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At least 955 records · Page 53
All sourcesNASA NTRSDOE OSTI
NASA NTRS1995

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

NASA Discipline Exobiology↗
DOE OSTI2022

Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing

Abstract CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Phage resistance mutations in a marine bacterium impact biogeochemically relevant cellular processes

Phage–bacteria interactions shape ecology and biogeochemistry across biomes. Resistance, arising from their evolutionary arms race, is well documented for receptor mutations, but other resistance mechanisms and their ecological implications remain unexplored. Here we isolated, sequenced and characterized 13 phage-resistant mutants of marine Cellulophaga baltica (Flavobacteriia). Mechanistically, mutations in surface proteins provided broad and complete extracellular resistance against multiple phages through decreased adsorption. Intracellular mutations affecting serine, glycine and threonine metabolism produced narrower resistance against a single phage, permitting viral DNA replication, and, in one mutant, were shown to be lipid mediated. Putative ecosystem impacts inferred from in vitro experiments include: (1) altered carbon utilization for all mutants, but especially by surface ones, (2) increased metabolite secretion for one modelled intracellular mutant (including experimentally verified acetate) and (3) increased ‘stickiness’ for all mutants, with surface mutants also sedimenting faster. Our findings highlight new resistance mechanisms and suggest that the phage–host arms race could result in ecosystem-level biogeochemical impacts in marine microorganisms.

Urvoy, Marion [Ohio State University]↗
NASA NTRS2017

Abiotic Synthesis of Nucleic Acids: Hypochromicity and Future Research

The earliest forms of life would likely have a protocellular form, with a membrane encapsulating some form of linear charged polymer. These polymers could have enzymatic as well as genetic properties. We can simulate plausible prebiotic conditions in the laboratory to test hypotheses related to this concept. In earlier work we have shown that mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of dehydration- rehydration cycles (Rajamani, 2008). If mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers DeGuzman, 2014). We have used conventional and novel techniques to demonstrate that secondary structures stabilized by hydrogen bonds may be present in the condensation products produced in dehydration- rehydration cycles that simulate hydrothermal fields that were present on the early Earth. Gel electrophoresis data corroborates the presence of up to 200-base pair length RNA fragments in products of Hydration-Dehydration experiments. Furthermore, hypochromicity measurements demonstrate a degree of hypochromicity found in single RNA strand of known sequence, as well as results that indicate this is true also for a sample of complementary strands of RNA. Analysis of ionic current signatures of known RNA hairpin molecule as measured using a nanopore detector indicate a significant variability in pattern, different from the signatures produced by DNA hairpin molecules. This informs how we may interpret nanopore data gathered from prebiotic simulations.

Glass, K.↗
DOE OSTI2020

Data from A Genetic Toolbox for Metabolic Engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis , which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis .

Conversion↗
NASA NTRS1986

Molecular replication

The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

Orgel, L. E.↗
NASA NTRS2019

A Lab with a View: Findings from NASA's Aircraft Bioaerosol Collector

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

bioaerosols↗
DOE OSTI2021

HNRNPUL1 inhibits cisplatin sensitivity of esophageal squamous cell carcinoma through regulating the formation of circMAN1A2

Highlights: • HNRNPUL1 is an independent prognostic factor of ESCC patients receiving platinum-based chemotherapy. • Knockdown of HNRNPUL1 enhances cisplatin sensitivity of ESCC cells. • HNRNPUL1 inhibits the cisplatin sensitivity of ESCC cells by regulating the formation of circMAN1A2. Cisplatin (CDDP) is widely used for chemotherapy of esophageal squamous cell carcinoma (ESCC) but the drug resistance limits its therapeutic benefit. Heterogeneous nuclear ribonucleoprotein U-like 1 (HNRNPUL1) belongs to the family of RNA-binding proteins (RBPs) and is involved in DNA damage repair. To investigate whether and how HNRNPUL1 affects CDDP resistance of ESCC, we evaluated the expression of HNRNPUL1 and found that it was associated with recurrence in ESCC patients receiving postoperative platinum-based chemotherapy and was an independent prognostic factor for disease-free survival (DFS). Besides, we showed that the reduced expression of HNRNPUL1 enhanced the CDDP sensitivity of ESCC cells. Furthermore, RNA immunoprecipitation coupled with high-throughput sequencing (RIP-seq) were performed and a range of HNRNPUL1-binding RNAs influenced by CDDP treatment were identified followed by bioinformatics analysis. In terms of mechanism, we found that HNRNPUL1 inhibited CDDP sensitivity of ESCC cells by regulating the CDDP sensitivity-inhibited circular RNA (circRNA) MAN1A2 formation. Taken together, our results first demonstrated the role of HNRNPUL1 in CDDP resistance of ESCC and suggested that HNRNPUL1 may be a potential target of ESCC chemotherapy.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1997

Conserved gene clusters in bacterial genomes provide further support for the primacy of RNA

Five complete bacterial genome sequences have been released to the scientific community. These include four (eu)Bacteria, Haemophilus influenzae, Mycoplasma genitalium, M. pneumoniae, and Synechocystis PCC 6803, as well as one Archaeon, Methanococcus jannaschii. Features of organization shared by these genomes are likely to have arisen very early in the history of the bacteria and thus can be expected to provide further insight into the nature of early ancestors. Results of a genome comparison of these five organisms confirm earlier observations that gene order is remarkably unpreserved. There are, nevertheless, at least 16 clusters of two or more genes whose order remains the same among the four (eu)Bacteria and these are presumed to reflect conserved elements of coordinated gene expression that require gene proximity. Eight of these gene orders are essentially conserved in the Archaea as well. Many of these clusters are known to be regulated by RNA-level mechanisms in Escherichia coli, which supports the earlier suggestion that this type of regulation of gene expression may have arisen very early. We conclude that although the last common ancestor may have had a DNA genome, it likely was preceded by progenotes with an RNA genome.

Non-NASA Center↗
DOE OSTI2022

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mycena genomes resolve the evolution of fungal bioluminescence

Mushroom-forming fungi in the order Agaricales represent an independent origin of bioluminescence in the tree of life; yet the diversity, evolutionary history, and timing of the origin of fungal luciferases remain elusive. We sequenced the genomes and transcriptomes of five bonnet mushroom species ( Mycena spp.), a diverse lineage comprising the majority of bioluminescent fungi. Two species with haploid genome assemblies ~150 Mb are among the largest in Agaricales, and we found that a variety of repeats between Mycena species were differentially mediated by DNA methylation. We show that bioluminescence evolved in the last common ancestor of mycenoid and the marasmioid clade of Agaricales and was maintained through at least 160 million years of evolution. Analyses of synteny across genomes of bioluminescent species resolved how the luciferase cluster was derived by duplication and translocation, frequently rearranged and lost in most Mycena species, but conserved in the Armillaria lineage. Luciferase cluster members were coexpressed across developmental stages, with the highest expression in fruiting body caps and stipes, suggesting fruiting-related adaptive functions. Our results contribute to understanding a de novo origin of bioluminescence and the corresponding gene cluster in a diverse group of enigmatic fungal species.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Validation and functional characterization of transcription factors in wheat using cell-free protein expression and high-throughput sequencing technologies

Transcription factors (TFs) are critical biomolecules that control and regulate gene expression in every organism. In wheat, there are 3,606 genes annotated as putative TFs which are primarily uncharacterized. In this work, using a DAP-seq approach, we have validated and characterized a select number of TFs (5bl and 5dl) belonging to GeBP family of proteins, responsible for plant cellular growth, development, and differentiation. We found that top DNA binding motifs for 5bl and 5dl are (G/T)N(T/G)GTGGT and (C/G)AA(C/G)AA respectively. These motifs/peaks are enriched in the promoter regions of the genes, which are associated with rRNA biogenesis and protein maturation pathways. This study is also a first demonstration that DAP-seq can be applied for such a complex and large genome such as wheat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Merging Fungal and Bacterial Community Profiles via an Internal Control

Integrated measurements of fungi and bacteria are critical to understand how interactions between these taxa drive key processes in ecosystems ranging from soils to animal guts. High-throughput amplicon sequencing is commonly used to census microbiomes, but the genetic markers targeted for fungi and bacteria (typically ribosomal regions) are domain-specific so profiling must be performed separately, obscuring relationships between these groups. To solve this problem, we developed a spike-in method with an internal control (IC) construct containing primer sites commonly used for bacterial and fungal taxonomic profiling. The internal control offers several advantages: estimation of absolute abundances, estimation of fungal to bacterial ratios (F:B), integration of bacterial and fungal profiles for holistic community analysis, and lower costs compared to other quantitation methods. To validate the IC as a scaling method, we compared IC-derived measures of F:B to measures from quantitative PCR (qPCR) using a commercial mock community (the ZymoBiomic Microbial Community DNA Standard II, containing two fungi and eight bacteria) and complex environmental samples. For both the mock community and the environmental samples, the IC produced F:B values that were statistically consistent with qPCR. Merging the environmental fungal and bacterial profiles based on the IC-derived F:B values revealed new relationships among samples in terms of community similarity. As a result, this IC method is the first spike-in method to employ a single construct for cross-domain amplicon sequencing, offering more reliable measurements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of Gabija anti-phage defence and viral immune evasion

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA–GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Isolation and characterization of 24 phages infecting the plant growth-promoting rhizobacterium Klebsiella sp. M5al

Bacteriophages largely impact bacterial communities via lysis, gene transfer, and metabolic reprogramming and thus are increasingly thought to alter nutrient and energy cycling across many of Earth’s ecosystems. However, there are few model systems to mechanistically and quantitatively study phage-bacteria interactions, especially in soil systems. Here, we isolated, sequenced, and genomically characterized 24 novel phages infectingKlebsiellasp. M5al, a plant growth-promoting, nonencapsulated rhizosphere-associated bacterium, and compared many of their features against all 565 sequenced, dsDNAKlebsiellaphage genomes. Taxonomic analyses revealed that theseKlebsiellaphages belong to three known phage families (Autographiviridae,Drexlerviridae, andStraboviridae) and two newly proposed phage families (CandidatusMavericviridaeand Ca.Rivulusviridae). At the phage family level, we found that core genes were often phage-centric proteins, such as structural proteins for the phage head and tail and DNA packaging proteins. In contrast, genes involved in transcription, translation, or hypothetical proteins were commonly not shared or flexible genes. Ecologically, we assessed the phages’ ubiquity in recent large-scale metagenomic datasets, which revealed they were not widespread, as well as a possible direct role in reprogramming specific metabolisms during infection by screening their genomes for phage-encoded auxiliary metabolic genes (AMGs). Even though AMGs are common in the environmental literature, only one of our phage families,Straboviridae, contained AMGs, and the types of AMGs were correlated at the genus level. Host range phenotyping revealed the phages had a wide range of infectivity, infecting between 1–14 of our 22 bacterial strain panel that included pathogenicKlebsiellaandRaoultellastrains. This indicates that not all capsule-independent Klebsiella phages have broad host ranges. Together, these isolates, with corresponding genome, AMG, and host range analyses, help build theKlebsiellamodel system for studying phage-host interactions of rhizosphere-associated bacteria.

Science & Technology - Other Topics↗
DOE OSTI2022

High throughput single cell multiomics platform [Abstract]

In this collaborative project, PNNL and Scienion will co-develop an integrated microfluidic technology to co-measure the transcriptome and proteome in single cells. The technology will enable us to efficiently separate proteins from mRNA transcripts between two microchips, barcode the molecules, and measure them separately with next-generation sequencing and mass spectrometry, respectively. Multicellular organisms contain diverse cell types and tremendous cell-to-cell heterogeneity that dictates a multitude of biological functions in both physiological and pathological environments. Even in the case of microbes, these genetically identical organisms can randomly differentiate into many subpopulations that assume different roles for the survival of the community. Bulk-scale measurements are insufficient to resolve such complexities. The development and applications of high throughput single-cell omics technologies have transformed our understanding of cellular heterogeneities and their differential responses to internal signaling events or external stimulations. Despite these advances, most current single-cell omic technologies provide information on only one type of biomolecule. Perse, such measurements provide incomplete information because the cell phenotype is determined by multiple layers of biomolecules and the interplay between genome, epigenome, transcriptome, and proteome. For example, mRNA abundance in one cell can not be precisely referred to the corresponding DNA and protein in other cells because of the potential subtle difference in genotype (e.g., somatic mutation or copy number variation) or phenotype (external microenvironment and cell-cell interactions). As such, parallel measurement of multiple biomolecules in the same single cells can offer unique advantages compared with measuring them separately in different single cells. Scienion is a world-leading biotech company focusing on precision liquid handling and its application in single-cell whole-genome sequencing and RNA sequencing. PNNL is the leading institution in ultrasensitive mass spectrometry, microfluidics, and untargeted single-cell proteomics (scProteomics). This collaboration will facilitate a unique fusion between scTranscriptomics capability at Scienion and scProteomics capabilities at PNNL to, for the first time, perform both untargeted transcriptomics and proteomics from the same single cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Detection of Candida DNA in peritoneal fluids by PCR assay optimizing the diagnosis and treatment for intra-abdominal candidiasis in high-risk ICU patients: A prospective cohort study

Background Intra-abdominal candidiasis (IAC) is the predominant type of invasive candidiasis with high mortality in critically ill patients. This study aimed to investigate whether the polymerase chain reaction (PCR) assay for detecting Candida DNA in peritoneal fluids (PF) is useful in diagnosing and management of IAC in high-risk patients in intensive care unit (ICU). Methods A prospective single-center cohort study of surgical patients at high risk for IAC was conducted in the ICU. PF was collected from the abdominal drainage tubes (within 24 h) or by percutaneous puncture. Direct PF smear microscopy, PF culture, blood culture, and serum (1–3)-β-D-glucan were performed in all patients. For Candida PCR assay, the ITS1/ITS4 primers that targeted the ITS1-5.8 s-ITS2 regions were used for PCR, and sequencing analysis was used to identify the pathogen at the species level. IAC was defined according to the 2013 European consensus criteria. Results Among 83 patients at high risk for IAC, the IAC criteria were present in 17 (20.5%). The sensitivity and specificity of the Candida PCR assay were 64.7 and 89.4%, respectively, and the area under the receiver operating characteristic curve was 0.77 (95% CI: 0.63–0.91). In this cohort, the positive predictive value and negative predictive value were 90.8% (95% CI: 80.3–96.2%) and 61.1% (95% CI: 36.1–81.7%), respectively. Diagnostic consistency was moderate (kappa 0.529, p < 0.001) according to the 2013 European consensus criteria. Conclusion Detection of Candida DNA in PF using PCR can be considered an adjunct to existing routine diagnostic tools which may optimize the diagnosis and antifungal treatment of IAC in high-risk patients in the ICU.

Xie, Min↗