Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance.

Regulation of virulence factor production in the pathogen Cryptococcus neoformans remains to be fully illustrated. We present here a finding that a gene, encoding the glycolysis enzyme phosphoglucose isomerase (Pgi1), is critical for the biosynthesis of melanin and capsule, cell wall integrity and resistance to stress conditions. A leaky mutant of the yeast, LZM19, resulted from an insertion of T-DNA in the PGI1 promoter region, expressed PGI1 at a level only 1.9% of the wild type. LZM19 could synthesize the pigment melanin in the presence of 2% glucose, suggesting a status of LAC1 derepression. Phenotypically, capsule biosynthesis in LZM19 was remarkably reduced. Integrity of the cell wall and plasma membrane of LZM19 were impaired based on its sensitivity to Congo red and SDS. Also, LZM19 exhibited hypersensitivity to osmotic stress generated by 2 M NaCl or 1 M KCl, indicating possible impairment in the HOG signaling pathway. Furthermore, LZM19 failed to utilize mannose and fructose, suggesting a possible involvement of Pgi1 in the breakdown of these two sugars. Our results revealed a crucial role of PGI1 in coordination of the production of virulence factors, cell wall integrity and stress response in C. neoformans.

Cocrystal of Lutein with Improved Stability and Bioavailability.

Lutein (LT) is a natural carotenoid and is widely used for its vision protection and antioxidant activity. However, the long-chain polyene structure makes lutein sensitive to light and oxygen and poses many difficulties in the production, processing, and storage. In addition, the special chemical structure of LT leads to low solubility and bioavailability. In this study, we propose an efficient solution to address these issues. A cocrystal of LT with adipic acid (LT-APC) was obtained for the first time. The cocrystals were fully characterized. After cocrystallization, the melting point of marketed LT was increased. The chemical stability of LT was significantly improved, and the influence of impurities on stability was limited. Dissolution experiments were performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) and the cocrystal generated a much higher apparent solubility. To deepen insight into the mechanisms underlying the cocrystal's improved solubility, wettability tests were performed by contact angle determination and film flotation methods. The cocrystal presented better wettability than the marketed LT. Finally, pharmacokinetic studies of marketed LT and its cocrystal were conducted in rats. The results showed that the cocrystal exhibited 3.4 times higher C max and 2.2 times higher AUC at a single dose compared with marketed LT.

Improving the Dissolution Rate and Bioavailability of Curcumin via Co-Crystallization.

Curcumin (CUR) is a natural polyphenolic compound with various pharmacological activities. Low water solubility and bioavailability limit its clinical application. In this work, to improve the bioavailability of CUR, we prepared a new co-crystal of curcumin and L-carnitine (CUR-L-CN) via liquid-assisted grinding. Both CUR and L-CN have high safe dosages and have a wide range of applications in liver protection and animal nutrition. The co-crystal was fully characterized and the crystal structure was disclosed. Dissolution experiments were conducted in simulated gastric fluids (SGF) and simulated intestinal fluids (SIF). CUR-L-CN exhibited significantly faster dissolution rates than those of pure CUR. Hirshfeld surface analysis and wettability testing indicate that CUR-L-CN has a higher affinity for water and thus exhibits faster dissolution rates. Pharmacokinetic studies were performed in rats and the results showed that compared to pure CUR, CUR-L-CN exhibited 6.3-times-higher AUC0-t and 10.7-times-higher Cmax.

Inhibition of Abl Kinase by Imatinib Can Rescue the Compromised Barrier Function of 22q11.2DS Patient-iPSC-Derived Blood-Brain Barriers.

We have previously established that the integrity of the induced blood-brain barrier (iBBB) formed by brain microvascular endothelial cells derived from the iPSC of 22q11.2 DS (22q11.2 Deletion Syndrome, also called DiGeorge Syndrome) patients is compromised. We tested the possibility that the haploinsufficiency of CRKL, a gene within the 22q11.2 DS deletion region, contributes to the deficit. The CRKL is a major substrate of the Abl tyrosine kinase, and the Abl/CRKL signaling pathway is critical for endothelial barrier functions. Imatinib, an FDA-approved drug, inhibits Abl kinase and has been used to treat various disorders involving vascular leakages. To test if imatinib can restore the compromised iBBB, we treated the patient's iBBB with imatinib. After treatment, both trans-endothelial electrical resistance and solute permeability returned to comparable levels of the control iBBB. Correspondingly, changes in tight junctions and endothelial glycocalyx of the iBBB were also restored. Western blotting showed that imatinib increased the level of active forms of the CRKL protein. A transcriptome study revealed that imatinib up-regulated genes in the signaling pathways responsible for the protein modification process and down-regulated those for cell cycling. The KEGG pathway analysis further suggested that imatinib improved the gene expression of the CRKL signaling pathway and tight junctions, which agrees with our expectations and the observations at protein levels. Our results indicate that the 22q11.2DS iBBB is at least partially caused by the haploinsufficiency of CRKL, which can be rescued by imatinib via its effects on the Abl/CRKL signaling pathway. Our findings uncover a novel disease mechanism associated with 22q11.2DS.

Requirement of a Tsp2-type tetraspanin for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.

Identification of a Zds-like gene ZDS3 as a new mediator of stress resistance, capsule formation and virulence of the human pathogenic yeast Cryptococcus neoformans.

The fungal Zds proteins are regulators of the serine/threonine phosphatase 2A (PP2A) and the protein kinase A. Here, we characterize a Zds-like gene ZDS3 that plays a broad range of roles in the basidiomycetous pathogenic yeast Cryptococcus neoformans. ZDS3 harbors the conserved activation domain ZDS_C of Zds proteins. By gene disruption, ZDS3 is shown to play roles in capsule production, cell wall integrity, growth at a high temperature, resistance to H(2)O(2) stress, osmotic pressures and glucose-dependent invasive growth on the agar. As a consequence, the disruption of ZDS3 resulted in complete loss of virulence in a mouse cryptococcosis model. The data suggest that ZDS3 is a novel mediator of the virulence of C. neoformans. Zds3 may serve as an antifungal drug target as no homologs are found in mammals.

EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity.

Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.

Cell-type-specific brain methylomes profiled via ultralow-input microfluidics.

Methylomic analyses typically require substantial amounts of DNA, thus hindering studies involving scarce samples. Here, we show that microfluidic diffusion-based reduced representative bisulfite sequencing (MID-RRBS) permits high-quality methylomic profiling with nanogram-to-single-cell quantities of starting DNA. We used the microfluidic device, which allows for efficient bisulfite conversion with high DNA recovery, to analyse genome-wide DNA methylation in cell nuclei isolated from mouse brains and sorted into NeuN+ (primarily neuronal) and NeuN- (primarily glial) fractions, and to establish cell-type-specific methylomes. Genome-wide methylation and methylation in low-CpG-density promoter regions showed distinct patterns for NeuN+ and NeuN- fractions from the mouse cerebellum. The identification of substantial variations in the methylomic landscapes of the NeuN+ fraction of the frontal cortex of mice chronically treated with an atypical antipsychotic drug suggests that this technology can be broadly used for cell-type-specific drug profiling and for the study of drug-methylome interactions.

Mammalian Brain Development is Accompanied by a Dramatic Increase in Bipolar DNA Methylation.

DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders.

Activation of Endogenous Retroviruses in Dnmt1(-/-) ESCs Involves Disruption of SETDB1-Mediated Repression by NP95 Binding to Hemimethylated DNA.

Repression of endogenous retroviruses (ERVs) in mammals involves several epigenetic mechanisms. Acute loss of the maintenance methyltransferase Dnmt1 induces widespread DNA demethylation and transcriptional activation of ERVs, including CpG-rich IAP (intracisternal A particle) proviruses. Here, we show that this effect is not due simply to a loss of DNA methylation. Conditional deletions reveal that both Dnmt1 and Np95 are essential for maintenance DNA methylation. However, while IAPs are derepressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these ERVs remain silenced when Np95 is deleted alone or in combination with Dnmt1. This paradoxical phenotype results from an ectopic interaction between NP95 and the H3K9 methyltransferase SETDB1. Normally, SETDB1 maintains silencing of IAPs, but in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA transiently disrupts SETDB1-dependent H3K9me3 deposition. Thus, our observations reveal an unexpected antagonistic interplay between two repressive pathways involved in retroviral silencing in mammalian cells.