Similar evolutionary trajectories in an environmental Cryptococcus neoformans isolate after human and murine infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Barley HOMOCYSTEINE METHYLTRANSFERASE 2 confers drought tolerance by improving polyamine metabolism.

Drought stress poses a serious threat to crop production worldwide. Genes encoding homocysteine methyltransferase (HMT) have been identified in some plant species in response to abiotic stress, but its molecular mechanism in plant drought tolerance remains unclear. Here, transcriptional profiling, evolutionary bioinformatics, and population genetics were conducted to obtain insight into the involvement of HvHMT2 from Tibetan wild barley (Hordeum vulgare ssp. agriocrithon) in drought tolerance. We then performed genetic transformation coupled with physio-biochemical dissection and comparative multiomics approaches to determine the function of this protein and the underlying mechanism of HvHMT2-mediated drought tolerance. HvHMT2 expression was strongly induced by drought stress in tolerant genotypes in a natural Tibetan wild barley population and contributed to drought tolerance through S-adenosylmethionine (SAM) metabolism. Overexpression of HvHMT2 promoted HMT synthesis and efficiency of the SAM cycle, leading to enhanced drought tolerance in barley through increased endogenous spermine and less oxidative damage and growth inhibition, thus improving water status and final yield. Disruption of HvHMT2 expression led to hypersensitivity under drought treatment. Application of exogenous spermine reduced accumulation of reactive oxygen species (ROS), which was increased by exogenous mitoguazone (inhibitor of spermine biosynthesis), consistent with the association of HvHMT2-mediated spermine metabolism and ROS scavenging in drought adaptation. Our findings reveal the positive role and key molecular mechanism of HvHMT2 in drought tolerance in plants, providing a valuable gene not only for breeding drought-tolerant barley cultivars but also for facilitating breeding schemes in other crops in a changing global climate.

Neurological manifestations of SARS-CoV-2 infection in children in Taiwan: A cross-section, multicenter study.

BACKGROUND: The SARS-CoV-2 virus has been a global public health threat since December 2019. This study aims to investigate the neurological characteristics and risk factors of coronavirus disease 2019 (COVID-19) in Taiwanese children, using data from a collaborative registry. METHODS: A retrospective, cross-sectional, multi-center study was done using an online network of pediatric neurological COVID-19 cohort collaborative registry. RESULTS: A total of 11160 COVID-19-associated emergency department (ED) visits and 1079 hospitalizations were analyzed. Seizures were the most common specific neurological symptom, while encephalitis and acute disseminated encephalomyelitis (ADEM) was the most prevalent severe involvement. In ED patients with neurological manifestations, severe neurological diagnosis was associated with visual hallucination, seizure with/without fever, behavior change, decreased GCS, myoclonic jerk, decreased activity/fatigue, and lethargy. In hospitalized patients with neurological manifestations, severe neurological diagnosis was associated with behavior change, visual hallucination, decreased GCS, seizure with/without fever, myoclonic jerk, fatigue, and hypoglycemia at admission. Encephalitis/ADEM was the only risk factor for poor neurological outcomes at discharge in hospitalized patients. CONCLUSION: Neurological complications are common in pediatric COVID-19. Visual hallucination, seizure, behavior change, myoclonic jerk, decreased GCS, and hypoglycemia at admission are the most important warning signs of severe neurological involvement such as encephalitis/ADEM.

Update current understanding of neurometabolic disorders related to lysine metabolism.

Lysine, as an essential amino acid, predominantly undergoes metabolic processes through the saccharopine pathway, whereas a smaller fraction follows the pipecolic acid pathway. Although the liver is considered the primary organ for lysine metabolism, it is worth noting that lysine catabolism also takes place in other tissues and organs throughout the body, including the brain. Enzyme deficiency caused by pathogenic variants in its metabolic pathway may lead to a series of neurometabolic diseases, among which glutaric aciduria type 1 and pyridoxine-dependent epilepsy have the most significant clinical manifestations. At present, through research, we have a deeper understanding of the multiple pathophysiological mechanisms related to these diseases, including intracerebral accumulation of neurotoxic metabolites, imbalance between GABAergic and glutamatergic neurotransmission, energy deprivation due to metabolites, and the dysfunction of antiquitin. Because of the complexity of these diseases, their clinical manifestations are also diverse. The early implementation of lysine-restricted diets and supplementation with arginine and carnitine has reported positive impacts on the neurodevelopmental outcomes of patients. Presently, there is more robust evidence supporting the effectiveness of these treatments in glutaric aciduria type 1 compared with pyridoxine-dependent epilepsy.

Melanin deposition in two Cryptococcus species depends on cell-wall composition and flexibility.

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.

CMAP changes upon symptom onset and during treatment in spinal muscular atrophy patients: lessons learned from newborn screening.

PURPOSE: Early identification and treatment of spinal muscular atrophy (SMA) are crucial but difficult. In this study, we aimed to assess the significance of compound motor action potential (CMAP) amplitude in patients identified through a newborn screening program. METHODS: We initiated a large-scale population newborn screening program for SMA in Taiwan in 2014. Patients had access to treatment through clinical trials or expanded use programs. Symptomatic patients were evaluated regularly, including CMAP exams. RESULTS: Among 364,000 screened newborns, 21 were diagnosed with SMA. The incidence of SMA was around 1 in 17,000 live births, and 70% developed SMA type 1. All infants with two SMN2 copies became symptomatic before the age of 1 month. CMAP amplitudes of 12 newborns were available, including 6 who were subsequently treated with nusinersen. We found that a rapid decrease of CMAP amplitude was an early predictor of symptom onset. Pretreatment CMAP and rapid increment of post-treatment CMAP could predict better treatment outcomes. CONCLUSION: This study prospectively demonstrated the incidence of SMA and its types. Our results imply the importance of pretreatment CMAP amplitude and rapid reversal of post-treatment CMAP amplitude with regard to disease presentation and also treatment outcomes.

Dragotcytosis: Elucidation of the Mechanism for Cryptococcus neoformans Macrophage-to-Macrophage Transfer.

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate-labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis ("Dragot" is a Greek surname meaning "sentinel"), as it represents sharing of a microbe between two sentinel cells of the innate immune system.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

The Outcome of the Cryptococcus neoformans-Macrophage Interaction Depends on Phagolysosomal Membrane Integrity.

Cryptococcus neoformans is a fungal pathogen with worldwide distribution. C. neoformans resides within mature phagolysosomes where it often evades killing and replicates. C. neoformans induces phagolysosomal membrane permeabilization (PMP), but the mechanism for this phenomenon and its consequences for macrophage viability are unknown. In this study, we used flow cytometry methodology in combination with cell viability markers and LysoTracker to measure PMP in J774.16 and murine bone marrow-derived macrophages infected with C. neoformans Our results showed that cells manifesting PMP were positive for apoptotic markers, indicating an association between PMP and apoptosis. We investigated the role of phospholipase B1 in C. neoformans induction of PMP. Macrophages infected with a C. neoformans Δplb1 mutant had reduced PMP compared with those infected with wild-type and phospholipase B1-complemented strains, suggesting a mechanism of action for this virulence factor. Capsular enlargement inside macrophages was identified as an additional likely mechanism for phagolysosomal membrane damage. Macrophages undergoing apoptosis did not maintain an acidic phagolysosomal pH. Induction of PMP with ciprofloxacin enhanced macrophages to trigger lytic exocytosis whereas nonlytic exocytosis was common in those without PMP. Our results suggest that modulation of PMP is a critical event in determining the outcome of C. neoformans-macrophage interaction.

The enigmatic role of fungal annexins: the case of Cryptococcus neoformans.

Annexins are multifunctional proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins play a myriad of critical and well-characterized roles in mammals, ranging from membrane repair to vesicular secretion. The role of annexins in the kingdoms of bacteria, protozoa and fungi have been largely overlooked. The fact that there is no known homologue of annexins in the yeast model organism Saccharomyces cerevisiae may contribute to this gap in knowledge. However, annexins are found in most medically important fungal pathogens, with the notable exception of Candida albicans. In this study we evaluated the function of the one annexin gene in Cryptococcus neoformans, a causative agent of cryptococcosis. This gene CNAG_02415, is annotated in the C. neoformans genome as a target of calcineurin through its transcription factor Crz1, and we propose to update its name to cryptococcal annexin, AnnexinC1. C. neoformans strains deleted for AnnexinC1 revealed no difference in survival after exposure to various chemical stressors relative to wild-type strain, as well as no major alteration in virulence or mating. The only alteration observed in strains deleted for AnnexinC1 was a small increase in the titan cells' formation in vitro. The preservation of annexins in many different fungal species suggests an important function, and therefore the lack of a strong phenotype for annexin-deficient C. neoformans indicates either the presence of redundant genes that can compensate for the absence of AnnexinC1 function or novel functions not revealed by standard assays of cell function and pathogenicity.

Response of Tibetan Wild Barley Genotypes to Drought Stress and Identification of Quantitative Trait Loci by Genome-Wide Association Analysis.

Tibetan wild barley has been identified to show large genetic variation and stress tolerance. A genome-wide association (GWA) analysis was performed to detect quantitative trait loci (QTLs) for drought tolerance using 777 Diversity Array Technology (DArT) markers and morphological and physiological traits of 166 Tibetan wild barley accessions in both hydroponic and pot experiments. Large genotypic variation for these traits was found; and population structure and kinship analysis identified three subpopulations among these barley genotypes. The average LD (linkage disequilibrium) decay distance was 5.16 cM, with the minimum on 6H (0.03 cM) and the maximum on 4H (23.48 cM). A total of 91 DArT markers were identified to be associated with drought tolerance-related traits, with 33, 26, 16, 1, 3, and 12 associations for morphological traits, H⁺K⁺-ATPase activity, antioxidant enzyme activities, malondialdehyde (MDA) content, soluble protein content, and potassium concentration, respectively. Furthermore, 7 and 24 putative candidate genes were identified based on the reference Meta-QTL map and by searching the Barleymap. The present study implicated that Tibetan annual wild barley from Qinghai⁻Tibet Plateau is rich in genetic variation for drought stress. The QTLs detected by genome-wide association analysis could be used in marker-assisting breeding for drought-tolerant barley genotypes and provide useful information for discovery and functional analysis of key genes in the future.

Genome-Wide Identification, Characterization and Expression Analysis of Xyloglucan Endotransglucosylase/Hydrolase Genes Family in Barley (Hordeum vulgare).

Xyloglucan endotransglucosylase/hydrolases (XTHs)-a family of xyloglucan modifying enzymes-play an essential role in the construction and restructuring of xyloglucan cross-links. However, no comprehensive study has been performed on this gene family in barley. A total of 24 HvXTH genes (named HvXTH1-24) and an EG16 member were identified using the recently completed genomic database of barley (Hordeum vulgare). Phylogenetic analysis showed that 24 HvXTH genes could be classified into three phylogenetic groups: (I/II, III-A and III-B) and HvXTH15 was in the ancestral group. All HvXTH protein members-except HvXTH15-had a conserved N-glycosylation site. The genomic location of HvXTHs on barley chromosomes showed that the 24 genes are unevenly distributed on the 7 chromosomes, with 10 of them specifically located on chromosome 7H. A structure-based sequence alignment demonstrates that each XTH possesses a highly conserved domain (ExDxE) responsible for catalytic activity. Expression profiles based on the barley genome database showed that HvXTH family members display different expression patterns in different tissues and at different stages. This study is the first systematic genomic analysis of the barley HvXTH gene family. Our results provide valuable information that will help to elucidate the roles of HvXTH genes in the growth and development of barley.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus Cryptococcus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be Cryptococcus neoformans and Cryptococcus gattii We compared the interaction of murine macrophages with three C. gattii species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one C. neoformans species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies of C. neoformans and C. gattii species complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with either C. gattii or C. neoformans Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.

Divalent Metal Cations Potentiate the Predatory Capacity of Amoeba for Cryptococcus neoformans.

Among the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that of Acanthamoeba castellanii and Cryptococcus neoformans The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg2+ and Ca2+ in phosphate-buffered saline (PBS), C. neoformans was consistently killed when incubated with A. castellaniiA. castellanii survived better in the presence of Mg2+ and Ca2+, even when incubated with C. neoformans In the absence of Mg2+ and Ca2+, C. neoformans survived when incubated with A. castellanii, and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg2+ and Ca2+ can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba. Of the two metals, Mg2+ had a stronger effect than Ca2+ The cations enhanced A. castellanii activity against C. neoformans via enhanced phagocytosis, which is the major mechanism by which amoebae kill fungal cells. We found no evidence that amoebae use extracellular killing mechanisms in their interactions with C. neoformans In summary, the presence of Mg2+ and Ca2+ enhanced the cell adhesion on the surfaces and the motility of the amoeba, thus increasing the chance for contact with C. neoformans and the frequency of phagocytosis. Our findings imply that the divalent cation concentration in soils could be an important variable for whether amoebae can control C. neoformans in the environment.IMPORTANCE The grazing of soil organisms by phagocytic predators such as amoebae is thought to select for traits that enable some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoebae and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the interaction between an amoeba and the pathogenic fungus C. neoformans is influenced by the presence in the assay of magnesium and calcium, which potentiate amoebae. The results may also have practical applications, since enriching soils with divalent cations may reduce C. neoformans numbers in contaminated soils.

Lipids Affect the Cryptococcus neoformans-Macrophage Interaction and Promote Nonlytic Exocytosis.

Many microbes exploit host cellular lipid droplets during the host-microbe interaction, but this phenomenon has not been extensively studied for fungal pathogens. In this study, we analyzed the role of lipid droplets during the interaction of Cryptococcus neoformans with macrophages in the presence and the absence of exogenous lipids, in particular, oleate. The addition of oleic acid increased the frequency of lipid droplets in both C. neoformans and macrophages. C. neoformans responded to oleic acid supplementation by faster growth inside and outside macrophages. Fungal cells were able to harvest lipids from macrophage lipid droplets. Supplementation of C. neoformans and macrophages with oleic acid significantly increased the rate of nonlytic exocytosis while having no effect on lytic exocytosis. The process for lipid modulation of nonlytic exocytosis was associated with actin changes in macrophages. In summary, C. neoformans harvests lipids from macrophages, and the C. neoformans-macrophage interaction is modulated by exogenous lipids, providing a new tool for studying nonlytic exocytosis.

JNK pathway in osteoarthritis: pathological and therapeutic aspects.

CONTEXT: Osteoarthritis (OA) is a common chronic degenerative joint disease resulting in physical disability and reduced quality of life. Different biochemical signaling pathways are involved in the progression of OA, including the c-Jun NH2-terminal kinase (JNK) signal transduction pathway. OBJECTIVE: In this study, we have reviewed the recent updates on the association of JNK pathway with OA. METHODS: In this review, we have explored the databases like PubMed, Google Scholar, Medline, Scopus, etc., and collected the most relevant papers of JNK signaling pathway involved in the pathogenesis and therapeutics of OA Results: JNK has been shown by scientific studies to be activated (phosphorylated) in OA that can play a key role in the cartilage destruction. Activation of JNK causes the phosphorylation of c-Jun that causes decreased proteoglycan synthesis and enhanced production of matrix metalloproteinase 13 (MMP-13). Overproduction of MMP-13 by chondrocytes plays a central role in cartilage degeneration in OA. Thus, targeting JNK pathway might be a promising therapeutic application for the prevention and treatment of OA. A number of JNK-inhibitors have been used in vitro and in vivo studies; however, not yet been translated into human use. CONCLUSIONS: This review study indicates that JNK pathway plays an important role in development and progression of OA, and targeting the JNK pathway might be a potential approach for the treatment of OA in future.

Molecular Identification of Diphyllobothrium latum from a Pediatric Case in Taiwan.

Human diphyllobothriasis is a parasitic disease caused by ingestion of larvae (plerocercoids) in raw or undercooked fish and commonly found in temperate areas. Rare cases were reported in tropical or subtropical areas especially in children. The first documented case of pediatric diphyllobothriasis in Taiwan had been reported 11 years ago. Here, we report another 8-year-old girl case who presented with a live noodle-like worm hanging down from her anus, with no other detectable symptoms. We pulled the worm out and found the strobila being 260 cm in length. Examination of gravid proglottids showed that they were wider than their lengths, containing an ovoid cirrus sac in the anterior side and the rosette-shaped uterus. Eggs extracted from the uterus were ovoid and operculated. Diphyllobothrium latum was confirmed by molecular analysis of the mitochondrial DNA cytochrome c oxidase subunit 1 (cox1) gene. The girl was treated with a single oral dose of praziquantel, and no eggs or proglottids were observed from her stool in the subsequent 3 months. The reemergence of human diphyllobothriasis in non-endemic countries is probably due to prevalent habit of eating imported raw fish from endemic areas. This pediatric case raised our concern that human diphyllobothriasis is likely underestimated because of unremarkable symptoms.

Endoscopic-assisted retrieval of retained lacrimal stent material in congenital nasolacrimal duct obstruction without dacryocystorhinostomy.

We report a 3-year-old child with history of congenital nasolacrimal duct obstruction who failed lacrimal probing and underwent bicanalicular intubation. Removal of stent from the superior punctum was unsuccessful and it was trapped in the lacrimal sac. We performed endonasal endoscopic-assisted retrieval of retained stent material without necessitating dacryocystorhinostomy (DCR). There was no recurrence of infection and nasolacrimal duct obstruction symptoms during 6 months of follow-up.

[Simultaneous determination of seven alkaloids and three flavonoids in Sophorae Tonkinensis Radix et Rhizoma by HPLC].

In this study, an HPLC method was developed for simultaneous determination of seven alkaloids (cytosine, oxymatrine, N-oxysophocarpine, N-methylcytisine, sophoranol, matrine, and sophocarpine) and three flavonoids (trifolirhizin, fermononetin, and maackiain) from different samples of Sophorae Tonkinensis Radix et Rhizoma. Samples were analyzed on a Welch XtimateTM C18 column (4. 6 mm× 250 mm, 5 µm) eluted with the mobile phase of acetonitrile (A) and 0.01 mol•L⁻¹ ammonium acetate solution (pH 8.0) (B) in a linear gradient mode as follows: 0-20 min,4%-14% A;20-30 min,14%-25% A;30-45 min,25%-40% A;45-65 min,40%-55% A;65-75 min,55% A. The flow rate of the mobile phase, the column temperature, and the PDA detector wavelength were set at 1.0 mL•min⁻¹, 30 ℃, and 225 nm, respectively. For the method validation, these ten compounds showed good separation and satisfactory linearity (r≥0.999 7) within the concentration ranges tested. The mean recoveries were in the range of 98.60% to 102.6% with the RSD (n=6) between 0.60% and 3.7%. This method was proved to be simple, accurate and repeatable. The quantitative results showed that there were significant differences in the contents of seven alkaloids and three flavonoids among the different samples. This result revealed that the quality of Sophorae Tonkinensis Radix et Rhizoma varied widely. This method could be used for the simultaneous determination of the multi-ingredients from Sophorae Tonkinensis Radix et Rhizoma, which might provide scientific evidences to evaluate/control the quality of Sophorae Tonkinensis Radix et Rhizoma, comprehensively.

Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy of the protozoan parasite Trichomonas vaginalis.

BACKGROUND: To establish an infection in the vagina, Trichomonas vaginalis must adapt to various environmental cues for survival and further replication. Nutrient competition by lactobacilli, the major normal vaginal flora, is one of the mechanisms to limit the growth of other microorganisms. Additionally, lactobacilli produce H2O2 that can reduce the genital infections caused by other pathogens. Thus, the ability to overcome the metabolic stresses, such as glucose restriction (GR), as well as the oxidative stresses, is critical for T. vaginalis to establish an infection. METHODS: To gain insights into the molecular mechanisms of adaptation to GR, we utilized next-generation RNA sequencing (RNA-seq) to quantify the gene expression changes upon GR. Autophagy, a cytoprotective response to starvation, was monitored by using autophagy-specific staining, autophagy inhibition assay, and co-localization of autophagosomes with lysosomes. RESULTS: We demonstrated that GR promotes the survival of T. vaginalis. Besides, GR-cultivated cells exhibit higher H2O2 resistance. Our RNA-seq data revealed that genes involved in general energy metabolism were downregulated, whereas genes encoding glutamate metabolism-related aminotransferases were strikingly upregulated under GR. Furthermore, autophagy was first identified and characterized in T. vaginalis under GR. CONCLUSIONS: These data suggest that GR induces a metabolic reprogramming, enhancing antioxidant ability and autophagy for cellular homeostasis to maintain survival. GENERAL SIGNIFICANCE: Our work not only led to significant advances in understanding the transcriptional changes in response to GR but also provided possible strategies elicited by GR for T. vaginalis to adapt to the vaginal microenvironment.