Genome-wide profiling of piggyBac transposon insertion mutants reveals loss of the F1F0 ATPase complex causes fluconazole resistance in Candida glabrata.

Invasive candidiasis caused by non-albicans species has been on the rise, with Candida glabrata emerging as the second most common etiological agent. Candida glabrata possesses an intrinsically lower susceptibility to azoles and an alarming propensity to rapidly develop high-level azole resistance during treatment. In this study, we have developed an efficient piggyBac (PB) transposon-mediated mutagenesis system in C. glabrata to conduct genome-wide genetic screens and applied it to profile genes that contribute to azole resistance. When challenged with the antifungal drug fluconazole, PB insertion into 270 genes led to significant resistance. A large subset of these genes has a role in the mitochondria, including almost all genes encoding the subunits of the F1F0 ATPase complex. We show that deleting ATP3 or ATP22 results in increased azole resistance but does not affect susceptibility to polyenes and echinocandins. The increased azole resistance is due to increased expression of PDR1 that encodes a transcription factor known to promote drug efflux pump expression. Deleting PDR1 in the atp3Δ or atp22Δ mutant resulted in hypersensitivity to fluconazole. Our results shed light on the mechanisms contributing to azole resistance in C. glabrata. This PB transposon-mediated mutagenesis system can significantly facilitate future genome-wide genetic screens.

In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

The transcription factor Rpn4 activates its own transcription and induces efflux pump expression to confer fluconazole resistance in Candida auris.

IMPORTANCE: Candida auris is a recently emerged pathogenic fungus of grave concern globally due to its resistance to conventional antifungals. This study takes a whole-genome approach to explore how C. auris overcomes growth inhibition imposed by the common antifungal drug fluconazole. We focused on gene disruptions caused by a "jumping genetic element" called transposon, leading to fluconazole resistance. We identified mutations in two genes, each encoding a component of the Ubr2/Mub1 ubiquitin-ligase complex, which marks the transcription regulator Rpn4 for degradation. When either protein is absent, stable Rpn4 accumulates in the cell. We found that Rpn4 activates the expression of itself as well as the main drug efflux pump gene CDR1 by binding to a PACE element in the promoter. Furthermore, we identified an amino acid change in Ubr2 in many resistant clinical isolates, contributing to Rpn4 stabilization and increased fluconazole resistance.

ggcoverage: an R package to visualize and annotate genome coverage for various NGS data.

BACKGROUND: Visualizing genome coverage is of vital importance to inspect and interpret various next-generation sequencing (NGS) data. Besides genome coverage, genome annotations are also crucial in the visualization. While different NGS data require different annotations, how to visualize genome coverage and add the annotations appropriately and conveniently is challenging. Many tools have been developed to address this issue. However, existing tools are often inflexible, complicated, lack necessary preprocessing steps and annotations, and the figures generated support limited customization. RESULTS: Here, we introduce ggcoverage, an R package to visualize and annotate genome coverage of multi-groups and multi-omics. The input files for ggcoverage can be in BAM, BigWig, BedGraph and TSV formats. For better usability, ggcoverage provides reliable and efficient ways to perform read normalization, consensus peaks generation and track data loading with state-of-the-art tools. ggcoverage provides various available annotations to adapt to different NGS data (e.g. WGS/WES, RNA-seq, ChIP-seq) and all the available annotations can be easily superimposed with ' + '. ggcoverage can generate publication-quality plots and users can customize the plots with ggplot2. In addition, ggcoverage supports the visualization and annotation of protein coverage. CONCLUSIONS: ggcoverage provides a flexible, programmable, efficient and user-friendly way to visualize and annotate genome coverage of multi-groups and multi-omics. The ggcoverage package is available at https://github.com/showteeth/ggcoverage under the MIT license, and the vignettes are available at https://showteeth.github.io/ggcoverage/ .

Endothelial Brg1 fine-tunes Notch signaling during zebrafish heart regeneration.

Myocardial Brg1 is essential for heart regeneration in zebrafish, but it remains unknown whether and how endothelial Brg1 plays a role in heart regeneration. Here, we found that both brg1 mRNA and protein were induced in cardiac endothelial cells after ventricular resection and endothelium-specific overexpression of dominant-negative Xenopus Brg1 (dn-xbrg1) inhibited myocardial proliferation and heart regeneration and increased cardiac fibrosis. RNA-seq and ChIP-seq analysis revealed that endothelium-specific overexpression of dn-xbrg1 changed the levels of H3K4me3 modifications in the promoter regions of the zebrafish genome and induced abnormal activation of Notch family genes upon injury. Mechanistically, Brg1 interacted with lysine demethylase 7aa (Kdm7aa) to fine-tune the level of H3K4me3 within the promoter regions of Notch family genes and thus regulated notch gene transcription. Together, this work demonstrates that the Brg1-Kdm7aa-Notch axis in cardiac endothelial cells, including the endocardium, regulates myocardial proliferation and regeneration via modulating the H3K4me3 of the notch promoters in zebrafish.

Biomechanics of a novel artificial cervical vertebra from an in vivo caprine cervical spine non-fusion model.

Objective: Anterior cervical corpectomy and fusion (ACCF) has been widely used in the treatment of cervical spondylotic myelopathy (CSM) but is accompanied by unavoidable motion loss and destruction of vertebra. We aim to evaluate the range of motion (ROM) of caprine cervical spine constructs implanted with cervical artificial disc and vertebra system (ADVS). The purpose of this study was to investigate the biomechanical properties of the ADVS from an in vivo caprine cervical spine non-fusion model. Methods: Twelve goats were randomly divided into ADVS or control group, with 6 animals in each group. The animals in the ADVS group were implanted with ADVS at the C4 level. The cervical spine constructs were harvested 6 months after the operation. The ROM of cervical spine specimens in the ADVS group was recorded. Biomechanical testing of the specimens in the control group were conducted to evaluate the ROM of the cervical spine specimens under intact and fixed condition (C3-C5) by an anterior plate, respectively. Results: The biomechanical outcomes showed that the ROM of the levels (C3-C5) implanted with ADVS was maintained. The ROM in the adjacent level (C2-3) did not increase significantly comparing with intact group. Conclusions: In general, ADVS could preserve the ROM of operative levels and could reconstruct the height of the vertebra. ADVS did not increase the ROM of upper adjacent level. This device provides a non-fusion method for the treatment of patients suffering from CSM. However, improvements on the design of ADVS are still needed. Translational potential statement: This study introduced a novel cervical spinal implant, which was designed to have the ability of motion preservation and vertebra construction. Our study provided a non-fusion procedure in the treatment of CSM after ACCF.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris.

The emergent fungal pathogen Candida auris exhibits high resistance to antifungal drugs and environmental stresses, impeding treatment and decontamination1-3. The fungal factors mediating this stress tolerance are largely unknown. In the present study, we performed piggyBac, transposon-mediated, genome-wide mutagenesis and genetic screening in C. auris, and identified a mutant that grew constitutively in the filamentous form. Mapping the transposon insertion site revealed the disruption of a long non-coding RNA, named DINOR for DNA damage-inducible non-coding RNA. Deletion of DINOR caused DNA damage and an upregulation of genes involved in morphogenesis, DNA damage and DNA replication. The DNA checkpoint kinase Rad53 was hyperphosphorylated in dinorΔ mutants, and deletion of RAD53 abolished DNA damage-induced filamentation. DNA-alkylating agents, which cause similar filamentous growth, induced DINOR expression, suggesting a role for DINOR in maintaining genome integrity. Upregulation of DINOR also occurred during exposure to the antifungal drugs caspofungin and amphotericin B, macrophages, H2O2 and sodium dodecylsulfate, indicating that DINOR orchestrates multiple stress responses. Consistently, dinorΔ mutants displayed increased sensitivity to these stresses and were attenuated for virulence in mice. Moreover, genome-wide genetic interaction studies revealed links between the function of DINOR and TOR signalling, an evolutionarily conserved pathway that regulates the stress response. Identification of the mechanism(s) by which DINOR regulates stress responses in C. auris may provide future opportunities for the development of therapeutics.

Recent advances in autophagic machinery: a proteomic perspective.

INTRODUCTION: Autophagy is an evolutionarily conserved cellular clearance process, by which cytosolic components are delivered to autolysosomes for breakdown and recycling to maintain cellular homeostasis. During the past decades, autophagy has been found to be tightly implicated in various physiological and pathological progresses. Unraveling the regulatory mechanisms of the autophagy process will contribute to the development of emerging autophagy-targeting strategies for the treatment of various diseases. Recently, the rapid development of proteomics approaches has enabled the use of large-scale unbiased strategies to unravel autophagy machinery. AREAS COVERED: In this review, we will highlight the recent contributions of proteomics strategies in clarifying the autophagy machinery, with an emphasis on the three different types of autophagy (namely macroautophagy, microautophagy, and chaperone-mediated autophagy). We will also discuss the emerging role of proteomics approaches in investigating the mechanism of the autophagy-based unconventional secretory pathway (secretory autophagy). EXPERT OPINION: Proteomics has provided an effective strategy for the comprehensive analysis of the autophagy process, which will broaden our understanding of autophagy machinery, and holds great promise for developing clinical therapies targeting autophagy.