From microbes to molecules: a review of microbial-driven antioxidant peptide generation.

Oxidative stress, arising from excess reactive oxygen species (ROS) or insufficient antioxidant defenses, can damage cellular components, such as lipids, proteins, and nucleic acids, resulting in cellular dysfunction. The relationship between oxidative stress and various health disorders has prompted investigations into potent antioxidants that counteract ROS's detrimental impacts. In this context, antioxidant peptides, composed of two to twenty amino acids, have emerged as a unique group of antioxidants and have found applications in food, nutraceuticals, and pharmaceuticals. Antioxidant peptides are sourced from natural ingredients, mainly proteins derived from foods like milk, eggs, meat, fish, and plants. These peptides can be freed from their precursor proteins through enzymatic hydrolysis, fermentation, or gastrointestinal digestion. Previously published studies focused on the origin and production methods of antioxidant peptides, describing their structure-activity relationship and the mechanisms of food-derived antioxidant peptides. Yet, the role of microorganisms hasn't been sufficiently explored, even though the production of antioxidant peptides frequently employs a variety of microorganisms, such as bacteria, fungi, and yeasts, which are recognized for producing specific proteases. This review aims to provide a comprehensive overview of microorganisms and their proteases participating in enzymatic hydrolysis and microbial fermentation to produce antioxidant peptides. This review also covers endogenous peptides originating from microorganisms. The information obtained from this review might guide the discovery of novel organisms adept at generating antioxidant peptides.

Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen, Cryptococcus neoformans.

Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.

Distinct and redundant roles of protein tyrosine phosphatases Ptp1 and Ptp2 in governing the differentiation and pathogenicity of Cryptococcus neoformans.

Protein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.

Sulphiredoxin plays peroxiredoxin-dependent and -independent roles via the HOG signalling pathway in Cryptococcus neoformans and contributes to fungal virulence.

Mechanisms of oxidative stress resistance are crucial virulence factors for survival and proliferation of fungal pathogens within the human host. In this study we have identified and functionally characterized the role of sulphiredoxin, Srx1, in oxidative stress resistance of Cryptococcus neoformans causing fungal meningoencephalitis and regulation of peroxiredoxins, Tsa1 and Tsa3, and thioredoxins, Trx1 and Trx2. The C. neoformans HOG (High Osmolarity Glycerol response) pathway was essential for the transcriptional regulation of SRX1 under peroxide stress conditions. A gene deletion study revealed that Srx1 was required for cells to counteract peroxide stress, but not other oxidative damaging agents. HOG1 was found to be essential for the induction of adaptive response to peroxide stress with concurrent repression of ergosterol biosynthesis in an SRX1-independent manner. Consistent with this, phosphorylation of C. neoformans Hog1 was modulated by both low and high doses of exogenous hydrogen peroxide treatment. Immunoblot analysis using the C. neoformans Tsa1 specific antibody revealed that both Srx1 and Trx1 were essential for recycling of oxidized Tsa1. In addition to its role in peroxide sensing and response C. neoformans Srx1 was also found to be required for a peroxiredoxin-independent function in promoting fungicide-dependent cell swelling and growth arrest. Finally we showed the importance of C. neoformans Srx1 in fungal pathogenesis by demonstrating its requirement for full virulence using a mouse infection model.

Stress signaling pathways for the pathogenicity of Cryptococcus.

Sensing, responding, and adapting to the surrounding environment are crucial for all living organisms to survive, proliferate, and differentiate in their biological niches. This ability is also essential for Cryptococcus neoformans and its sibling species Cryptococcus gattii, as these pathogens have saprobic and parasitic life cycles in natural and animal host environments. The ability of Cryptococcus to cause fatal meningoencephalitis is highly related to its capability to remodel and optimize its metabolic and physiological status according to external cues. These cues act through multiple stress signaling pathways through a panoply of signaling components, including receptors/sensors, small GTPases, secondary messengers, kinases, transcription factors, and other miscellaneous adaptors or regulators. In this minireview, we summarize and highlight the importance of several stress signaling pathways that influence the pathogenicity of Cryptococcus and discuss future challenges in these areas.

Sensing and responding to host-derived stress signals: lessons from fungal meningitis pathogen.

The sophisticated ability of living organisms to sense and respond to external stimuli is critical for survival. This is particularly true for fungal pathogens, where the capacity to adapt and proliferate within a host is essential. To this end, signaling pathways, whether evolutionarily conserved or unique, have been refined through interactions with the host. Cryptococcus neoformans, an opportunistic fungal pathogen, is responsible for over 190,000 cases and an estimated 147,000 annual deaths globally. Extensive research over the past decades has shed light on the signaling pathways underpinning the pathogenicity of C. neoformans, as well as the host's responses during infection. In this context, we delineate the regulatory mechanisms employed by C. neoformans to detect and react to stresses derived from the host.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Comparative genomics of Deinococcus radiodurans: unveiling genetic discrepancies between ATCC 13939K and BAA-816 strains.

The Deinococcus genus is renowned for its remarkable resilience against environmental stresses, including ionizing radiation, desiccation, and oxidative damage. This resilience is attributed to its sophisticated DNA repair mechanisms and robust defense systems, enabling it to recover from extensive damage and thrive under extreme conditions. Central to Deinococcus research, the D. radiodurans strains ATCC BAA-816 and ATCC 13939 facilitate extensive studies into this remarkably resilient genus. This study focused on delineating genetic discrepancies between these strains by sequencing our laboratory's ATCC 13939 specimen (ATCC 13939K) and juxtaposing it with ATCC BAA-816. We uncovered 436 DNA sequence differences within ATCC 13939K, including 100 single nucleotide variations, 278 insertions, and 58 deletions, which could induce frameshifts altering protein-coding genes. Gene annotation revisions accounting for gene fusions and the reconciliation of gene lengths uncovered novel protein-coding genes and refined the functional categorizations of established ones. Additionally, the analysis pointed out genome structural variations due to insertion sequence (IS) elements, underscoring the D. radiodurans genome's plasticity. Notably, ATCC 13939K exhibited a loss of six ISDra2 elements relative to BAA-816, restoring genes fragmented by ISDra2, such as those encoding for α/ß hydrolase and serine protease, and revealing new open reading frames, including genes imperative for acetoin decomposition. This comparative genomic study offers vital insights into the metabolic capabilities and resilience strategies of D. radiodurans.

Effects of Gamma Irradiation on Changes in Chemical Composition and Antioxidant Activity of Euphorbia maculata Callus.

In this study, we investigated the effects of gamma irradiation on the antioxidant activity and metabolite profiles of Euphorbia maculata calli (PC3012). Gamma irradiation at various doses (0, 0.05, 0.5, and 10 kGy) significantly enhanced the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+) radical scavenging activities of the callus extracts of PC3012 in a dose-dependent manner. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) analyses revealed that irradiation increased the lysophospholipid content, although no new antioxidant compounds were formed. Furthermore, a PLS-DA analysis revealed evident metabolic differences between non-irradiated and irradiated samples, which were further verified by statistical validation. These findings suggest that gamma irradiation induces specific biochemical modifications that enhance the bioactive properties of PC3012 calli. This technology exhibits potential for utilization in the natural product and food sectors, particularly in the development of functional foods and nutraceuticals with improved health benefits.

Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans.

In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen Cryptococcus neoformans has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by HXL1 (HAC1 and XBP1-Like gene 1). C. neoformans HXL1 encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of HXL1 and its unconventional splicing is widely conserved in C. neoformans strains of divergent serotypes. Notably, both C. neoformans ire1 and hxl1 mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the ire1 mutant were suppressed by the spliced active form of Hxl1, supporting that HXL1 mRNA is a downstream target of Ire1. Interestingly, however, the ire1 and hxl1 mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in C. neoformans. Finally, Ire1 and Hxl1 were shown to be critical for virulence of C. neoformans, suggesting UPR signaling as a novel antifungal therapeutic target.

Functional Characterization of DNA N-Glycosylase Ogg1 and Ntg1 in DNA Damage Stress of Cryptococcus neoformans.

Reactive oxygen species induce DNA strand breaks and DNA oxidation. DNA oxidation leads to DNA mismatches, resulting in mutations in the genome if not properly repaired. Homologous recombination (HR) and non-homologous end-joining (NHEJ) are required for DNA strand breaks, whereas the base excision repair system mainly repairs oxidized DNAs, such as 8-oxoguanine and thymine glycol, by cleaving the glycosidic bond, inserting correct nucleotides, and sealing the gap. Our previous studies revealed that the Rad53-Bdr1 pathway mainly controls DNA strand breaks through the regulation of HR- and NHEJ-related genes. However, the functional roles of genes involved in the base excision repair system remain elusive in Cryptococcus neoformans. In the present study, we identified OGG1 and NTG1 genes in the base excision repair system of C. neoformans, which are involved in DNA oxidation repair. The expression of OGG1 was induced in a Hog1-dependent manner under oxidative stress. On the other hand, the expression of NTG1 was strongly induced by DNA damage stress in a Rad53-independent manner. We demonstrated that the deletion of NTG1, but not OGG1, resulted in elevated susceptibility to DNA damage agents and oxidative stress inducers. Notably, the ntg1Δ mutant showed growth defects upon antifungal drug treatment. Although deletion of OGG1 or NTG1 did not increase mutation rates, the mutation profile of each ogg1Δ and ntg1Δ mutant was different from that of the wild-type strain. Taken together, we found that DNA N-glycosylase Ntg1 is required for oxidative DNA damage stress and antifungal drug resistance in C. neoformans.

Two cation transporters Ena1 and Nha1 cooperatively modulate ion homeostasis, antifungal drug resistance, and virulence of Cryptococcus neoformans via the HOG pathway.

Maintenance of cation homeostasis is essential for survival of all living organisms in their biological niches. It is also important for the survival of human pathogenic fungi in the host, where cation concentrations and pH will vary depending on different anatomical sites. However, the exact role of diverse cation transporters and ion channels in virulence of fungal pathogens remains elusive. In this study we functionally characterized ENA1 and NHA1, encoding a putative Na(+)/ATPase and Na(+)/H(+) antiporter, respectively, in Cryptococcus neoformans, a basidiomycete fungal pathogen which causes fatal meningoencephalitis. Expression of NHA1 and ENA1 is induced in response to salt and osmotic shock mainly in a Hog1-dependent manner. Phenotypic analysis of the ena1Δ, nha1Δ, and ena1Δnha1Δ mutants revealed that Ena1 controls cellular levels of toxic cations, such as Na(+) and Li(+) whereas both Ena1 and Nha1 are important for controlling less toxic K(+) ions. Under alkaline conditions, Ena1 was highly induced and required for growth in the presence of low levels of Na(+) or K(+) salt and Nha1 played a role in survival under K(+) stress. In contrast, Nha1, but not Ena1, was essential for survival at acidic conditions (pH 4.5) under high K(+) stress. In addition, Ena1 and Nha1 were required for maintenance of plasma membrane potential and stability, which appeared to modulate antifungal drug susceptibility. Perturbation of ENA1 and NHA1 enhanced capsule production and melanin synthesis. However, Nha1 was dispensable for virulence of C. neoformans although Ena1 was essential. In conclusion, Ena1 and Nha1 play redundant and discrete roles in cation homeostasis, pH regulation, membrane potential, and virulence in C. neoformans, suggesting that these transporters could be novel antifungal drug targets for treatment of cryptococcosis.

Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans.

A balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts. IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

New damage model for simulating radiation-induced direct damage to biomolecular systems and experimental validation using pBR322 plasmid.

In this work, we proposed a new damage model for estimating radiation-induced direct damage to biomolecular systems and validated its the effectiveness for pBR322 plasmids. The proposed model estimates radiation-induced damage to biomolecular systems by: (1) simulation geometry modeling using the coarse-grained (CG) technique to replace the minimum repeating units of a molecule with a single bead, (2) approximation of the threshold energy for radiation damage through CG potential calculation, (3) calculation of cumulative absorption energy for each radiation event in microscopic regions of CG models using the Monte Carlo track structure (MCTS) code, and (4) estimation of direct radiation damage to biomolecular systems by comparing CG potentials and absorption energy. The proposed model replicated measured data with an average error of approximately 14.2% in the estimation of radiation damage to pBR322 plasmids using the common MCTS code Geant4-DNA. This is similar to the results of previous simulation studies. However, in existing damage models, parameters are adjusted based on experimental data to increase the reliability of simulation results, whereas in the proposed model, they can be determined without using empirical data. Because the proposed model proposed is applicable to DNA and various biomolecular systems with minimal experimental data, it provides a new method that is convenient and effective for predicting damage in living organisms caused by radiation exposure.

Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway.

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.

Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans.

The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Delta, gpa1Delta, cac1Delta, aca1Delta, and pka1Delta pka2Delta mutants. The aca1Delta, gpa1Delta, cac1Delta, and pka1Delta pka2Delta mutants displayed similar transcriptome patterns, whereas the ras1Delta mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Delta and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.

Functional Roles of Homologous Recombination and Non-Homologous End Joining in DNA Damage Response and Microevolution in Cryptococcus neoformans.

DNA double-strand breaks (DSBs) are the most deleterious type of DNA lesions because they cause loss of genetic information if not properly repaired. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are required for DSB repair. However, the relationship of HR and NHEJ in DNA damage stress is unknown in the radiation-resistant fungus Cryptococcus neoformans. In this study, we found that the expression levels of HR- and NHEJ-related genes were highly induced in a Rad53-Bdr1 pathway-dependent manner under genotoxic stress. Deletion of RAD51, which is one of the main components in the HR, resulted in growth under diverse types of DNA damage stress, whereas perturbations of KU70 and KU80, which belong to the NHEJ system, did not affect the genotoxic stresses except when bleomycin was used for treatment. Furthermore, deletion of both RAD51 and KU70/80 renders cells susceptible to oxidative stress. Notably, we found that deletion of RAD51 induced a hypermutator phenotype in the fluctuation assay. In contrast to the fluctuation assay, perturbation of KU70 or KU80 induced rapid microevolution similar to that induced by the deletion of RAD51. Collectively, Rad51-mediated HR and Ku70/Ku80-mediated NHEJ regulate the DNA damage response and maintain genome stability.

Characterizing the role of RNA silencing components in Cryptococcus neoformans.

The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNA-dependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans.

A Signature-Tagged Mutagenesis (STM)-based murine-infectivity assay for Cryptococcus neoformans.

Signature-tagged mutagenesis (STM) is a high-throughput genetic technique that can be used to investigate the function of genes by constructing a large number of mutant strains with unique DNA identification tags, pooling them, and screening them for a particular phenotypic trait. STM was first designed for the identification of genes that contribute to the virulence or infectivity of a pathogen in its host. Recently, this method has also been applied for the identification of mutants with specific phenotypes, such as antifungal drug resistance and proliferation. In the present study, we describe an STM method for the identification of genes contributing to the infectivity of Cryptococcus neoformans using a mutant library, in which each strain was tagged with a unique DNA sequence.

Carbon nanotube sponges as an enrichment material for aromatic volatile organic compounds.

Sensitive methods are required for in situ monitoring of volatile organic compounds (VOCs). Herein, carbon nanotube (CNT) sponges were investigated as a new type of adsorbent for enriching trace aromatic VOCs. A square pillar configuration (3 mm × 3 mm × 45 mm, 5 mg) of a CNT sponge was enclosed in a glass tube (4 mm i.d.). After accumulating the sample vapor, a direct current pulse (26 V, 0.5-3.0 s) through the CNT sponge allowed narrow desorption bandwidths of 0.48-0.84 s (with a photoionization detector) and 1.2 s (with a flame ionization detector) and high desorption efficiency (>96.5%). Gas chromatographic analysis of a nine-component VOC mixture (100 mL adsorption volume) gave enrichment factors of 88 (benzene) to 323 (toluene and m-xylene) with detection limits in the range of 0.9-2.6 ppb (v/v). These results demonstrate that CNT sponges are a promising preconcentrator material for trace detection of VOCs. The adsorption breakthrough experiments exhibited good correlation with the kinetic adsorption and Langmuir isotherm models. The maximum adsorption capacities of the CNT sponge increased in the order benzene (0.13 mg/g) < toluene (2.45 mg/g) < ethylbenzene (13.90 mg/g) < o-xylene (14.31 mg/g), with R2 values of >0.95. The rollup phenomena observed during multicomponent adsorption were explained by the competitive displacement or adsorption affinities of aromatic VOCs. The feasibility of the CNT sponge preconcentrator in a real environment was tested for interfering species (NO2 and NH3), laboratory air, and a human breath sample and demonstrated similar performance as in the controlled nine-component tests.