 48 hr postinfection in biological triplicates and were compared to uninfected kidney controls (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 Data</a>). Mouse genes that represent early and late expression classes are presented. We define early genes as those with significantly higher expression in 12 hr postinfection samples compared to uninfected samples. We define late genes as those with no significant difference in expression between uninfected and 12 hr postinfection samples, but with significantly higher expression in 48 hr postinfection samples compared to 24 hr postinfection samples. An asterisk indicates significant differences (<i>p</i>-value < 0.05) between successive time-course samples (uninfected samples versus 12 hr, 24 hr versus 12 hr, 48 hr versus 24 hr). (B). Mice were infected with the indicated transcription factor mutants and respective complemented strains; RNA levels for 46 mouse immune response genes were determined by nanoString at 24h postinfection (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 Data</a>). Induction of four early genes (the same genes shown in panel A for the wild-type strain) are shown here. All four genes were induced significantly more strongly by complemented strains than the respective mutants (<i>p</i>-value < 0.05), as indicated by the bracket and asterisk. <i>P</i>-values for expression of each gene are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 Data</a>. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Rim101-dependent gene regulation during invasive infection.

<p>RNA levels for 148 <i>C</i>. <i>albicans</i> environmental response genes were determined by nanoString at 24 hr postinfection for the <i>rim101</i> mutant and complemented strains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>). (A). Expression ratios are plotted for each gene in <i>rim101Δ/Δ</i> versus wild type (X axis) and <i>rim101Δ/Δ</i> versus complemented strain (Y axis). Three genes (red data points) have significantly different expression ratios in the two comparisons. (B). Expression ratios are presented for all genes significantly down-regulated in the <i>rim101Δ/Δ</i> strain relative to the complemented strain during kidney infection (blue bars; ≥2-fold change and <i>p</i>-value < 0.05). The expression ratios for the same genes in the same strains during intra-abdominal candidiasis (IAC) infection (red bars; reported in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]) or during in vitro growth in Spider medium (green bars) are displayed. Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>. (C). Expression ratios are presented for all genes significantly up-regulated in the <i>rim101Δ/Δ</i> strain relative to the complemented strain during kidney infection (blue bars; ≥2-fold change and <i>p</i>-value < 0.05), during abdominal infection (red bars; reported in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]) or during in vitro growth in Spider medium (green bars) are displayed. Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Gene expression response to caspofungin treatment during infection.

<p>(A). Changes in expression levels for 248 <i>C</i>. <i>albicans</i> environmentally responsive genes are presented for caspofungin treated versus untreated cells at 24 hr postinfection (“Kidney,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>), in vitro in YPD at 30°C (“YPD,” from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref030" target="_blank">30</a>]), and in vitro in RPMI at 37°C (“RPMI,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). These environmentally responsive genes are the same ones for which expression was measured during the time-course of infection depicted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.g001" target="_blank">Fig. 1</a>. For comparison, the expression ratios of the same genes at 12 hr postinfection relative to the inoculum are shown (“12 hr/0 hr,” from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.g001" target="_blank">Fig. 1</a>). The data are presented in heat map format. Regions “1” and “2” are expanded on the right to make gene names legible. (B). Expression levels for 231 <i>C</i>. <i>albicans</i> transcription factor genes were measured for caspofungin treated versus untreated cells at 24 hr postinfection (“In vivo caspo-induced,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>) and in vitro in RPMI at 37°C (“In vitro caspo-induced,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). Significantly up-regulated transcription factor genes are listed (≥2-fold change and <i>p</i> < 0.05). For comparison, the significantly down-regulated transcription factor genes at 12 hr postinfection are listed (“Early down-regulated,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Gene expression during murine infection.

<p>Expression levels for 114 genes are compared in three murine infection models: oropharyngeal candidiasis (48 hr postinfection; oropharyngeal candidiasis [OPC]) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref013" target="_blank">13</a>], kidney infection (12, 24, and 48 hr [this study]), and intra-abdominal infection (48 hr postinfection; IAC) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]. Expression levels are presented as ratios to levels in the inoculum samples used in this study (stationary phase, YPD), and shown as a heat map. Expanded portions illustrate genes induced during oral infection, during all three types of infection, and during abdominal infection. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p