Higher temperature variability in deforested mountain regions impacts the competitive advantage of nocturnal species.

Deforestation is a major contributor to biodiversity loss, yet the impact of forest loss on daily microclimate variability and its implications for species with different daily activity patterns remain poorly understood. Using a recently developed microclimate model, we investigated the effects of deforestation on the daily temperature range (DTR) in low-elevation tropical regions and high-elevation temperate regions. Our results show that deforestation substantially increases DTR in these areas, suggesting a potential impact on species interactions. To test this hypothesis, we studied the competitive interactions between nocturnal burying beetles and all-day-active blowfly maggots in forested and deforested habitats in Taiwan. We show that deforestation leads to increased DTR at higher elevations, which enhances the competitiveness of blowfly maggots during the day and leads to a higher failure rate of carcass burial by the beetles at night. Thus, deforestation-induced temperature variability not only modulates exploitative competition between species with different daily activity patterns, but also likely exacerbates the negative impacts of climate change on nocturnal organisms. In order to limit potential adverse effects on species interactions and their ecological functions, our study highlights the need to protect forests, especially in areas where deforestation can greatly alter temperature variability.

Seeking policy solutions in a complex system: experimentalist governance in China's healthcare reform.

The proliferation of "wicked" policy problems in complex systems requires an experimental approach of problem-solving. Experimentalist governance offers a conducive framework through which to seek policy solutions amidst high levels of complexity in a multilevel governance structure. This study conceptualizes four distinctive experimental modalities based on varying levels of technical complexity and interest complexity, both of which represent salient constraints for policy reforms in a complex system, the health sector in particular. Trail-blazing pilots, crowdsourcing pilots, managed pilots, and road-testing pilots are all associated with distinct mechanisms of experimentation in a multilevel governance structure. Through four illustrative cases from China's massive experimental program of public hospital reform, this study demonstrates how experimentalist governance seeks policy solutions in the health sector. Should governance arrangements, policy capacity, pragmatism, and informational devices become aligned in a conducive way, experimentalist governance can play an instrumental role in seeking solutions for difficult problems in a complex policy system. A governance structure capable of policy learning and adaptive management is the key.

The Relationship Between Smoker Identity and Smoking Cessation Among Young Smokers: The Role of Smoking Rationalization Beliefs and Cultural Value of Guanxi.

Although the relationship between smoker identity and smoking cessation behavior has been confirmed, the role of smoking-related beliefs and cultural values in this relationship for young smokers is little known. The present study aimed to examine whether the relationship between smoker identity and smoking cessation behavior would be mediated by smoking rationalization beliefs and/or intention to quit smoking and whether the effect of smoker identity on smoking cessation behavior was moderated by cultural value of guanxi. A total of 708 young smokers participated in the study and completed questionnaires that measured smoker identity, smoking rationalization beliefs, intention to quit smoking, smoking cessation behavior and cultural value of guanxi. The results showed: (1) the relationship between smoker identity and smoking cessation behavior was negative and significant. (2) The mediating effect of intention to quit smoking and the serial mediating effect of "smoking rationalization beliefs → intention to quit smoking" on the relationship between smoker identity and smoking cessation behavior was significant. (3) Both the serial mediating effect of "smoking rationalization beliefs → intention to quit smoking" and the direct effect of smoker identity on smoking cessation behavior were moderated by cultural value of guanxi. The current findings increased understanding of psychosocial mechanisms underlying the hindering effect of smoker identity on smoking cessation and suggested the role of smoking rationalization beliefs and cultural value of guanxi should be considered in smoking cessation interventions for young smokers.

Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth.

Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.

Immunoprotection against Cryptococcosis Offered by Znf2 Depends on Capsule and the Hyphal Morphology.

Systemic cryptococcosis is fatal without treatment. Globally, this disease kills 180,000 of the 225,000 infected people each year, even with the use of antifungal therapies. Currently, there is no vaccine to prevent cryptococcosis. Previously, we discovered that Znf2, a morphogenesis regulator that directs Cryptococcus yeast-to-hyphal 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, either in live or heat-inactivated form, offers significant protection to the host from a subsequent challenge by the otherwise lethal wild-type H99 strain. We hypothesize that cellular components enriched in ZNF2oe cells are immunoprotective. Here, we discovered that serum from protected animals vaccinated with inactivated ZNF2oe cells recognizes cryptococcal antigens that reside within the capsule. Consistently, capsule is required for immunoprotection offered by ZNF2oe cells. Interestingly, the serum from protective animals recognizes antigens in both wild-type yeast cells and ZNF2oe cells, with higher abundance in the latter. Consequently, even the heat-inactivated wild-type cells become immunoprotective with an increased vaccination dose. We also found that disruption of a chromatin remodeling factor Brf1, which is important for initiation of filamentation by Znf2, reduces the antigen level in ZNF2oe cells. Deletion of BRF1 drastically reduces the protective effect of ZNF2oe cells in both live and heat-killed forms even though the ZNF2oebrf1Δ strain itself is avirulent. Collectively, our findings underscore the importance of identifying the subset of cryptococcal surface factors that are beneficial in host protection. IMPORTANCE Cryptococcosis claims close to 200,000 lives annually. There is no vaccine clinically available for this fungal disease. Many avirulent mutant strains do not provide protection against cryptococcosis. We previously discovered that hyphal ZNF2oe strains elicit protective host immune responses both in the live and heat-inactivated forms. Here we seek to understand the mechanism underlying the host protection provided by ZNF2oe cells. We discovered increased accumulation of antigens located within the caspusle of ZNF2oe cells and consequently the requirement of the capsule for ZNF2oe strain-elicited host protection. Furthermore, genetically blocking the ability of ZNF2oe cells to grow in the hyphal form significantly reduces antigen accumulation and impairs the ability of ZNF2oe strain to provide host protection. Our findings highlight the importance of identifying the Znf2-regulated capsular surface factors that are fundamental in host protection.

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.

Author Correction: Locally-adapted reproductive photoperiodism determines population vulnerability to climate change in burying beetles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Locally-adapted reproductive photoperiodism determines population vulnerability to climate change in burying beetles.

Understanding how phenotypic traits vary among populations inhabiting different environments is critical for predicting a species' vulnerability to climate change. Yet, little is known about the key functional traits that determine the distribution of populations and the main mechanisms-phenotypic plasticity vs. local adaptation-underlying intraspecific functional trait variation. Using the Asian burying beetle Nicrophorus nepalensis, we demonstrate that mountain ranges differing in elevation and latitude offer unique thermal environments in which two functional traits-thermal tolerance and reproductive photoperiodism-interact to shape breeding phenology. We show that populations on different mountain ranges maintain similar thermal tolerances, but differ in reproductive photoperiodism. Through common garden and reciprocal transplant experiments, we confirm that reproductive photoperiodism is locally adapted and not phenotypically plastic. Accordingly, year-round breeding populations on mountains of intermediate elevation are likely to be most susceptible to future warming because maladaptation occurs when beetles try to breed at warmer temperatures.

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.

In vitro modulation of mercury-induced rat liver mitochondria dysfunction.

Mercury (Hg) is a toxic environmental pollutant that exerts its cytotoxic effects as cations by targeting mitochondria. In our work, we determined different mitochondrial toxicity factors using specific substrates and inhibitors following the addition of Hg2+ to the mitochondria isolated from Wistar rat liver in vitro. We found that Hg2+ induced marked changes in the mitochondrial ultrastructure accompanied by mitochondrial swelling, mitochondrial membrane potential collapse, mitochondrial membrane fluidity increase and Cytochrome c release. Additionally, the effects of Hg2+ on heat production of mitochondria were investigated using microcalorimetry; simultaneously, the effects on mitochondrial respiration were determined by Clark oxygen-electric methods. Microcalorimetry could provide detailed kinetic and thermodynamic information which demonstrated that Hg2+ had some biotoxicity effect on mitochondria. The inhibition of energy metabolic activities suggested that high concentrations of Hg2+ could induce mitochondrial ATP depletion under MPT and mitochondrial respiration inhibition. These results help us learn more about the toxicity of Hg2+ at the subcellular level.

Transcription factor CBF-1 is critical for circadian gene expression by modulating WHITE COLLAR complex recruitment to the frq locus.

Transcription of the Neurospora crassa circadian clock gene frequency (frq) is an essential process in the negative feedback loop that controls circadian rhythms. WHITE COLLAR 1 (WC-1) and WHITE COLLAR 2 (WC-2) forms the WC complex (WCC) that is the main activator of frq transcription by binding to its promoter. Here, we show that Centromere Binding Factor 1 (CBF-1) is a critical component of the N. crassa circadian clock by regulating frq transcription. Deletion of cbf-1 resulted in long period and low amplitude rhythms, whereas overexpression of CBF-1 abolished the circadian rhythms. Loss of CBF-1 resulted in WC-independent FRQ expression and suppression of WCC activity. As WCC, CBF-1 also binds to the C-box at the frq promoter. Overexpression of CBF-1 impaired WCC binding to the C-box to suppress frq transcription. Together, our results suggest that the proper level of CBF-1 is critical for circadian clock function by suppressing WC-independent FRQ expression and by regulating WCC binding to the frq promoter.

Plant Homeodomain Genes Play Important Roles in Cryptococcal Yeast-Hypha Transition.

Cryptococcus neoformans is a major opportunistic fungal pathogen. Like many dimorphic fungal pathogens, C. neoformans can undergo morphological transition from the yeast form to the hypha form, and its morphotype is tightly linked to its virulence. Although some genetic factors controlling morphogenesis have been identified, little is known about the epigenetic regulation in this process. Proteins with the plant homeodomain (PHD) finger, a structurally conserved domain in eukaryotes, were first identified in plants and are known to be involved in reading and effecting chromatin modification. Here, we investigated the role of the PHD finger family genes in Cryptococcus mating and yeast-hypha transition. We found 16 PHD finger domains distributed among 15 genes in the Cryptococcus genome, with two genes, ZNF1α and RUM1α, located in the mating type locus. We deleted these 15 PHD genes and examined the impact of their disruption on cryptococcal morphogenesis. The deletion of five PHD finger genes dramatically affected filamentation. The rum1αΔ and znf1αΔ mutants showed enhanced ability to initiate filamentation but impaired ability to maintain filamentous growth. The bye1Δ and the phd11Δ mutants exhibited enhanced filamentation, while the set302Δ mutants displayed reduced filamentation. Ectopic overexpression of these five genes in the corresponding null mutants partially or completely restored the defect in filamentation. Furthermore, we demonstrated that Phd11, a suppressor of filamentation, regulates the yeast-hypha transition through the known master regulator Znf2. The findings indicate the importance of epigenetic regulation in controlling dimorphic transition in C. neoformansIMPORTANCE Morphotype is known to have a profound impact on cryptococcal interaction with various hosts, including mammalian hosts. The yeast form of Cryptococcus neoformans is considered the virulent form, while its hyphal form is attenuated in mammalian models of cryptococcosis. Although some genetic regulators critical for cryptococcal morphogenesis have been identified, little is known about epigenetic regulation in this process. Given that plant homeodomain (PHD) finger proteins are involved in reading and effecting chromatin modification and their functions are unexplored in C. neoformans, we investigated the roles of the 15 PHD finger genes in Cryptococcus mating and yeast-hypha transition. Five of them profoundly affect filamentation as either a suppressor or an activator. Phd11, a suppressor of filamentation, regulates this process via Znf2, a known master regulator of morphogenesis. Thus, epigenetic regulation, coupled with genetic regulation, controls this yeast-hypha transition event.

Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex.

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this 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.

Asymmetric synthesis of heteroaryl atropisomers via a gold-catalyzed cycloisomerization-amination cascade reaction.

The chiral gold(i) complex enables enantioselective cycloisomerization-amination of 2-(alkynyl)phenyl boronic acids and diazenes in high yields. A wide scope of substrates bearing various functional groups was tolerated to generate structurally different hydrazide derivatives as a new type of atropisomer.