Biology subjects

Liu, N.-N.

Publications and source records attributed to Liu, N.-N..

6 recordsLinked to original sources

bioRxiv2024

Single-cell analysis of the somatic mutational landscape in human chondrocytes during aging and in osteoarthritis

Somatic mutation is now recognized as a cause of multiple human diseases other than cancer. Osteoarthritis (OA), a highly prevalent age-related disease, has been associated with increased chromosomal abnormalities in articular cartilage of OA patients. Thus far no systematic attempt has been made to characterize the somatic mutational landscape of chondrocytes during normal aging and in affected cartilage of OA patients. Here we used single-cell whole genome sequencing to quantitatively analyze single-nucleotide variants (SNVs) and small insertions and deletions (InDels) in 100 single chondrocytes isolated from the cartilage of hip femoral heads of 17 subjects aged from 26 to 90 years, including 9 OA patients and 8 non-OA donors. Both SNVs and InDels were found to accumulate with age in chondrocytes with a clock-like mutational signature. Surprisingly, the age-related accumulation rate of these mutations was found to be lower in OA chondrocytes compared with chondrocytes from non-OA control subjects.

genomics↗

bioRxiv2021

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells expressing a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing a wild type TOR1 allele from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. While consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats were capable of utilizing non-fermentable as well as fermentable carbon sources and of growth during severe acid stress, but were uniquely unable to grow on lactate. Genome-wide expression analysis showed paradoxical simultaneous activation of anabolic- and starvation responses in cells lacking Tor1 N-terminal HEAT repeats, with misregulation of carbon metabolism and of translational machinery biosynthesis. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

microbiology↗

bioRxiv2020

Identification of microbial markers across populations in early detection of colorectal cancer

Several studies have investigated the association between microbial and colorectal cancer (CRC). However, the replicable markers for early stage adenoma diagnosis across multiple populations remain elusive. Here, a meta-analysis of six studies, comprising a total of 1057 fecal samples, was performed to identify candidate markers. By adjusting the potential confounders, 11 and 26 markers (P<0.05) were identified and separately applied into constructing Random Forest classifier models to discriminate adenoma from control, and adenoma from CRC, achieving robust diagnostic accuracy with AUC = 0.80 and 0.89, respectively. Moreover, these markers demonstrated high diagnostic accuracy in independent validation cohorts. Pooled functional analysis and targeted qRT-PCR based genetic profiles reveal that the altered microbiome triggers different pathways of ADP-heptose and menaquinone biosynthesis (P<0.05) in adenoma vs. control and adenoma vs. CRC sequences respectively. The combined analysis of heterogeneous studies confirm adenoma-specific but universal markers across multi-populations, which improves early diagnosis and prompt treatment of CRC.

pathology↗

bioRxiv2020

Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans

Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to -ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% ([~]110 mM) to 0.2% ([~]11 mM), or when glycerol is used as carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. Consistently, gdh2-/- strains are as virulent as wildtype in fly and murine models of systemic candidiasis. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence, demonstrating that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate description of the microenvironment within the phagosomal compartment of macrophages and the metabolic events underlying the survival of phagocytosed C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome. Author SummaryCandida albicans is a commensal component of the human microflora and the most common fungal pathogen. The incidence of candidiasis is low in healthy populations. Consequently, environmental factors, such as interactions with innate immune cells, play critical roles. Macrophages provide the first line of defense and rapidly internalize C. albicans cells within specialized intracellular compartments called phagosomes. The microenvironment within phagosomes is dynamic and ill defined, but has a low pH, and contains potent hydrolytic enzymes and oxidative stressors. Despite the inhospitable conditions, phagocytized C. albicans cells catabolize amino acids to obtain energy to survive and grow. Here, we have critically examined amino acid catabolism and ammonia extrusion in C. albicans, the latter is thought to neutralize the phagosomal pH and induce the switch of morphologies from round "yeast-like" to elongated hyphal cells that can pierce the phagosomal membrane leading to escape from macrophages. We report that Gdh2, which catalyzes the deamination of glutamate to -ketoglutarate, is responsible for the observed environmental alkalization when C. albicans catabolize amino acids. Strikingly, Gdh2 is dispensable for escape from macrophages and virulent growth. These results provide new insights into host-pathogen interactions that determine the pathogenic outcome of C. albicans infections.

microbiology↗

bioRxiv2018

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

microbiology↗

bioRxiv2017

Phosphate Is The Third Nutrient Monitored By TOR In Candida albicans And Provides A Target For Fungal-Specific Indirect TOR Inhibition

The TOR pathway regulates morphogenesis and responses to host cells in the fungal pathogen Candida albicans. Eukaryotic TOR complex 1 (TORC1) induces growth and proliferation in response to nitrogen and carbon source availability. Our unbiased genetic approach seeking new components of TORC1 signaling in C. albicans revealed that the phosphate transporter Pho84 is required for normal TORC1 activity. We found that mutants in PHO84 are hypersensitive to rapamycin and, in response to phosphate feeding, generate less phosphorylated ribosomal protein S6 (P-S6) than wild type. The small GTPase Gtr1, a component of the TORC1-activating EGO complex, links Pho84 to TORC1. Mutants in Gtr1, but not in another TORC1-activating GTPase, Rhb1, are defective in the P-S6 response to phosphate. Overexpression of Gtr1 and of a constitutively active Gtr1Q67L mutant suppress TORC1-related defects. In S. cerevisiae pho84 mutants, constitutively active Gtr1 suppresses a TORC1 signaling defect but does not rescue rapamycin hypersensitivity. Hence connections from phosphate homeostasis to TORC1 may differ between C. albicans and S. cerevisiae. The converse direction of signaling, from TORC1 to the phosphate homeostasis (PHO) regulon, previously observed in S. cerevisiae, was genetically demonstrated in C. albicans using conditional TOR1 alleles. A small molecule inhibitor of Pho84, an FDA-approved drug, inhibits TORC1 signaling and potentiates the activity of the antifungals amphotericin B and micafungin. Anabolic TORC1-dependent processes require significant amounts of phosphate. Our study demonstrates that phosphate availability is monitored and also controlled by TORC1, and that TORC1 can be indirectly targeted by inhibiting Pho84.\n\nSignificanceThe human fungal pathogen Candida albicans uses the TOR signaling pathway to contend with varying host environments and thereby regulate cell growth. Seeking novel components of the C. albicans TOR pathway we identified a cell-surface phosphate importer, Pho84, and its molecular link to TOR complex 1 (TORC1). Since phosphorus is a critical element for anabolic processes like DNA replication, ribosome biogenesis, translation and membrane biosynthesis, TORC1 monitors its availability in regulating these processes. By depleting the central kinase in the TORC1 pathway, we showed that TORC1 signaling modulates regulation of phosphate acquisition. An FDA-approved small-molecule inhibitor of Pho84 inhibits TORC1 signaling and potentiates the activity of the gold-standard antifungal amphotericin B and the echinocandin micafungin.

microbiology↗