Understanding the mechanism that controls CO2 hydrogenation selectivity is of great significance for designing efficient catalysts in carbon-neutral energy conversion systems. It has been established that strong metal-support interactions (SMSI) and SMSI-induced catalyst encapsulation can regulate the selectivity and activity of the metal-oxide composite catalysts in CO2 reduction; however, the detailed underlying mechanism remains elusive. In this study, we employed theoretical investigations to rationalize CO2 hydrogenation selectivity reversal on Ru@TiOx catalysts, with a focus on the roles of encapsulation-induced electronic modulation and interfacial effects. Density functional theory (DFT) calculations were performed to examine three representative structures: unencapsulated, interface-rich, and fully encapsulated Ru@TiOx. The calculation results reveal that encapsulation of Ru by the TiOx layer markedly alters the electronic structure and distribution of active sites, triggering a reversal in product selectivity from CH4 to CO. Microkinetic modelling analysis further indicates that constructing interfacial sites between Ru and TiOx enhances the CH4 formation rate by an order of magnitude, while encapsulating Ru sites with the TiOx layer enables precise regulation of product selectivity. These findings provide atomic-level insights into the roles of SMSI and interfacial evolution in tuning CO2 hydrogenation selectivity, and offer theoretical guidance for the rational design of efficient Ru-based catalysts via interface and encapsulation engineering.

Single-atom catalysts (SACs) exhibit significant potential in Fenton-like systems. However, precisely regulating isolated active sites to enhance non-radical processes remains a critical challenge. Herein, a Se-modulated single-atom Fe catalyst coupled with ZnSe was constructed for selective non-radical peroxymonosulfate (PMS) activation. Accordingly, Se modulated the coordination environment of Fe sites, driving coordination reconstruction and yielding the Fe-N2C2-Se2 coordination structure. Meanwhile, ZnSe modulated the electronic structure of these Fe sites. These effects contributed to a synergistic PMS activation effect. The resulting catalyst achieved 98.3% tetracycline (TC) removal within 60 min, predominantly via nonradical pathways involving electron transfer process (ETP) and singlet oxygen (1O2). Electrochemical measurements and electron paramagnetic resonance (EPR) analyses demonstrated that Se incorporation increased the electrochemically active surface area (ECSA), decreased the Tafel slope, and significantly enhanced 1O2 generation, indicating improved electron-transfer kinetics. Furthermore, density functional theory (DFT) calculations revealed that, compared with Fe-NC, Se modulation in FeSe-NC induced dual-shell coordination, which enhanced electron delocalization and upshifted the Fe d-band center (from −0.99 to −0.65 eV), thereby optimizing the electronic structure. Correspondingly, PMS adsorption was strengthened (−2.86 eV compared to −2.42 eV), accompanied by OO bond elongation (1.478 to 1.495 Å) and increased Bader charge transfer (0.84 to 0.94 e), ultimately promoting a more efficient and selective non-radical activation pathway. This work establishes a clear structure-electronic structure-reactivity relationship and provides mechanistic insights into nonmetal-modulated SACs design for PMS activation in wastewater treatment.

Single-atom catalysts (SACs) have emerged as a promising frontier in Fenton-like processes. However, optimizing their isolated and dispersed active sites remains a major challenge. Herein, a Mn-modulated dual-Fe-site catalyst (FeMn-NC) featuring both Fe single atoms and Fe nanoclusters was developed. Mn incorporation promoted the formation of Fe nanoclusters composed of Fe0 and Fe1.8Mn1.2C species. The catalyst achieved 97.1% tetracycline (TC) degradation via peroxymonosulfate (PMS) activation, with a reaction rate constant (k) of 0.1152 min-1, arising from the synergistic interplay between Fe single atoms and Fe nanoclusters. Mechanistically, Fe single atoms dominated the non-radical pathway, whereas Fe nanoclusters enhanced electron transfer and introduced a complementary radical route. Density functional theory (DFT) calculations revealed that the coexistence of Fe single atoms and Fe nanoclusters optimized charge distribution and the Fe d-band center, thereby enhancing electron delocalization and promoting PMS adsorption, O-O bond activation, and interfacial Bader charge transfer. Additionally, Fukui function calculation, intermediate products analysis, and wheat germination assays confirmed the reduced environmental toxicity of pollutants after catalytic degradation. This work provides new insights into the rational design of SACs with multiple active sites for the efficient catalytic PMS activation.

Efficient separation of palladium (Pd) from high-level liquid waste (HLLW) is critically important, considering its scarcity in natural resources and associated issues regarding nuclear waste disposal. However, this remains challenging due to the extreme compositional complexity and high acidity of HLLW. Inspired by the square-planar coordination preference of Pd2+ with soft donors, we propose a coordination microenvironment-driven strategy to design a series of bis(pyridyl triazole) pincer ligands (LI-LIII). These ligands feature a tetradentate coordination cavity with four inwardly oriented nitrogen donor atoms, enabling exceptional Pd2+ extraction across a wide acidity range (0.1-5 M HNO3) and high selectivity over 16 competing elements (SFPd/M > 78) as well as the minor actinide americium (SFPd/Am > 793). Varying the alkyl substituents significantly alters the extraction performance, and LIII, bearing the longest alkyl chain, exhibits the highest efficiency. Mechanism studies indicate that extraction proceeds predominantly via a 1:1 metal-ligand complex alongside a minor 1:2 species. Spectroscopic and density functional theory (DFT) results further reveal that in monophasic organic solutions, both 1:1 and 2:1 complexes coexist. The quasi-square-planar coordination geometry of Pd2+ is verified by single-crystal X-ray analysis of Pd(LI)·(NO3)2·(HNO3)2 complex. This work provides a feasible design strategy for advanced extractants through tailoring the metal coordination microenvironment.

Being a program written primarily in Python that strictly adheres to modern object-oriented software engineering and parallel programming practices, VeloxChem is shown to be suitable for the development of (semi)automatized workflows that extend its scope from first-principles quantum chemical purism to hybrid quantum-classical interoperability and some degree of semiempiricism. Methods are presented for building complex systems such as metal-organic frameworks, constructing molecular mechanics and interpolation mechanics force fields, conformer searches, system solvation, determining free energies of solvation, and determining free energy profiles of reaction pathways using the empirical valence bond method. The implementations are made intuitive with opportunities for interactive plotting and 3D molecular structure illustrations through the use of Jupyter notebooks.

Background:Pediatric sepsis is a complex and heterogeneous condition resulting from a dysregulated immune response to infection. Pyroptosis, a newly recognized form of programmed cell death, has been implicated in the progression of various inflammatory diseases. However, the role of pyroptosis-related genes in pediatric sepsis remains unclear. Methods:Based on the GSE13904 dataset, we explored the pyroptosis-related differentially expressed genes (DEGs) in pediatric sepsis. We analyzed the molecular clusters based on pyroptosis-related DEGs. The WGCNA algorithm was performed to identify cluster-specific DEGs. The optimal machine model was identified by multiple machine learning methods (RF, SVM, GLM, XGB). The diagnostic value of hub genes in pediatric sepsis was verified in the training (GSE13904) and validation set (GSE26440) through ROC. qRT-PCR was used to verify the expression levels of 5 hub genes in whole blood between the pediatric sepsis and the control. Results:The dysregulated pyroptosis-related DEGs were identified in pediatric sepsis. Three pyroptosis-related molecular clusters were determined in pediatric sepsis. SVM presented the best discriminative performance with relatively lower residual and root mean square error. The nomogram, calibration curve, and decision curve analysis indicated the accuracy of SVM model to predict pediatric sepsis. 5 hub genes based on SVM presented satisfactory performance in the training and validation sets. These hub genes expression levels in pediatric sepsis were significantly higher than those in healthy controls in clinical samples. Conclusion:Our study systematically analyzed the relationship between pyroptosis and pediatric sepsis, and constructed a promising predictive model to evaluate the risk of pediatric sepsis.

BACKGROUND:Diabetes coexisting with infections (DCI) significantly increases the risk of severe outcomes and mortality in patients. This study proposes that RPL7P1, an uncharacterized pseudogene, plays a role in the pathogenesis of DCI. METHODS:Using a multifaceted approach, we employed experimental datasets from ENCODE to identify key genes. Drug repositioning was performed using gene network analysis with z-scores and the ROCR package. Network expansion was facilitated by NetworkAnalyst's core algorithms, with disease validation through the NHGRI GWAS Catalog. Cytoscape was utilized for network visualization. RESULTS:Our findings reveal RPL7P1's potential involvement in DCI through modulation of CBL and STXBP3 by sequestering hsa-miR-144-3p, interaction with IGF2BP2 protein, and crosstalk with ATP6V1E1 RNA. Single-cell profiling pinpointed endothelial cells as a potential signaling nexus. Therapeutic agents targeting the RPL7P1-centric network showed promise in managing infections in diabetic patients. Additionally, we identified key molecular players, the m6A modification of RPL7P1, and its role in cuproptosis-a novel form of cell death. CONCLUSION:This research elucidates the potential role of the novel pseudogene RPL7P1 in DCI, highlighting the importance of pseudogenes in complex diseases and providing novel insights into the epigenetic modulation of diabetes complicated by infections. Insight Box Diabetes and infections often coexist, complicating our understanding of their shared mechanisms. To address this, we employed a comprehensive suite of bioinformatics methods, including pathway mapping, ceRNA analysis, PPI evaluation, single-cell dissection, network modeling, and drug repositioning strategies. Our research revealed that RPL7P1 modulates signaling pathways by sponging miR-144-3p, interacting with IGF2BP2 and ATP6V1E1 RNA, and influencing cuproptosis-a novel form of cell death-through m6A modification, a key RNA modification. We identified endothelial cells as key mediators of the RPL7P1 network in the liver. Additionally, our integrative approach uncovered five potential therapeutic drugs targeting the RPL7P1 network. These findings provide novel insights into coexisting diabetes and infections, underscoring the value of multidisciplinary strategies in uncovering coexisting disease mechanisms and treatment opportunities.

The photothermal catalysis of carbon dioxide (CO2) reduction into value-added solar fuels represents a promising approach to addressing the energy crisis and mitigating global warming. Recent experimental findings indicate that Ru-HxMoO3-y is capable of completely converting CO2 into methane (CH4). In contrast, Pt-HxMoO3-y has been observed to produce a range of products, with carbon monoxide (CO) being the most prevalent. A comprehensive understanding of the reaction mechanism is essential to elucidate the different sensitivities of catalysts and to facilitate the development of HxMoO3-y-based catalytic systems. This study employs density functional theory to examine the mechanism of CO2 reduction on Ru-HxMoO3-y. The d(7) configuration of Ru enables the transfer of d electrons from the Ru-HxMoO3-y catalyst to CO via pi-back-bond, which results in the weakening of the C-O bond and the preferential formation of CH4. Moreover, Ru-HxMoO3-y has been identified as a promising candidate for the production of ethylene (C2H4), with its selectivity being adjustable through variations in reaction temperature and pressure. Our findings demonstrate that the performance of C-C coupling in HxMoO3-y-based catalysts is significantly influenced by the d configuration of the metal cluster. The theoretically designed Fe/Ru-HxMoO3-y exhibits the most favorable catalytic activity. These insights offer critical mechanistic guidance for the design of advanced photocatalysts to convert CO2 into solar fuels.

The catalytic conversion of carbon dioxide (CO2) into valuable products represents a promising strategy for mitigating the greenhouse effect. However, developing catalysts that exhibit both high activity and selectivity remains a considerable challenge. Composite catalysts based on metal clusters supported on MXene have emerged as a promising candidate for CO2 reduction. A thorough understanding of the catalytic mechanisms of CO2 reduction utilizing MXene-based catalysts is crucial for advancing and optimizing these catalysts. In this study, the detailed reaction mechanism of CO2 reduction on composite Run@Mo2TiC2O2 catalysts was investigated theoretically, employing the four-atom Ru4 cluster as the model system. Electronic structure analysis revealed that the orbital interactions between the Ru4 cluster and CO2 play a pivotal role in determining the adsorption configuration of the CO2 molecule. Specifically, CO2 exhibits distinct adsorption modes on planar and tetrahedral Ru4 clusters, leading to divergent CO2 reduction pathways. Density functional theory (DFT) calculations suggest that the tetrahedral Ru4 cluster demonstrates higher selectivity for the reduction of CO2 to carbon monoxide (CO) compared to the planar configuration. However, the results indicate that although the planar Ru4 cluster is predominant on the Mo2TiC2O2 surface at room temperature, there is a coexistence of planar and tetrahedral Ru4 clusters at the higher temperature used in photothermal catalysis. It suggests that the configuration of Ru4@Mo2TiC2O2 can be modulated by manipulating the reaction temperature, thereby adjusting the selectivity. This finding provides comprehensive insights into the CO2 reduction mechanism facilitated by MXene-supported metal cluster catalysts. This work also highlights that the impact of temperature on the catalytic activity and selectivity of catalysts comprising metal clusters should be subjected to more rigorous investigation.

gamma-Butyrobetaine hydroxylase (BBOX) is a non-heme FeII/2OG dependent enzyme that is able to perform two different kinds of catalytic reactions on 3-(2,2,2-trimethylhydrazinium) propionate (THP) to produce distinct catalytic products. Although the structure of BBOX complexed with THP has been resolved, the details of its catalytic mechanism are still elusive. In this study, by employing molecular dynamics (MD) simulations and density functional theory (DFT) calculations, the mechanism of the THP oxidative rearrangement reactions catalysed by BBOX was investigated. Our calculations revealed how the enzyme undergoes a conformational conversion to initiate the catalytic reactions. In the first catalytic step, BBOX performs hydrogen abstraction from the substrate THP as a common non-heme iron enzyme. Due to the structure of the substrate stabilizing the radical species and polarizing the adjacent N-N bond, in the next step, THP takes the pathway for N-N bond homolysis but not regular hydroxyl rebounding. The cleaved ammonium radical could either react with the hydroxyl group on the iron centre of the enzyme or recombine with the other cleaved fragment of the substrate to generate the rearranged product. This study revealed the catalytic mechanism of BBOX, detailing how the enzyme and the substrate regulated the hydroxyl rebound process to generate various products. gamma-Butyrobetaine hydroxylase (BBOX) is able to perform two different kinds of catalytic reactions on gamma-butyrobetaine (gamma-BB) and 3-(2,2,2-trimethylhydrazinium) propionate (THP) to produce distinct catalytic products.

Herein, we proposed a novel metal-organic gel (YTU-G-1) for efficient adsorption and portable sensing of gaseous iodine. YTU-G-1 exhibits an unprecedentedly high detection sensitivity (KSV = 2.21 x 106 L mol-1) and an extremely low limit of detection (LOD) down to the pmol level (481 pmol L-1). YTU-G-1 also shows a marked iodine adsorption capacity of 1.398 g g-1. A wearable membrane was successfully fabricated via the electrospinning technique, which exhibits excellent skin-compatibility and serves as a portable tool for sensitive response to potential on-site nuclear emergencies. An AIEgen-functionalized luminescent metal-organic gel (YTU-G-1) with an extremely high luminescence quantum yield (95.5%) was prepared for gaseous iodine capture (1.398 g g-1) and detection (LOD: 481 pmol L-1).

The spliceosome, a large complex containing five conserved small ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6, plays important roles in precursor messenger RNA splicing. However, the function and mechanism of the spliceosomal snRNPs have not been thoroughly studied in the pathogenic yeast Cryptococcus deneoformans. In this study, we identified a U2A' homologous protein as a component of the cryptococcal U2 snRNP, which was encoded by the LEA1 gene. Using the "suicide" CRISPR-Cas9 tool, we deleted the LEA1 gene in C. deneoformans JEC21 strain and obtained the disruption mutant lea1 Delta. The mutant showed a hypersensitivity to 0.03 % sodium dodecyl sulfate, as well as disordered chitin distribution in cell wall observed with Calcofluor White staining, which collectively illustrated the function of U2A' in maintenance of cell wall integrity. Further examination showed that lea1 Delta displayed a decreased tolerance to lower or elevated temperatures, osmotic pressure and oxidative stress. The lea1 Delta still exhibited susceptibility to geneticin and 5-flucytosine, and increased resistance to ketoconazole. Even, the mutant had a reduced capsule, and the virulence of lea1 Delta in the Galleria mellonella model was decreased. Our results indicate that the U2A'-mediated RNA-processing has a particular role in the processing of gene products involved in response to stresses and virulence. (c) 2023 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights reserved.

The catalytic mechanisms for the wild-type and the mutated Cu-only superoxide dismutase were studied using the hybrid density functional B3LYP and a quantum chemical cluster approach. Optimal protonation states of the active site were examined for each stage of the catalytic cycle. For both the reductive and the oxidative half-reactions, the arrival of the substrate O-2(center dot-) was found to be accompanied by a chargecompensating H+ with exergonicities of -15.4 kcal center dot mol and -4.7 kcal center dot mol, respectively. The second-sphere Glu-110 and first-sphere His-93 were suggested to be the transient protonation site for the reductive and the oxidative half-reactions, respectively, which collaborates with the hydrogen bonding water chain to position the substrate near the redox-active copper center. For the reductive half-reaction, the rate-limiting step was found to be the inner-sphere electron transfer from the partially coordinated O-2(center dot-) to Cu-II with a barrier of 8.1 kcal center dot mol. The formed O-2 is released from the active site with an exergonicity of -14.9 kcal center dot mol. For the oxidative half-reaction, the inner-sphere electron transfer from CuI to the partially coordinated O-2(center dot-) was found to be accompanied by the proton transfer from the protonated His-93 and barrierless. The rate-limiting step was found to be the second proton transfer from the protonated Glu-110 to HO2 with a barrier of 7.3 kcal center dot mol. The barriers are reasonably consistent with experimental activities, and a proton-transfer rate-limiting step in the oxidative half-reaction could explain the experimentally observed pH-dependence. For the E110Q CuSOD, Asp-113 was suggested to be likely to serve as the transient protonation site in the reductive half-reaction. The rate-limiting barriers were found to be 8.0 and 8.6 kcal center dot mol, respectively, which could explain the slightly lower performance of E110X mutants. The results were found to be stable, with respect to the percentage of exact exchange in B3LYP.

The leading mechanism for the formation of O2 in photosystem II (PSII) has, during the past decade, been established as the so-called oxyl-oxo mechanism. In that mechanism, O2 is formed from a binding between an oxygen radical (oxyl) and a bridging oxo group. For the case of higher plants, that mechanism has recently been criticized. Instead, a nucleophilic attack of an oxo group on a five-coordinated Mn(V)═O group forming O2 has been suggested in a so-called water-unbound (WU) mechanism. In the present study, the WU mechanism has been investigated. It is found that the WU mechanism is just a variant of a previously suggested mechanism but with a reactant and a transition state that have much higher energies. The addition of a water molecule on the empty site of the Mn(V)═O center is very exergonic and leads back to the previously suggested oxyl-oxo mechanism.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

The yeast SKI (superkiller) complex was originally identified from cells that were infected by the M 'killer' virus. Ski2, as the core of the SKI complex, is a cytoplasmic cofactor and regulator of RNA-degrading exosome. The putative RNA helicase Ski2 was highly conserved from yeast to animals and has been demonstrated to play a key role in the regulation of RNA surveillance, temperature sensitivity, and growth in several yeasts but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Ski2 protein, named SKI2, in the fungal pathogen C. neoformans. To obtain insights into the function of Ski2, we created a mutant strain, ski2Δ, with the CRISPR-Cas9 editing tool. Disruption of SKI2 impaired cell wall integrity. Further investigations revealed the defects of the ski2Δ mutant in resistance to osmotic stresses and extreme growth temperatures. However, significantly, the ability to undergo invasive growth under nutrient-depleted conditions was increased in the ski2Δ mutant. More importantly, our results showed that the ski2Δ mutant exhibited slightly lower virulence and severe susceptibility to anti-ribosomal drugs by comparison to the wild type, but it developed multidrug resistance to azoles and flucytosine. By constructing the double deletion strain ski2Δafr1Δ, we verified that increased Afr1 in ski2Δ contributed to the azole resistance, which might be influenced by nonclassical small interfering RNA. Our work suggests that Ski2 plays critical roles in drug resistance and regulation of gene transcription in the yeast pathogen C. neoformans.

Abstract The biological function of RNAi machinery in fungi appears puzzling. In yeast Cryptococcus neoformans, we observed that RNAi-deficient mutants displayed diminished ribosome function. We conducted deep sequencing and bioinformatics analysis for siRNAs derived from rRNAs (sirRNAs), and discovered a novel class of sirRNAs with unique structure, e.g. a nucleotide C at 5’end, and a size of 19 or 20 nt. Two sirRNAs, 001 and 006, had the largest reads of all sRNAs and were located on 25S rRNA close to each other with a 4-nt spacer. Sequencing data and Northern blotting manifested sirRNAs were generated by RNAi machinery, rather by random degradation. Multiple RNAi pathways could produce sirRNAs, a long pathway consisting of Dcrs, Agos and Rdp and a short pathway through Dcrs and Agos solely. We found a 120-nt 25S rRNA fragment was the common precursor of sirRNAs 001/006 which were split into two pri-sirRNAs, 55-nt and 60-nt, respectively. The pri-sirRNAs were further processed merely by RNAi machinery to form pre-sirRNAs, subsequently the final products sirRNAs 001 and 006. Silencing assays suggested that sirRNAs-guided RNAi suppressed the expression of the reporters, URA5 and CLC1. On the other hand, we found loss of RNAi caused a significant decrease of 25S rRNA, but a dramatically increase of the natural antisense transcripts of rRNAs (NAT-rRNAs), suggesting RNAi machinery played a positive role in maintaining 25S rRNA level, while an antagonistic suppression on NAT-rRNAs which was demonstrated by sequencing and blotting. Considering the presence of sirRNAs, we speculate the yeast has evolved a protective mechanism for rRNA homeostasis through sirRNAs-guided RNAi against NAT-rRNAs. When RNAi machinery was absent, NAT-rRNAs were accumulated to form with rRNAs double-stranded RNA molecules and caused rRNAs to be degraded, which impaired ribosomal function. This work reveals that RNAi machinery maintains rRNA homeostasis and ribosome function.

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H2O2 as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol−1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release–rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a “gate” construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157–Trp198 “gate” from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.

The yeast noncanonical polyadenylation polymerase Cid14 was originally identified from fission yeast and plays a critical role in the TRAMP complex. This protein is a cytoplasmic cofactor and regulator of RNA-degrading exosomes. Cid14 is highly conserved from yeast to animals and has been demonstrated to play key roles in the regulation of RNA surveillance, nutrition metabolism, and growth in model organisms, but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Cid14 protein, named CID14, in the fungal pathogen C. neoformans. To obtain insights into the function of Cid14, we created a mutant strain, cid14Δ, with the CRISPR-Cas9 editing tool. Disruption of CID14 impaired cell membrane stability. Further investigations revealed the defects of the cid14Δ mutant in resistance to low carbohydrate levels. Meanwhile, significantly, the ability to grow under flucytosine stress was decreased in the cid14Δ mutant. More importantly, our results showed that the cid14Δ mutant does not affect yeast virulence but exhibits multidrug resistance to azole. Our work is the first to suggest that Cid14 plays critical roles in azole resistance by affecting Afr1, which is chiefly responsible for azole excretion in the ABC (ATP-binding cassette) transporter.