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Background Aetiology detection is crucial in the diagnosis and treatment of ventilator-associated pneumonia (VAP). However, the detection method needs improvement. In this study, we used Nanopore sequencing to build a quick detection protocol and compared the efficiency of different methods for detecting 7 VAP pathogens. Methods The endotracheal aspirate (ETA) of 83 patients with suspected VAP from Peking University Third Hospital (PUTH) was collected, saponins were used to deplete host genomes, and PCR- or non-PCR-amplified library construction methods were used and compared. Sequence was performed with MinION equipment and local data analysis methods were used for sequencing and data analysis. Results Saponin depletion effectively removed 11 of 12 human genomes, while most pathogenic bacterial genome results showed no significant difference except for S. pneumoniae. Moreover, the average sequence time decreased from 19.6 h to 3.62 h. The non-PCR amplification method and PCR amplification method for library build has a similar average sensitivity (85.8% vs. 86.35%), but the non-PCR amplification method has a better average specificity (100% VS 91.15%), and required less time. The whole method takes 5–6 h from ETA extraction to pathogen classification. After analysing the 7 pathogens enrolled in our study, the average sensitivity of metagenomic sequencing was approximately 2.4 times higher than that of clinical culture (89.15% vs. 37.77%), and the average specificity was 98.8%. Conclusions Using saponins to remove the human genome and a non-PCR amplification method to build libraries can be used for the identification of pathogens in the ETA of VAP patients within 6 h by MinION, which provides a new approach for the rapid identification of pathogens in clinical departments.

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens., Author summary Many human fungal pathogens can undergo dimorphic transition between yeast and hyphal forms in response to different external stimuli, and this morphological transition is generally and critically linked with their infections. In Cryptococcus neoformans, a model pathogenic fungus, the yeast-to-hypha transition can be elicited by mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. Here, we show that GlcN specifically evokes a unique hyperactive cell wall synthetic response, which determines GlcN-induced filamentation (GIF) as a key commitment event. The Mpk1-directed cell wall signaling pathway as a core and conserved cascade connects the cell wall synthetic response and GIF activation in different Cryptococcus pathogens. Overall, the findings reveal a previously unrecognized function of GlcN in stimulating cell wall signaling and biosynthetic machinery, which enables a unique dimorphism commitment mechanism underlying the signal specificity of the mating-independent yeast-to-hypha transition in Cryptococcus pathogens.

In fungi, the sex-determination program universally directs sexual development and syngamy (the fusion of gametes) that underlies pre-meiotic diploidization. However, the contribution of sex-determination to syngamy-independent sexual cycle, which requires autopolyploidization as an alternative approach to elevate ploidy before meiosis, remains unclear in fungi and other eukaryotes. The human fungal pathogenCryptococcus neoformans, as a model organism for studying fungal sexual reproduction, can undergo syngamy-dependent bisexual and syngamy-independent solo unisexual reproduction, in which endoreplication is considered to enable pre-meiotic self-diploidization. Here, by characterizing a mutant lacking all the core sex-determination factors, we show that sex-determination plays a central role in bisexual syngamy but is not strictly required for unisexual development and self-diploidization. This implies an unknown circuit, rather than the sex-determination program, for specifically coordinatingCryptococcusunisexual cycle. We reveal that syngamy and self-diploidization are both governed by the Qsp1-directed paracrine system via two regulatory branches, Vea2 and Cqs2. Vea2 directs bisexual syngamy through the sex-determination program; conversely, Cqs2 is dispensable for bisexual syngamy but activates unisexual endoreplication. Through functional profiling of 41 transcription factors documented to regulateCryptococcussexual development, we reveal that only Cqs2 can drive and integrate all unisexual phases and ensure the production of meiospore progenies. Furthermore, ChIP-seq analysis together with genetic evaluation indicate that Cqs2 induces unisexual self-diploidization through its direct control ofPUM1, whose expression is sufficient to drive autopolyploidization. Therefore, Cqs2 serves as the critical determinant that orchestratesCryptococcusmultistage unisexual cycle that does not strictly require the sexual-determination program.

Bacterial quorum sensing is a well-characterized communication system that governs a large variety of collective behaviours. By comparison, quorum sensing regulation in eukaryotic microbes remains poorly understood, especially its functional role in eukaryote-specific behaviours, such as sexual reproduction. Cryptococcus neoformans is a prevalent fungal pathogen that has two defined sexual cycles (bisexual and unisexual) and is a model organism for studying sexual reproduction in fungi. Here, we show that the quorum sensing peptide Qsp1 serves as an important signalling molecule for both forms of sexual reproduction. Qsp1 orchestrates various differentiation and molecular processes, including meiosis, the hallmark of sexual reproduction. It activates bisexual mating, at least in part through the control of pheromone, a signal necessary for bisexual activation. Notably, Qsp1 also plays a major role in the intercellular regulation of unisexual initiation and coordination, in which pheromone is not strictly required. Through a multi-layered genetic screening approach, we identified the atypical zinc finger regulator Cqs2 as an important component of the Qsp1 signalling cascade during both bisexual and unisexual reproduction. The absence of Cqs2 eliminates the Qsp1-stimulated mating response. Together, these findings extend the range of behaviours governed by quorum sensing to sexual development and meiosis.

Background Circular RNAs (circRNAs) are implicated in various biological processes. As a layer of the gene regulatory network, circRNA expression is also an intermediate phenotype bridging genetic variation and phenotypic changes. Thus, analyzing circRNA expression variation will shed light on molecular fundamentals of complex traits and diseases. Results We systematically characterize 10,559 high-quality circRNAs in 589 human dorsolateral prefrontal cortex samples. We identify biological and technical factors contributing to expression heterogeneity associated with the expression levels of many circRNAs, including the well-known circRNA CDR1as. Combining the expression levels of circRNAs with genetic cis-acting SNPs, we detect 196,255 circRNA quantitative trait loci (circQTLs). By characterizing circQTL SNPs, we find that partial circQTL SNPs might influence circRNA formation by altering the canonical splicing site or the reverse complementary sequence match. Additionally, we find that a subset of these circQTL SNPs is highly linked to genome-wide association study signals of complex diseases, especially schizophrenia, inflammatory bowel disease, and type II diabetes mellitus. Conclusions Our results reveal technical, biological, and genetic factors affecting circRNA expression variation among individuals, which lead to further understanding of circRNA regulation and thus of the genetic architecture of complex traits or diseases. Electronic supplementary material The online version of this article (10.1186/s13059-019-1701-8) contains supplementary material, which is available to authorized users.

Figure S1. Relative circRNA expression level in 14 cell lines, 29 tissues, and DLPFC. Figure S2. The Sanger sequencing results for circRNAs from AKT3, HOOK3, and MDGA2. Figure S3. Distributions of the exon numbers (A) and lengths (B) of exonic circRNAs. Figure S4. Pie charts indicated the fraction of circRNAs produced from one host gene. Figure S5. LIB-associated circRNAs show higher expression levels and shorter median lengths. Figure S6. Comparison of distributions of effect size of circRNAs (red line) and genes (blue line) in detecting SCZ-control differential expression in CMC dataset. (DOCX 1163 kb)

Aims 14,15-Epoxyeicosatrienoic acid (14,15-EET) is abundantly expressed in brain and exerts protective effects against ischaemia. 14,15-EET is hydrolysed by soluble epoxide hydrolase (sEH). sEH−/− mice show a higher level of 14,15-EET in the brain. Astrocytes play a pivotal role in neuronal survival under ischaemic conditions. However, it is unclear whether the neuroprotective effect of 14,15-EET is associated with astrocytes. Methods A mouse model of focal cerebral ischaemia was induced by middle cerebral artery occlusion. Oxygen–glucose deprivation/reoxygenation (OGD/R) was performed on cultured murine astrocytes, neurons and a human cell line. Cell viabilities were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The mRNA expressions were quantified by real-time PCR. Brain derived neurotrophic factor (BDNF) concentration was measured by ELISA. Protein expressions were quantified by Western blotting. BDNF and peroxisome proliferators-activated receptor gamma (PPAR-γ) expressions were analysed by confocal microscopy. Results Decreased infarct volumes, elevated BDNF expression and increased numbers of BDNF/GFAP Glial Fibrillary Acidic Protein double-positive cells were observed in the ischaemic penumbra of sEH−/− mice. The decreased infarct volumes of sEH−/− mice were diminished by intracerebroventricular injection of a blocker of BDNF receptor. 14,15-EET increases BDNF expression and cell viability of murine astrocytes and U251 cells by BDNF-TrkB Tyrosine receptor kinase-B-extracellular signal-regulated kinase 1/2 signalling during OGD/R. 14,15-EET protects neurons from OGD/R by stimulating the production of astrocyte-derived BDNF. 14,15-EET stimulates the production of astrocyte-derived BDNF through PPAR-γ/p-cAMP-response element binding protein signal pathways. Conclusions Our study demonstrates the importance of 14,15-EET-mediated production of astrocyte-derived BDNF for enhancing viability of astrocytes and protecting neurons from the ischaemic injury and provides insights into the mechanism by which 14,15-EET is involved in neuroprotection.