Nickel tolerance is channeled through C-4 methyl sterol oxidase Erg25 in the sterol biosynthesis pathway.

Nickel (Ni) is an abundant element on Earth and it can be toxic to all forms of life. Unlike our knowledge of other metals, little is known about the biochemical response to Ni overload. Previous studies in mammals have shown that Ni induces various physiological changes including redox stress, hypoxic responses, as well as cancer progression pathways. However, the primary cellular targets of nickel toxicity are unknown. Here, we used the environmental fungus Cryptococcus neoformans as a model organism to elucidate the cellular response to exogenous Ni. We discovered that Ni causes alterations in ergosterol (the fungal equivalent of mammalian cholesterol) and lipid biosynthesis, and that the Sterol Regulatory Element-Binding transcription factor Sre1 is required for Ni tolerance. Interestingly, overexpression of the C-4 methyl sterol oxidase gene ERG25, but not other genes in the ergosterol biosynthesis pathway tested, increases Ni tolerance in both the wild type and the sre1Δ mutant. Overexpression of ERG25 with mutations in the predicted binding pocket to a metal cation cofactor sensitizes Cryptococcus to nickel and abolishes its ability to rescue the Ni-induced growth defect of sre1Δ. As overexpression of a known nickel-binding protein Ure7 or Erg3 with a metal binding pocket similar to Erg25 does not impact on nickel tolerance, Erg25 does not appear to simply act as a nickel sink. Furthermore, nickel induces more profound and specific transcriptome changes in ergosterol biosynthetic genes compared to hypoxia. We conclude that Ni targets the sterol biosynthesis pathway primarily through Erg25 in fungi. Similar to the observation in C. neoformans, Ni exposure reduces sterols in human A549 lung epithelial cells, indicating that nickel toxicity on sterol biosynthesis is conserved.

A high-fat diet promotes cancer progression by inducing gut microbiota-mediated leucine production and PMN-MDSC differentiation.

A high-fat diet (HFD) is a high-risk factor for the malignant progression of cancers through the disruption of the intestinal microbiota. However, the role of the HFD-related gut microbiota in cancer development remains unclear. This study found that obesity and obesity-related gut microbiota were associated with poor prognosis and advanced clinicopathological status in female patients with breast cancer. To investigate the impact of HFD-associated gut microbiota on cancer progression, we established various models, including HFD feeding, fecal microbiota transplantation, antibiotic feeding, and bacterial gavage, in tumor-bearing mice. HFD-related microbiota promotes cancer progression by generating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Mechanistically, the HFD microbiota released abundant leucine, which activated the mTORC1 signaling pathway in myeloid progenitors for PMN-MDSC differentiation. Clinically, the elevated leucine level in the peripheral blood induced by the HFD microbiota was correlated with abundant tumoral PMN-MDSC infiltration and poor clinical outcomes in female patients with breast cancer. These findings revealed that the "gut-bone marrow-tumor" axis is involved in HFD-mediated cancer progression and opens a broad avenue for anticancer therapeutic strategies by targeting the aberrant metabolism of the gut microbiota.

Host populations, challenges, and commercialization of cryptococcal vaccines.

Vaccines are one of the most effective public health tools to prevent and manage infectious diseases. Since the first clinical use of vaccines in the late 18th century, many vaccines have been successfully developed to combat bacterial and viral infections, including the most recent Coronavirus Disease 2019 (COVID-19) pandemic. However, there remains no vaccine that is clinically available to treat or prevent invasive fungal diseases, including cryptococcal meningoencephalitis. This fungal disease is uniformly fatal without treatment and has a global mortality rate of over 70%. Despite a dire need for an effective cryptococcal vaccine, there are many scientific and economic challenges to overcome prior to making it a reality. Here, we discuss some of these challenges as well as steps that the community is taking for commercialization of effective cryptococcal vaccines.

Dectin-3-targeted antifungal liposomes efficiently bind and kill diverse fungal pathogens.

DectiSomes are anti-infective drug-loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C-type lectin (CTL) pathogen receptors Dectin-1, Dectin-2, and DC-SIGN to target DectiSomes to the extracellular oligoglycans surrounding diverse pathogenic fungi and kill them. Dectin-3 (also known as MCL, CLEC4D) is a CTL pathogen receptor whose known cognate ligands are partly distinct from other CTLs. We expressed and purified a truncated Dectin-3 polypeptide (DEC3) comprised of its carbohydrate recognition domain and stalk region. We prepared amphotericin B (AmB)-loaded pegylated liposomes (AmB-LLs) and coated them with this isoform of Dectin-3 (DEC3-AmB-LLs), and we prepared control liposomes coated with bovine serum albumin (BSA-AmB-LLs). DEC3-AmB-LLs bound to the exopolysaccharide matrices of Candida albicans, Rhizopus delemar (formerly known as R. oryzae), and Cryptococcus neoformans from one to several orders of magnitude more strongly than untargeted AmB-LLs or BSA-AmB-LLs. The data from our quantitative fluorescent binding assays were standardized using a CellProfiler program, AreaPipe, that was developed for this purpose. Consistent with enhanced binding, DEC3-AmB-LLs inhibited and/or killed C. albicans and R. delemar more efficiently than control liposomes and significantly reduced the effective dose of AmB. In conclusion, Dectin-3 targeting has the potential to advance our goal of building pan-antifungal DectiSomes.

Life Cycle of Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous environmental fungus and an opportunistic pathogen that causes fatal cryptococcal meningitis. Advances in genomics, genetics, and cellular and molecular biology of C. neoformans have dramatically improved our understanding of this important pathogen, rendering it a model organism to study eukaryotic biology and microbial pathogenesis. In light of recent progress, we describe in this review the life cycle of C. neoformans with a special emphasis on the regulation of the yeast-to-hypha transition and different modes of sexual reproduction, in addition to the impacts of the life cycle on cryptococcal populations and pathogenesis.

Metabolic subtypes and immune landscapes in esophageal squamous cell carcinoma: prognostic implications and potential for personalized therapies.

BACKGROUND: This study aimed to identify metabolic subtypes in ESCA, explore their relationship with immune landscapes, and establish a metabolic index for accurate prognosis assessment. METHODS: Clinical, SNP, and RNA-seq data were collected from 80 ESCA patients from the TCGA database and RNA-seq data from the GSE19417 dataset. Metabolic genes associated with overall survival (OS) and progression-free survival (PFS) were selected, and k-means clustering was performed. Immune-related pathways, immune infiltration, and response to immunotherapy were predicted using bioinformatic algorithms. Weighted gene co-expression network analysis (WGCNA) was conducted to identify metabolic genes associated with co-expression modules. Lastly, cell culture and functional analysis were performed using patient tissue samples and ESCA cell lines to verify the identified genes and their roles. RESULTS: Molecular subtypes were identified based on the expression profiles of metabolic genes, and univariate survival analysis revealed 163 metabolic genes associated with ESCA prognosis. Consensus clustering analysis classified ESCA samples into three distinct subtypes, with MC1 showing the poorest prognosis and MC3 having the best prognosis. The subtypes also exhibited significant differences in immune cell infiltration, with MC3 showing the highest scores. Additionally, the MC3 subtype demonstrated the poorest response to immunotherapy, while the MC1 subtype was the most sensitive. WGCNA analysis identified gene modules associated with the metabolic index, with SLC5A1, NT5DC4, and MTHFD2 emerging as prognostic markers. Gene and protein expression analysis validated the upregulation of MTHFD2 in ESCA. MTHFD2 promotes the progression of ESCA and may be a potential therapeutic target for ESCA. CONCLUSION: The established metabolic index and identified metabolic genes offer potential for prognostic assessment and personalized therapeutic interventions for ESCA, underscoring the importance of targeting metabolism-immune interactions in ESCA. MTHFD2 promotes the progression of ESCA and may be a potential therapeutic target for ESCA.

Combined modified Valsalva maneuver with adenosine supraventricular tachycardia: A comparative study.

BACKGROUND AND IMPORTANCE: Paroxysmal supraventricular tachycardia (PSVT) is an arrhythmia commonly seen in the emergency department. Both modified Valsalva maneuver (MVM) and intravenous adenosine are the first line treatment, of which the former has e lower success rate while the latter has a higher success rate but some risks and adverse effects. Given both of these reverse rhythms quickly, combining them may achieve a better effect. OBJECTIVE: The objective of this study is to evaluate the success rate and potential risk of combining the use of intravenous adenosine while patients were doing MVM as a treatment for paroxysmal supraventricular tachycardia(pSVT). DESIGN, SETTINGS AND PARTICIPANTS: We recruited patients with pSVT from 2017 to 2022, and randomly assigned them into 3 groups, MVM group, intravenous adenosine group, and combination therapy group, in which MVM was allowed to be performed twice, while intravenous adenosine was given in a titration manner to repeat three times, recorded the success rate and side effects in each group. MAIN RESULTS: The success rate of the MVM group, adenosine group, and combination group are 42.11%, 75.00 and 86.11%, respectively. The success rate of the adenosine group and combination group is significantly higher than the n MVSM group (p < 0.01, p < 0.001), while the success rate of the combination group is higher than the adenosine group, it has no significant difference (p = 0.340). In terms of safety, the longest RR durations (asystole period) are 1.61 s, 1.60s, and 2.27 s, there is a statistical difference among the three groups (p < 0.01) and between the adenosine and combination group (0.018). CONCLUSION: Therefore, we can conclude that combination therapy has a relatively high success rate and good safety profile, but the current study failed to show its superiority to adenosine.

Vaccination with a ZNF2oe Strain of Cryptococcus Provides Long-Lasting Protection against Cryptococcosis and Is Effective in Immunocompromised Hosts.

Systemic cryptococcosis is fatal without treatment. Even with the current antifungal therapies, this disease kills 180,000 of 225,000 infected people annually. Exposure to the causative environmental fungus Cryptococcus neoformans is universal. Either reactivation of a latent infection or an acute infection after high exposure to cryptococcal cells can result in cryptococcosis. Currently, there is no vaccine to prevent cryptococcosis. Previously, we discovered that Znf2, a transcription factor that directs Cryptococcus yeast-to-hypha transition, profoundly affects cryptococcal interaction with the host. Overexpression of ZNF2 drives filamentous growth, attenuates cryptococcal virulence, and elicits protective host immune responses. Importantly, immunization with cryptococcal cells overexpressing ZNF2, in either live or heat-inactivated form, offers significant protection to the host from a subsequent challenge by the otherwise lethal clinical isolate H99. In this study, we found that the heat-inactivated ZNF2oe vaccine offered long-lasting protection with no relapse upon challenge with the wild-type H99. Vaccination with heat-inactivated ZNF2oe cells provides partial protection in hosts with preexisting asymptomatic cryptococcal infection. Importantly, once animals have been vaccinated with heat-inactivated or live short-lived ZNF2oe cells, they are protected against cryptococcosis even when their CD4+ T cells are depleted at the time of fungal challenge. Remarkably, vaccination with live, short-lived ZNF2oe cells in CD4-depleted hosts still provides strong protection to these hosts with preexisting immunodeficiency at the time of vaccination. This work raises hope for developing effective vaccines with long-lasting protection for individuals who are immunocompromised or could become immunocompromised later in life.

Revealing Atomic Configuration and Synergistic Interaction of Single-Atom-Based Zn-Co-Fe Trimetallic Sites for Enhancing Oxygen Reduction and Evolution Reactions.

Anchoring single metal atom to carbon supports represents an exceptionally effective strategy to maximize the efficiency of catalysts. Recently, dual-atom catalysts (DACs) emerge as an intriguing candidate for atomic catalysts, which perform better than single-atom catalysts (SACs). However, the clarification of the polynary single-atom structures and their beneficial effects remains a daunting challenge. Here, atomically dispersed triple Zn-Co-Fe sites anchored to nitrogen-doped carbon (ZnCoFe-N-C) prepared by one-step pyrolysis of a designed metal-organic framework precursor are reported. The atomically isolated trimetallic configuration in ZnCoFe-N-C is identified by annular dark-field scanning transmission electron microscopy and spectroscopic techniques. Benefiting from the synergistic effect of trimetallic single atoms, nitrogen, and carbon, ZnCoFe-N-C exhibits excellent catalytic performance in bifunctional oxygen reduction/evolution reactions in an alkaline medium, outperforming other SACs and DACs. The ZnCoFe-N-C-based Zn-air battery exhibits a high specific capacity (liquid state: 931.8 Wh kgZn -1 ), power density (liquid state: 137.8 mW cm-2 ; all-solid-state: 107.9 mW cm-2 ), and good cycling stability. Furthermore, density-functional theory calculations rationalize the excellent performance by demonstrating that the ZnCoFe-N-C catalyst has upshifted d-band center that enhances the adsorption of the reaction intermediates.

Aiming for a bull's-eye: Targeting antifungals to fungi with dectin-decorated liposomes.

Globally, there are several million individuals with life-threatening invasive fungal diseases such as candidiasis, aspergillosis, cryptococcosis, Pneumocystis pneumonia (PCP), and mucormycosis. The mortality rate for these diseases generally exceeds 40%. Annual medical costs to treat these invasive fungal diseases in the United States exceed several billion dollars. In addition to AIDS patients, the risks of invasive mycoses are increasingly found in immune-impaired individuals or in immunosuppressed patients following stem cell or organ transplant or implantation of medical devices. Current antifungal drug therapies are not meeting the challenge, because (1) at safe doses, they do not provide sufficient fungal clearance to prevent reemergence of infection; (2) most become toxic with extended use; (3) drug-resistant fungal isolates are emerging; and (4) only one new class of antifungal drugs has been approved for clinical use in the last 2 decades. DectiSomes represent a novel design of drug delivery to drastically increase drug efficacy. Antifungals packaged in liposomes are targeted specifically to where the pathogen is, through binding to the fungal cell walls or exopolysaccharide matrices using the carbohydrate recognition domains of pathogen receptors. Relative to untargeted liposomal drug, DectiSomes show order of magnitude increases in the binding to and killing of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus in vitro and similarly improved efficacy in mouse models of pulmonary aspergillosis. DectiSomes have the potential to usher in a new antifungal drug treatment paradigm.

Degradation of imidacloprid and acetamiprid in tea (Camellia sinensis) infusion by ultraviolet light irradiation.

The degradation of imidacloprid and acetamiprid in tea infusion by ultraviolet (UV) light irradiation was investigated in this study. Results showed that the influence of UV light irradiation on the quality of tea infusion was controllable and UV light irradiation was effective on the degradation of both pesticides. The maximum removal rates were 75.2% for imidacloprid and 17.6% for acetamiprid after irradiation (650 µW/cm,2 120 min). The degradation of both pesticides followed the first-order kinetics model. Three degradation products were identified for imidacloprid and one for acetamiprid based on liquid chromatography-tandem mass spectrometry analysis. The degradation pathway of imidacloprid involved in the cleavage of C-C bond with the loss of nitro group followed by the hydrogenation, oxidation and hydrolysis, while the degradation of acetamiprid involved in the oxidation at the chlorine atom with the bonding of C atoms at positions 1 and 4 on the pyridine ring. Simultaneously, the toxicity of both pesticides was mitigated by UV light irradiation according to LO2 cell toxicity evaluation. The study provided a low-cost and effective way to reduce imidacloprid and acetamiprid from tea infusion, and it has the potential to be applied to the ready-to drink tea beverage production in industrial scale.

Non-coding RNAs rewire cancer metabolism networks.

Non-coding RNAs (ncRNAs) are functional RNAs with limited or no protein-coding ability. These interact with their target molecules and participate in the precise regulation of disease development. Metabolic reprogramming is a hallmark in cancer, and is considered essential in meeting increased macromolecular biosynthesis and energy generation of tumors. Recent studies have revealed the involvement of ncRNAs in several metabolic regulations of cancer through direct modulation of metabolic enzyme activities or participation of metabolism-related signaling pathways. Elucidation of how ncRNAs regulate metabolic reprogramming of cancers has opened up a novel intention to understand the mechanism of metabolic rewiring and also the opportunities of utilizing ncRNA-based therapeutics for targeting the metabolism in cancer treatment.

DectiSomes: Glycan Targeting of Liposomal Drugs Improves the Treatment of Disseminated Candidiasis.

Candida albicans causes life-threatening disseminated candidiasis. Individuals at greatest risk have weakened immune systems. An outer cell wall, exopolysaccharide matrix, and biofilm rich in oligoglucans and oligomannans help Candida spp. evade host defenses. Even after antifungal treatment, the 1-year mortality rate exceeds 25%. Undoubtedly, there is room to improve drug performance. The mammalian C-type lectin pathogen receptors Dectin-1 and Dectin-2 bind to fungal oligoglucans and oligomannans, respectively. We previously coated amphotericin B-loaded liposomes, AmB-LLs, pegylated analogs of AmBisome, with the ligand binding domains of these two Dectins. DectiSomes, DEC1-AmB-LLs and DEC2-AmB-LLs, showed two distinct patterns of binding to the exopolysaccharide matrix surrounding C. albicans hyphae grown in vitro. Here we showed that DectiSomes were preferentially associated with fungal colonies in the kidneys. In a neutropenic mouse model of candidiasis, DEC1-AmB-LLs and DEC2-AmB-LLs delivering only one dose of 0.2 mg/kg AmB reduced the kidney fungal burden several fold relative to AmB-LLs. DEC1-AmB-LLs and DEC2-AmB-LLs increased the percent of surviving mice 2.5-fold and 8.3-fold, respectively, relative to AmB-LLs. Dectin-2 targeting of anidulafungin loaded liposomes, DEC2-AFG-LLs, and of commercial AmBisome, DEC2-AmBisome, reduced fungal burden in the kidneys several fold over their untargeted counterparts. The data herein suggest that targeting of a variety of antifungal drugs to fungal glycans may achieve lower safer effective doses and improve drug efficacy against a variety of invasive fungal infections.

DiLeuPMP: A Multiplexed Isobaric Labeling Method for Quantitative Analysis of O-Glycans.

As one of the most important post-translational modifications, glycosylation plays a pivotal role in many essential physiological functions, including cell recognition, signaling, and immune response. Thus, various qualitative and quantitative analytical strategies for glycomic profiling have been developed in recent decades. However, while extensive efforts have been devoted to the analysis of N-glycans, high-throughput quantitative analysis of O-glycans is often overlooked and underexplored. This is partially due to the lack of a universal enzyme for the release of O-glycans from the protein backbone. Furthermore, the traditional chemical releasing method suffers from severe side reactions and involves tedious sample preparation procedures. Here, a multiplexed isobaric labeling method enabled by N,N-dimethyl leucine containing pyrazolone analogue (DiLeuPMP) is introduced. This method combines the release and labeling of O-glycans in a one-pot reaction and achieves accurate MS2-based relative quantification with the ability to process four samples at a time. The method has been applied to core-1 O-glycan standard and three glycoproteins first, and the results demonstrated its validity. Following this proof-of-principle demonstration, we analyzed more complex biological specimen using human serum samples. Overall, this method provides an effective and reliable approach for the profiling and high-throughput quantitative analysis of O-glycans in complex samples.

MOF Structure Engineering to Synthesize CoNC Catalyst with Richer Accessible Active Sites for Enhanced Oxygen Reduction.

Single-atom cobalt-based CoNC are promising low-cost electrocatalysts for oxygen reduction reaction (ORR). However, further increasing the single cobalt-based active sites and the ORR activity remain a major challenge. Herein, an acetate (OAc) assisted metal-organic framework (MOF) structure-engineering strategy is developed to synthesize hierarchical accordion-like MOF with higher loading amount and better spatial isolation of Co and much higher yield when compared with widely reported polyhedron MOF. After pyrolysis, the accordion-structured CoNC (CoNC (A)) is loaded with denser CoN4 active sites (Co: 2.88 wt%), approximately twice that of Co in the CoNC reported. The presence of OAc in MOF also induces the generation of big pores (5-50 nm) for improving the accessibility of active sites and mass transfer during catalytic reactions. Consequently, the CoNC (A) catalyst shows an admirable ORR activity with a E1/2 of 0.89 V (40 mV better than Pt/C) in alkaline electrolytes, outstanding durability, and absolute tolerance to methanol in both alkaline and acidic media. The CoNC-based Zn-air battery exhibits a high specific capacity (976 mAh g-1 Zn ), power density (158 mW cm-2 ), rate capability, and long-term stability. This work demonstrates a reliable approach to construct single atom doped carbon catalysts with denser accessible active sites through MOF structure engineering.

Microbe Profile: Cryptococcus neoformans species complex.

Cryptococcus neoformans is a lethal fungus disguised in a polysaccharide coat. It can remain dormant in the host for decades prior to reactivation, causing systemic cryptococcosis in humans and other mammals. Cryptococcus deploys a multitude of traits to adapt to and survive within the host, including immunosuppression, an ability to replicate intra- and extra-cellularly in phagocytes, changes in morphology and ploidy, a predilection to infect the CNS, and the capacity to utilize neurotransmitters and unique carbon sources available in the brain. These pathogenic strategies displayed by this fungus might have evolved through its interactions with microbial predators in the environment.

An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.

Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free "safe haven" (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.

Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-Cas9 expression (TRACE) system.

The basidiomycete Cryptococcus neoformans is not only a clinically important pathogen, but also a model organism for studying microbial pathogenesis and eukaryotic biology. One key factor behind its rise as a model organism is its genetic amenability. The widely used methods for transforming the C. neoformans species complex are Agrobacterium-mediated transformation (AMT) for random insertional mutagenesis and biolistic transformation for targeted mutagenesis. Electroporation was introduced to C. neoformans in early 1990s. Although electroporation is economic and yields a large number of transformants, introduced DNA rarely integrates into cryptococcal genome, which limits its use. Biolistic transformation, although costly and inefficient, has been the only method used in targeted mutagenesis in the past two decades. Several modifications, including the use of a donor DNA with split markers, a drug-resistant selection marker, and a recipient strain deficient in non-homologous end joining (NHEJ), have since modestly increased the frequency of genome integration and the rate of homologous replacement of the DNA introduced by electroporation. However, electroporation was not the method of choice for transformation until the recent adoption of CRISPR-Cas9 systems. We have developed a Transient CRISPR-Cas9 coupled with Electroporation System (TRACE), which dramatically facilitates targeted mutagenesis in the Cryptococcus species complex. TRACE combines the high transformation efficiency of electroporation with the high rates of DNA integration due to the transiently expressed CRISPR-Cas9. Here, we briefly discussed the history of electroporation for Cryptococcus transformation and provided detailed procedures for electroporation and the cassettes construction of the TRACE system for various genetic manipulations.

An intergenic "safe haven" region in Aspergillus fumigatus.

Aspergillus fumigatus is the most common opportunistic human fungal pathogen responsible for invasive aspergillosis. Gene manipulation is critical for the investigation of A. fumigatus biology and pathogenesis at the molecular level, and it often requires integration of the introduced DNA into the fungal genome. Here we have searched and identified two potential "safe haven" regions, SH1 and SH2, based on A. fumigatus genome annotation and transcriptome data. When a DNA fragment carrying a fluorescent protein gene mNeonGreen (mNG) and a drug selection marker was inserted into SH1 or SH2, the expression of mNeonGreen was easily detected, indicating that SH1 and SH2 are not surpressive genetic regions. We found that insertion of this DNA fragment into SH1 did not cause any significant changes in the expression of neighboring genes. Insertion of this DNA into either SH1 or SH2 did not significantly alter any of the phenotypes that we analyzed comparing to the wild type control. By comparison, transformants with random ectopic integration of the same DNA fragment showed a wider range of variation in mNeonGreen expression and in virulence in an insect infection model. Having identified predetermined "safe-haven" regions in A. fumigatus could therefore help reduce experimental variations and increase reproducibility, as it has been for the C. neoformans field.

Pheromone independent unisexual development in Cryptococcus neoformans.

The fungus Cryptococcus neoformans can undergo a-α bisexual and unisexual reproduction. Completion of both sexual reproduction modes requires similar cellular differentiation processes and meiosis. Although bisexual reproduction generates equal number of a and α progeny and is far more efficient than unisexual reproduction under mating-inducing laboratory conditions, the α mating type dominates in nature. Population genetic studies suggest that unisexual reproduction by α isolates might have contributed to this sharply skewed distribution of the mating types. However, the predominance of the α mating type and the seemingly inefficient unisexual reproduction observed under laboratory conditions present a conundrum. Here, we discovered a previously unrecognized condition that promotes unisexual reproduction while suppressing bisexual reproduction. Pheromone is the principal stimulus for bisexual development in Cryptococcus. Interestingly, pheromone and other components of the pheromone pathway, including the key transcription factor Mat2, are not necessary but rather inhibitory for Cryptococcus to complete its unisexual cycle under this condition. The inactivation of the pheromone pathway promotes unisexual reproduction despite the essential role of this pathway in non-self-recognition during bisexual reproduction. Nonetheless, the requirement for the known filamentation regulator Znf2 and the expression of hyphal or basidium specific proteins remain the same for pheromone-dependent or independent sexual reproduction. Transcriptome analyses and an insertional mutagenesis screen in mat2Δ identified calcineurin being essential for this process. We further found that Znf2 and calcineurin work cooperatively in controlling unisexual development in this fungus. These findings indicate that Mat2 acts as a repressor of pheromone-independent unisexual development while serving as an activator for a-α bisexual development. The bi-functionality of Mat2 might have allowed it to act as a toggle switch for the mode of sexual development in this ubiquitous eukaryotic microbe.