Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

A Review: Cytochrome P450 in Alcoholic and Non-Alcoholic Fatty Liver Disease.

Alcoholic fatty liver disease (FALD) and non-alcoholic fatty liver disease (NAFLD) have similar pathological spectra, both of which are associated with a series of symptoms, including steatosis, inflammation, and fibrosis. These clinical manifestations are caused by hepatic lipid synthesis and metabolism dysregulation and affect human health. Despite having been studied extensively, targeted therapies remain elusive. The Cytochrome P450 (CYP450) family is the most important drug-metabolising enzyme in the body, primarily in the liver. It is responsible for the metabolism of endogenous and exogenous compounds, completing biological transformation. This process is relevant to the occurrence and development of AFLD and NAFLD. In this review, the correlation between CYP450 and liver lipid metabolic diseases is summarised, providing new insights for the treatment of AFLD and NAFLD.

Aneuploidy enables cross-tolerance to unrelated antifungal drugs in Candida parapsilosis.

Candida parapsilosis is an emerging major human fungal pathogen. Echinocandins are first-line antifungal drugs for the treatment of invasive Candida infections. In clinical isolates, tolerance to echinocandins in Candida species is mostly due to point mutations of FKS genes, which encode the target protein of echinocandins. However, here, we found chromosome 5 trisomy was the major mechanism of adaptation to the echinocandin drug caspofungin, and FKS mutations were rare events. Chromosome 5 trisomy conferred tolerance to echinocandin drugs caspofungin and micafungin and cross-tolerance to 5-flucytosine, another class of antifungal drugs. The inherent instability of aneuploidy caused unstable drug tolerance. Tolerance to echinocandins might be due to increased copy number and expression of CHS7, which encodes chitin synthase. Although copy number of chitinase genes CHT3 and CHT4 was also increased to the trisomic level, the expression was buffered to the disomic level. Tolerance to 5-flucytosine might be due to the decreased expression of FUR1. Therefore, the pleiotropic effect of aneuploidy on antifungal tolerance was due to the simultaneous regulation of genes on the aneuploid chromosome and genes on euploid chromosomes. In summary, aneuploidy provides a rapid and reversible mechanism of drug tolerance and cross-tolerance in C. parapsilosis.

Aneuploidy Mediates Rapid Adaptation to a Subinhibitory Amount of Fluconazole in Candida albicans.

Candida albicans is a prevalent, opportunistic, human fungal pathogen. Antifungal drug resistance and tolerance are two distinct mechanisms of adaptation to drugs. Studies of mechanisms of drug resistance are limited to the applications of high doses of drugs. Few studies have investigated the effects of subinhibitory amounts of drugs on the development of drug resistance or tolerance. In this study, we found that growth in a subinhibitory amount of fluconazole (FLC), a widely used antifungal drug, for just a short time was sufficient to induce aneuploidy in C. albicans. Surprisingly, the aneuploids displayed fitness loss in the presence of subinhibitory FLC, but a subpopulation of cells could tolerate up to 128 µg/mL FLC. Particular aneuploidy (ChrR trisomy) caused tolerance, not resistance, to FLC. In the absence of FLC, the aneuploids were unstable. Depending on the karyotype, aneuploids might become completely euploid or maintain particular aneuploidy, and, accordingly, the tolerance would be lost or maintained. Mechanistically, subinhibitory FLC was sufficient to induce the expression of several ERG genes and as well as the drug efflux gene MDR1. Aneuploids had a constitutive high-level expression of genes on and outside the aneuploid chromosomes, including most of the ERG genes as well as the drug efflux genes MDR1 and CDR2. Therefore, aneuploids were prepared for FLC challenges. In summary, aneuploidy provides a rapid and reversible strategy of adaptation when C. albicans is challenged with subinhibitory concentrations of FLC. IMPORTANCE Genome instability is a hallmark of C. albicans. Aneuploidy usually causes fitness loss in the absence of stress but confers better fitness under particular stress conditions. Therefore, aneuploidy is considered to be a double-edged sword. Here, we extend the understanding of aneuploidy. We found that aneuploidy arose under weak stress conditions but that it did not confer better fitness to the stress. Instead, it was less fit than its euploid counterparts. If the stress was withdrawn, aneuploidy spontaneously reverted to euploidy. If the stress became stronger, aneuploidy enabled subpopulation growth in a dose-independent manner of the stress. Therefore, we posit that aneuploidy enables the rapid and reversible development of drug tolerance in C. albicans. Further studies are required to investigate whether this is a general mechanism in human fungal pathogens.

Novel strategies to reverse chemoresistance in colorectal cancer.

Colorectal cancer (CRC) is a common gastrointestinal malignancy with high morbidity and fatality. Chemotherapy, as traditional therapy for CRC, has exerted well antitumor effect and greatly improved the survival of CRC patients. Nevertheless, chemoresistance is one of the major problems during chemotherapy for CRC and significantly limits the efficacy of the treatment and influences the prognosis of patients. To overcome chemoresistance in CRC, many strategies are being investigated. Here, we review the common and novel measures to combat the resistance, including drug repurposing (nonsteroidal anti-inflammatory drugs, metformin, dichloroacetate, enalapril, ivermectin, bazedoxifene, melatonin, and S-adenosylmethionine), gene therapy (ribozymes, RNAi, CRISPR/Cas9, epigenetic therapy, antisense oligonucleotides, and noncoding RNAs), protein inhibitor (EFGR inhibitor, S1PR2 inhibitor, and DNA methyltransferase inhibitor), natural herbal compounds (polyphenols, terpenoids, quinones, alkaloids, and sterols), new drug delivery system (nanocarriers, liposomes, exosomes, and hydrogels), and combination therapy. These common or novel strategies for the reversal of chemoresistance promise to improve the treatment of CRC.

Interactions between Candida albicans and the resident microbiota.

Candida albicans is a prevalent, opportunistic human fungal pathogen. It usually dwells in the human body as a commensal, however, once in its pathogenic state, it causes diseases ranging from debilitating superficial to life-threatening systemic infections. The switch from harmless colonizer to virulent pathogen is, in most cases, due to perturbation of the fungus-host-microbiota interplay. In this review, we focused on the interactions between C. albicans and the host microbiota in the mouth, gut, blood, and vagina. We also highlighted important future research directions. We expect that the evaluation of these interplays will help better our understanding of the etiology of fungal infections and shed new light on the therapeutic approaches.

Sangxingtang inhibits the inflammation of LPS-induced acute lung injury in mice by down-regulating the MAPK/NF-κB pathway.

In the present study, we investigated anti-inflammatory effects of Sangxingtang (SXT) on acute lung injury using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The cell counting in the bronchoalveolar lavage fluid (BALF) was performed. The degree of lung edema was evaluated by measuring the wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activities were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were assayed by the enzyme-linked immunosorbent assay methods. Pathological changes of lung tissues were observed by Hematoxylin and eosin (HE) staining. The inflammatory signaling pathway-related proteins nuclear factor mitogen activated protein kinases (P38MAPK), extracellular regulated protein kinases (Erk), c-Jun N-terminal kinase (Jnk) and nuclear transcription factor (NF-κB) p65 expressions were measured by Western blotting. Our results showed that the treatment with the SXT markedly attenuated the inflammatory cell numbers in the BALF, decreased the levels of P-P38MAPK, P-Erk, P-Jnk and P-NF-κB p65 and the total protein levels in lungs, improved the SOD activity and inhibited the MPO activity. Histological studies demonstrated that SXT substantially reduced the LPS-induced neutrophils in lung tissues, compared with the untreated LPS group. In conclusion, our results indicated that SXT had protective effects on LPS-induced ALI in mice.

Novel triazolyl berberine derivatives prepared via CuAAC click chemistry: synthesis, anticancer activity and structure-activity relationships.

To search for novel anticancer agents, we designed and synthesized a series of new triazolyl berberine derivatives. The evaluation of all the synthesized compounds and their anticancer activities against a panel of four human cancer cell lines including MCF-7 (breast), MCF-7/ADR (breast), SW-1990 (pancreatic), SMMC-7721 (liver) and the noncancer cell line HUVEC (human umbilical vein endothelial cell). The results showed that most of the compounds displayed better anticancer activities against MCF-7 and SMMC-7721 compared with berberine. Among these derivatives, compounds 5p and 5a exhibited the most potent inhibitory activities against the SMMC-7721 and SW-1990 cell lines with IC50 values of 14.861 ± 2.4 µM and 16.798 ± 3.4 µM. Furthermore, compounds 5p, 5a and 5n exhibited much better selectivity toward the normal cell line HUVEC than berberine.

Novel Triazolyl berberine derivatives prepared via CuAAC click chemistry: Synthesis, Anticancer Activity and Structure-Activity Relationships.

To search for novel anticancer agents, we designed and synthesized a series of new triazolyl berberine derivatives. The evaluation of all the synthesized compounds and their anticancer activities against a panel of four human cancer cell lines including MCF-7 (breast), MCF-7/ADR (breast), SW-1990 (pancreatic), SMMC-7721 (liver) and the non-cancer cell line HUVEC (human umbilical vein endothelial cell). The results showed that most of the compounds displayed better anticancer activities against MCF-7 and SMMC-7721 compared with berberine. Among these derivatives, compounds 5p and 5a exhibited the most potent inhibitory activities against the SMMC-7721 and SW-1990 cell lines with IC50 values of 14.861 ± 2.4 µM and 16.798 ± 3.4 µM. Furthermore, compounds 5p, 5a and 5n exhibited much better selectivity toward the normal cell line HUVEC than berberine.

Design, synthesis, and anticancer activity of novel berberine derivatives prepared via CuAAC "click" chemistry as potential anticancer agents.

A series of novel derivatives of phenyl-substituted berberine triazolyls has been designed and synthesized via copper-catalyzed azide-alkyne cycloaddition click chemistry in an attempt to develop antitumor agents. All of the compounds were evaluated for anticancer activity against a panel of three human cancer cell lines, including MCF-7 (breast), SW-1990 (pancreatic), and SMMC-7721 (liver) and the noncancerous human umbilical vein endothelial cell (HUVEC) cell lines. The results indicated that most of the compounds displayed notable anticancer activities against the MCF-7 cells compared with berberine. Among these derivatives, compound 16 showed the most potent inhibitory activity against the SW-1990 and SMMC-7721 cell lines, with half-maximal inhibitory concentration (IC50) values of 8.54±1.97 µM and 11.87±1.83 µM, respectively. Compound 36 exhibited the most potent inhibitory activity against the MCF-7 cell line, with an IC50 value of 12.57±1.96 µM. Compound 16 and compound 36 exhibited low cytotoxicity in the HUVEC cell line, with IC50 values of 25.49±3.24 µM and 30.47±3.47 µM. Furthermore, compounds 14, 15, 16, 17, 18, 32, and 36 exhibited much better selectivity than berberine toward the normal cell line HUVEC.

Anti-asthma effects of synthetic salidroside through regulation of Th1/Th2 balance.

AIM: The aim of the study was to investigate the effect and mechanism of action of synthetic salidroside in an ovalbumin (OVA)-induced asthma model in mice. METHOD: BALB/c mice were sensitized with an intraperitoneal injection of ovalbumin (OVA) to induce a mouse model of asthma in paracmasis. The mice were treated with dexamethasone as the positive control. At the end of the study, respiratory reactivity was detected, the numbers of various kinds of white blood cells in the bronchoalveolar lavage fluid (BALF) were counted, and the levels of IL-4 and INF-γ in BALF were determined. Quantitative PCR was used to detect the mRNA contents of IL-4 and INF-γ in lung tissue. Histologic examination was performed to observe inflammatory cellular infiltration. RESULTS: Salidroside treatment virtually eliminated airway hyper-reactivity, markedly reduced the eosinophil percent, obviously reduced the levels of IL-4 and raised INF-γ in the bronchoalveolar lavage fluid (BALF) compared with the sham-treated group. Quantitative PCR on the mRNA content of IL-4 and INF-γ provided confirmation. Lung histologic observations showed that salidroside reduced inflammation and edema. These effects were equivalent to the effects of dexamethasone. CONCLUSION: Synthetic salidroside exhibits an anti-asthma effect which is related to the regulation of Th1/Th2 balance. This provides a new possibility for treatment of allergic asthma.

Pretreatment with antiasthmatic drug ibudilast ameliorates Aß 1-42-induced memory impairment and neurotoxicity in mice.

Amyloid-ß peptide (Aß) is thought to be associated with the progressive neuronal death observed in Alzheimer's disease (AD). However, effective neuroprotective approaches against Aß neurotoxicity are unavailable. Here, we investigated possible preventive effects of ibudilast, as a pharmacologic phosphodiesterase inhibitor, currently used for treatment of inflammatory diseases such as asthma, on Aß 1-42-induced neuroinflammatory, apoptotic responses and memory impairment. We found that pretreatment with ibudilast (4 or 12 mg/kg, i.p.) significantly ameliorated impaired spatial learning and memory in intracerebroventricularly (ICV) Aß 1-42-injected mice, as evidenced by decrease in escape latency during acquisition trials and increase in exploratory activities in the probe trial in Morris water maze (MWM) task, and by increase in the number of correct choices and decrease in latency to enter the shock-free compartment in Y-maze test. Further study showed that ibudilast prevented generation of pro-inflammatory cytokines such as NF-κB p65 and TNF-α as well as pro-apoptotic molecule caspase-3 activation and anti-apoptotic protein Bcl-2 downregulation in both hippocampus and cortex of ICV Aß 1-42-injected mice. Taken together, our findings suggest that ibudilast has preventive effects on Aß-induced cognitive impairment via inhibiting neuroinflammatory and apoptotic responses.

Pioglitazone ameliorates memory deficits in streptozotocin-induced diabetic mice by reducing brain ß-amyloid through PPARγ activation.

AIM: To examine the effects of pioglitazone, a PPARγ agonist, on memory performance and brain amyloidogenesis in streptozotocin (STZ)-induced diabetic mice. METHODS: ICR male mice were injected with STZ (150 mg/kg, iv) to induce experimental diabetes. Pioglitazone (9 and 18 mg·kg(-1)·d(-1), po) was administered for 6 weeks. Passive avoidance and Morris water maze (MWM) tests were used to evaluate cognitive function. The blood glucose and serum insulin levels were detected using the glucose oxidase method and an ELISA assay, respectively. ß-amyloid (Aß), ß-amyloid precursor protein (APP), ß-amyloid precursor protein cleaving enzyme 1 (BACE1), NF-κB p65, the receptor for advanced glycation end products (RAGE) and PPARγ in the brains were analyzed using Western blotting assays. RESULTS: The STZ-induced diabetic mice characterized by hyperglycemia and hypoinsulinemia performed poorly in both the passive avoidance and MWM tests, accompanied by increased Aß1-40/Aß1-42, APP, BACE1, NF-κB p65 and RAGE levels and decreased PPARγ level in the hippocampus and cortex. Chronic pioglitazone treatment significantly ameliorated the memory deficits and amyloidogenesis of STZ-induced diabetic mice, and suppressed expression of APP, BACE1, RAGE and NF-κB p65, and activated PPARγ in the hippocampus and cortex. However, pioglitazone did not significantly affect blood glucose and insulin levels. CONCLUSION: Pioglitazone ameliorates memory deficits in STZ-induced diabetic mice by reducing brain Aß level via activation of PPARγ, which is independent of its effects on blood glucose and insulin levels. The results suggest that pioglitazone may be used for treating the cognitive dysfunction in type 1 diabetes mellitus.

High-frequency genetic contents variations in clinical Candida albicans isolates.

Genome plasticity is a hallmark of Candida albicans and is believed to be an adaptation strategy. But the extent of such genomic variability is not well investigated. In this study, genetic contents of clinical C. albicans isolates were investigated at whole-genome level with array-based comparative genomic hybridization (array CGH) technology. It was revealed that C. albicans possessed variations of genetic contents, as well as aneuploidy. The variable genes were scattered across the chromosomes, as well clustered in particular regions, including sub-telomeric regions, retrotransposon-insertion sites and a variable region on chromosome 6.

TOP2 gene disruption reduces drug susceptibility by increasing intracellular ergosterol biosynthesis in Candida albicans.

In this study the role of the TOP2 gene in fungal drug susceptibility was investigated by disrupting and overexpressing the gene in Candida albicans. MIC determination and a spot assay showed that a top2Delta/Delta null mutant (strain T2bc) was more resistant to the antifungals tested than the wild-type (strain CAI4). Real-time RT-PCR and rhodamine 6G efflux examination showed that TOP2 did not influence the activity of drug efflux pumps. Sterol analysis with GC/high-resolution MS indicated that the intracellular ergosterol composition of the top2Delta/Delta mutant was significantly increased. Subsequently, fluorescence polarization measurements also revealed that Top2-deprived cells displayed a decrease in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Quantitative real-time RT-PCR analysis further confirmed that the ERG11 gene, an essential gene in ergosterol biosynthesis, was upregulated. These results demonstrate a close relationship between the TOP2 gene and drug susceptibility in C. albicans.