Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

Quadratic strong coupling in AlN Kerr cavity solitons.

Photonic platforms with χ(2) nonlinearity offer new degrees of freedom for Kerr frequency comb development. Here, we demonstrate Kerr soliton generation at 1550 nm with phase-matched quadratic coupling to the 775 nm harmonic band in a single AlN microring and thus the formation of dual-band mode-locked combs. In the strong quadratic coupling regime where the χ(2) phase-matching window overlaps the pump mode, the pump-to-harmonic-comb conversion efficiency is optimized. However, the strong quadratic coupling also drastically modifies the Kerr comb generation dynamics and decreases the probability of soliton generation. By engineering the χ(2) phase-matching wavelength, we are able to achieve a balance between high conversion efficiency and high soliton formation rate under the available pump power and microring quality factors. Our numerical simulations confirm the experimental observations. These findings provide guidance on tailoring single-cavity dual-band coherent comb sources.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis.

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major drawback to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size exclusion chromatography-based approach. Advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

RNA-based therapeutics to treat human fungal infections.

In recent decades, RNA-based therapeutics have transitioned from a near impossibility to a compelling treatment alternative for genetic disorders and infectious diseases. The mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are truly groundbreaking, and new adaptations are already being proposed to fight other microbes. Unfortunately, the potential of RNA-based therapeutics to treat human fungal infections has remained mostly absent from the conversation, despite the fact that invasive fungal infections kill as many per year as tuberculosis and even more than malaria. Here, we argue that RNA-based therapeutics should be investigated for the treatment of human fungal infections and discuss several major roadblocks and potential circumventions that may allow for the realization of RNA-based therapies against human fungal pathogens.

Aluminum nitride nanophotonics for beyond-octave soliton microcomb generation and self-referencing.

Frequency microcombs, alternative to mode-locked laser and fiber combs, enable miniature rulers of light for applications including precision metrology, molecular fingerprinting and exoplanet discoveries. To enable frequency ruling functions, microcombs must be stabilized by locking their carrier-envelope offset frequency. So far, the microcomb stabilization remains compounded by the elaborate optics external to the chip, thus evading its scaling benefit. To address this challenge, here we demonstrate a nanophotonic chip solution based on aluminum nitride thin films, which simultaneously offer optical Kerr nonlinearity for generating octave soliton combs and quadratic nonlinearity for enabling heterodyne detection of the offset frequency. The agile dispersion control of crystalline aluminum nitride photonics permits high-fidelity generation of solitons with features including 1.5-octave spectral span, dual dispersive waves, and sub-terahertz repetition rates down to 220 gigahertz. These attractive characteristics, aided by on-chip phase-matched aluminum nitride waveguides, allow the full determination of the offset frequency. Our proof-of-principle demonstration represents an important milestone towards fully integrated self-locked microcombs for portable optical atomic clocks and frequency synthesizers.

Photonic Dissipation Control for Kerr Soliton Generation in Strongly Raman-Active Media.

Microcavity solitons enable miniaturized coherent frequency comb sources. However, the formation of microcavity solitons can be disrupted by stimulated Raman scattering, particularly in the emerging crystalline microcomb materials with high Raman gain. Here, we propose and implement dissipation control-tailoring the energy dissipation of selected cavity modes-to purposely raise or lower the threshold of Raman lasing in a strongly Raman-active lithium niobate microring resonator and realize on-demand soliton mode locking or Raman lasing. Numerical simulations are carried out to confirm our analyses and agree well with experiment results. Our work demonstrates an effective approach to address strong stimulated Raman scattering for microcavity soliton generation.

Induction of protein aggregation and starvation response by tRNA modification defects.

Posttranscriptional modifications of anticodon loops contribute to the decoding efficiency of tRNAs by supporting codon recognition and loop stability. Consistently, strong synthetic growth defects are observed in yeast strains simultaneously lacking distinct anticodon loop modifications. These phenotypes are accompanied by translational inefficiency of certain mRNAs and disturbed protein homeostasis resulting in accumulation of protein aggregates. Different combinations of anticodon loop modification defects were shown to affect distinct tRNAs but provoke common transcriptional changes that are reminiscent of the cellular response to nutrient starvation. Multiple mechanisms may be involved in mediating inadequate starvation response upon loss of critical tRNA modifications. Recent evidence suggests protein aggregate induction to represent one such trigger.

Ultraviolet to mid-infrared supercontinuum generation in single-crystalline aluminum nitride waveguides.

We demonstrate ultrabroadband supercontinuum generation from ultraviolet to mid-infrared wavelengths in single-crystalline aluminum nitride waveguides. Tunable dispersive waves are observed at the mid-infrared regime by precisely controlling the waveguide widths. In addition, ultraviolet light is generated through cascaded second-harmonic generation in the modal phase-matched waveguides. Numerical simulation indicates a high degree of coherence of the generated spectrum at around the telecom pump and two dispersive waves. Our results establish a reliable path for multiple octave supercontinuum comb generation in single-crystalline aluminum nitride to enable applications including precision frequency metrology and spectroscopy.

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications.

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.

Beyond 100 THz-spanning ultraviolet frequency combs in a non-centrosymmetric crystalline waveguide.

Ultraviolet frequency combs enable applications ranging from precision spectroscopy to atomic clocks by addressing electronic transitions of atoms and molecules. Access to ultraviolet light via integrated nonlinear optics is usually hampered by the strong material dispersion and large waveguide attention in ultraviolet regions. Here we demonstrate a simple route to chip-scale ultraviolet comb generators, simultaneously showing a gap-free frequency span of 128 terahertz and high conversion efficiency. This process relies on adiabatic quadratic frequency translation of a near-visible supercontinuum sourced by an ultrafast fiber laser. The simultaneous cubic and quadratic nonlinear processes are implemented in single-crystalline aluminum nitride thin films, where chirp-modulated taper waveguides are patterned to ensure a broad phase matching. The heterodyne characterization suggests that both the near-visible and ultraviolet supercontinuum combs maintain high coherence. Our approach is also adaptable to other non-centrosymmetric photonic platforms for ultrafast nonlinear optics with scalable bandwidth.

Soliton microcomb generation at 2 µm in z-cut lithium niobate microring resonators.

Chip-based soliton frequency combs have been demonstrated on various material platforms, offering broadband, mutually coherent, and equally spaced frequency lines desired for many applications. Lithium niobate (LN), possessing both second- and third-order optical nonlinearities, as well as integrability on insulating substrates, has emerged as a novel source for microcomb generation and controlling. Here we demonstrate mode-locked soliton microcombs generated around 2 µm in a high-Q z-cut LN microring resonator. The intracavity photorefractive effect is found to be still dominant over the thermal effect in the 2 µm region, which facilitates direct accessing soliton states in the red-detuned regime, as reported in the telecom band. We also find that intracavity stimulated Raman scattering is greatly suppressed when moving the pump wavelength from the telecom band to 2 µm, thus alleviating Raman-Kerr comb competition. This Letter expands mode-locked LN microcombs to 2 µm, and could enable a variety of potential applications based on LN nanophotonic platform.

Phonon Coupling between a Nanomechanical Resonator and a Quantum Fluid.

Owing to their extraordinary sensitivity to external forces, nanomechanical systems have become an important tool for studying mesoscopic physics and realizing hybrid quantum systems. While nanomechanics has been widely applied in solid-state systems, its use in liquid receives less attention. There it finds unique applications such as biosensing, rheological sensing, and studying both classical and quantum fluid dynamics in unexplored regimes. In this work, we demonstrate efficient coupling of a nano-optomechanical resonator to a bosonic quantum fluid, superfluid 4He, through ultrahigh-frequency phonons (i.e., sound waves) approaching gigahertz frequencies. A high phonon exchange efficiency >92% and minimum excitation rate of 0.25 phonons per oscillations period, or equivalently kB T/ hfm Qm = 0.044 ≪ 1, are achieved. Based on our experimental results, we further predict that strong coupling between a nanomechanical resonator and superfluid cavity phonons with cooperativity up to 880 can be achieved. Our study opens new opportunities in controlling and manipulating superfluid at the nanoscale and low-excitation level.

Unfolded Protein Response Suppression in Yeast by Loss of tRNA Modifications.

Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyze whether this includes the activation of the unfolded protein response (UPR), which is commonly triggered by protein aggregation within the endoplasmic reticulum (ER). We demonstrate that two different aggregation prone tRNA modification mutants (elp6 ncs2; elp3 deg1) lacking combinations of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s²U: elp3; elp6; ncs2) and pseudouridine (Ψ: deg1) reduce, rather than increase, splicing of HAC1 mRNA, an event normally occurring as a precondition of UPR induction. In addition, tunicamycin (TM) induced HAC1 splicing is strongly impaired in the elp3 deg1 mutant. Strikingly, this mutant displays UPR independent resistance against TM, a phenotype we found to be rescued by overexpression of tRNAGln(UUG), the tRNA species usually carrying the mcm5s²U34 and Ψ38 modifications. Our data indicate that proper tRNA anticodon loop modifications promote rather than impair UPR activation and reveal that protein synthesis and homeostasis defects in their absence do not routinely result in UPR induction but may relieve endogenous ER stress.

High-fidelity cavity soliton generation in crystalline AlN micro-ring resonators.

Chip-scale mode-locked dissipative Kerr solitons have been realized on various materials platforms, making it possible to achieve a miniature, highly coherent frequency comb source with high repetition rates. Aluminum nitride (AlN), an appealing nonlinear optical material having both Kerr (χ3), and Pockels (χ2) effects, has immerse potential for comb self-referencing without the need for external harmonic generators. However, cavity soliton states have not yet been achieved in AlN microresonators. Here, we demonstrate mode-locked Kerr cavity soliton generation in a crystalline AlN microring resonator. By utilizing ultrafast tuning of the pump frequency through single-sideband modulation, in combination with an optimized wavelength scan and pump power-ramp patterns, we can deterministically elongate a ∼400 ns short-lived soliton to a time span as long as we wish to hold it.

Cooperativity between different tRNA modifications and their modification pathways.

Ribonucleotide modifications perform a wide variety of roles in synthesis, turnover and functionality of tRNA molecules. The presence of particular chemical moieties can refine the internal interaction network within a tRNA molecule, influence its thermodynamic stability, contribute novel chemical properties and affect its decoding behavior during mRNA translation. As the lack of specific modifications in the anticodon stem and loop causes disrupted proteome homeostasis, diminished response to stress conditions, and the onset of human diseases, the underlying modification cascades have recently gained particular scientific and clinical interest. Nowadays, a complicated but conclusive image of the interconnectivity between different enzymatic modification cascades and their resulting tRNA modifications emerges. Here we summarize the current knowledge in the field, focusing on the known instances of cross talk among the enzymatic tRNA modification pathways and the consequences on the dynamic regulation of the tRNA modificome by various factors. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications.

Recently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm5U or s2U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNALysUUU dependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm5U and s2U leads to increased ribosome pausing at tRNALysUUU dependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm5U and s2U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNALysUUU. Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t6A) to its cyclic derivative (ct6A) at the anticodon adjacent position 37. Since loss of ct6A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t6A to ct6A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast.

Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppressed either at elevated cultivation temperatures or under sulfur starvation. In line with this, mutants with sulfur transfer defects that are linked to enzymes (Tum1, Uba4) required for Urm1 activation by thiocarboxylation (Urm1-COSH) were found to maintain drastically reduced levels of Ahp1 urmylation and mcm5s2U34 modification. Moreover, as revealed by site specific mutagenesis, the S-transfer rhodanese domain (RHD) in the E1-like activator (Uba4) crucial for Urm1-COSH formation is critical but not essential for protein urmylation and tRNA thiolation. In sum, sulfur supply, transfer and activation chemically link protein urmylation and tRNA thiolation. These are features that distinguish the ubiquitin-like modifier system Uba4•Urm1 from canonical ubiquitin family members and will help elucidate whether, in addition to their mechanistic links, the protein and tRNA modification branches of the URM1 pathway may also relate in function to one another.