Proteomic Perspective of Azole Resistance in Aspergillus fumigatus Biofilm Extracellular Matrix in Response to Itraconazole.

Azole-resistant Aspergillus fumigatus makes a major challenge to the chemotherapy for invasive aspergillosis, whereas cyp51A gene mutation is the most dominant mechanism for azole resistance. Moreover, biofilm contributes to drug resistance for A. fumigatus, and extracellular matrix (ECM) is essential to protect live cells from antifungal drugs. Therefore, we performed a comparative proteomic study on the biofilm ECM of both the wild-type and azole-resistant strains of A. fumigatus under azole pressure. In total, 2377 proteins were identified, of which 480 and 604 proteins with differential expression were obtained from the wild-type and azole-resistant A. fumigatus in exposure to itraconazole respectively (fold change > 2 or < 0.5, P-value < 0.05). We found that a high proportion of regulated proteins were located in cytoplasm, nucleus, and mitochondria. Meanwhile, GO and KEGG analyses revealed that metabolic process and ribosome pathway were significantly enriched. Particularly, differentially expressed proteins in response to azole pressure of both the wild-type and resistant strains were further analyzed. Our results indicated that these changes in biofilm ECM proteins were related to ergosterol synthesis, oxidative stress, efflux pumps, DNA repair, DNA replication, and transcription.

A comparative proteomic study on the biofilm between wild-type and azole-resistant strains of A. fumigatus under drug pressure found that changes in biofilm ECM proteins were related to ergosterol synthesis, oxidative stress, efflux pumps, DNA repair, DNA replication, and transcription.

Repression of the Antioxidant Pyrroloquinoline Quinone in Skin Aging Induced by Bmi-1 Deficiency.

It is uncertain whether Bmi-1 deficiency could lead to skin aging by redox imbalance and DNA damage. In this study, we first confirmed that Bmi-1 had a relatively high expression level in the skin and Bmi-1 expression levels gradually decreased with age. Then, we studied the role of Bmi-1 in the skin using a Bmi-1-/- mouse model. Bmi-1-/- mice were supplemented with or without pyrroloquinoline quinone (PQQ) for 5 weeks, and their skin phenotypes were compared with Bmi1-/- and wild-type littermates. Our results showed that Bmi-1-/- mice displayed decreased vertical thickness of skin, sparse hair follicles, and thinner and more irregular collagen bundles. Mechanistically, increased oxidative stress with reducing antioxidant capacity and induced DNA damage occurred in Bmi-1-/- mice. Subsequently, this would lead to reduced cell proliferation, increased cell senescence and matrix metalloproteinases (MMPs), and the degradation of fibroblast function and further reduce collagen synthesis. All pathological alterations in the skin of Bmi-1-/- mice were alleviated by PQQ supplementation. These results demonstrated that Bmi-1 might play a key role in protection from skin aging by maintaining redox balance and inhibiting DNA damage response and will be a novel and potential target for preventing skin aging.

Effects of Itraconazole and Micafungin on Aspergillus fumigatus Biofilms.

Aspergillus fumigatus (A. fumigatus) is the most common airborne opportunistic fungal pathogen. Biofilm formation is one of the main pathogenic mechanisms of A. fumigatus. During the past decades, A. fumigatus azole resistance has become prevalent due to the medical and agricultural use of antifungal drugs and fungicides. Until now, the role of fungal biofilms in azole resistance of A. fumigatus remains unclear. In the present study, we compared biofilm drug susceptibility and biofilm formation under itraconazole of azole-resistant strains, sensitive strains, and standard strains, separately. The biofilm viability and matrix thickness at the early and the late stage were measured by XTT assay and Calcofluor white. Our results showed that the sessile minimum inhibitory concentration of itraconazole, which describing the inhibition of drugs on fungi sessile with biofilm, was much higher than the traditional minimal inhibitory concentration of itraconazole. Additionally, low concentrations of itraconazole inhibited biofilm formation of A. fumigatus strains. Notably, biofilm formation by azole-resistant strains could not be inhibited by high concentrations of itraconazole but could be effectively restrained by low concentrations of micafungin, revealing the efficacy of a cell-wall inhibitor to disrupt A. fumigatus biofilm formation. However, late-stage biofilms of both azole-resistant strains and standard strains were hard to disrupt using itraconazole. We found that itraconazole was effective to prevent A. fumigatus biofilm formation at the early stage. For the treatment of A. fumigatus biofilm, our findings suggest that an early-stage preventive strategy is preferred and micafungin is effective to control the azole-resistant strain infection.

Uncovering New Mutations Conferring Azole Resistance in the Aspergillus fumigatus cyp51A Gene.

The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.

Triazoles bind the C-terminal domain of SMO: Illustration by docking and molecular dynamics simulations the binding between SMO and triazoles.

Itraconazole is an antagonist of the component Smoothened of Hedgehog pathway, which can inhibit the growth of medulloblastoma, basal cell carcinoma, and melanoma, etc. To research the binding mechanism of the Smoothened and triazoles, we used docking and molecular dynamics simulations on the Smoothened crystal structure and six triazoles. Unlike vismodegib, itraconazole can effectively bind into the pocket in the C-terminal domain of the Smoothened crystal structure instead of the N-terminal domain. The binding of itraconazole can change the conformation of the N-terminal domain even although itraconazole only had limited area contacting with N-terminal domain of the Smoothened. Besides, the binding of Itraconazole will not affect the binding of vismodegib. The strong binding affinity could be demonstrated between itraconazole and the Smoothened. Posaconazole and ketoconazole also had the strong binding affinity and the similar binding mode with the Smoothened crystal structure.

Itraconazole exerts its anti-melanoma effect by suppressing Hedgehog, Wnt, and PI3K/mTOR signaling pathways.

Malignant melanoma is the deadliest form of all skin cancers. Itraconazole, a commonly used systemic antifungal drug, has been tested for its anti-tumor effects on basal cell carcinoma, prostate cancer, and non-small cell lung cancer. Whether itraconazole has any specific anti-tumor effect on melanoma remains unknown. However, the goal of this study is to investigate the effect of itraconazole on melanoma and to reveal some details of its underlying mechanism. In the in vivo xenograft mouse model, we find that itraconazole can inhibit melanoma growth and extend the survival of melanoma xenograft mice, compared to non-itraconazole-treated mice. Also, itraconazole can significantly inhibit cell proliferation, as demonstrated by Ki-67 staining in itraconazole-treated tumor tissues. In in vitro, we show that itraconazole inhibits the proliferation and colony formation of both SK-MEL-28 and A375 human melanoma cells. Moreover, we demonstrate that itraconazole significantly down-regulates Gli-1, Gli-2, Wnt3A, ß-catenin and cyclin D1, while it up-regulates Gli-3 and Axin-1, indicating potent inhibitory effects of itraconazole on Hedgehog (Hh) and Wnt signaling pathways. Furthermore, itraconazole significantly suppresses the PI3K/mTOR signaling pathway - indicated by the down-regulated phosphorylation of p70S6K, 4E-BP1 and AKT - but has no effect on the phosphorylation of MEK or ERK. Our data suggest that itraconazole inhibits melanoma growth through an interacting regulatory network that includes Hh, Wnt, and PI3K/mTOR signaling pathways. These results suggest that this agent has several potent anti-melanoma features and may be useful in the synergesis of other anti-cancer drugs via blockage of the Hh, Wnt and PI3K/mTOR signaling pathways.

cyp51A-based mechanism of azole resistance in Aspergillus fumigatus: Illustration by a new 3D Structural Model of Aspergillus fumigatus CYP51A protein.

Mutations of CYP51A protein (Cytochrome P450 14-α Sterol demethylase) play a central role in the azole resistance of Aspergillus fumigatus The available structural models of CYP51A protein ofA. fumigatus are built based on that of Homo sapiens and that of Mycobacterium tuberculosis, of which the amino acid homology is only 38% and 29% compared with CYP51A protein ofA. fumigatus, respectively. In the present study, we constructed a new 3D structural model ofA. fumigatus CYP51A protein based on a recently resolved crystal structure of the homologous protein in the fungus S. cerevisiae, which shares 50% amino acid homology with A. fumigatus CYP51A protein. Three azole molecules, itraconazole, voriconazole, and posaconazole, were docked to the wild-type and the mutant A. fumigatus CYP51A protein models, respectively, to illustrate the impact of cyp51A mutations to azole-resistance. We found the mutations that occurred at L98, M220, and Y431 positions would decrease the binding affinity of azoles to the CYP51A protein and therefore would reduce their inhibitory effects. Additionally, the mutations of L98 and G432 would reduce the stability of the protein, which might lead to conformational change of its binding pocket and eventually the resistance to azoles.

Multiple cyp51A-based mechanisms identified in azole-resistant isolates of Aspergillus fumigatus from China.

Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

Causes of death among active leprosy patients in China.

We analyzed the causes of death among active leprosy patients in China, to better understand the disease and improve the quality of services, by performing a retrospective study involving 24 provinces of China. Information about patients with active leprosy who were not clinically cured and died between January 1, 2000, and December 31, 2005, was collected by professionals at county levels. A total of 524 deaths were analyzed. The leading cause of death was suicide. There were 86 patients (16%) who died at a mean of 21 ± 19 months after starting multi-drug therapy (MDT). The second and third leading causes of death were cardiovascular disease and organ failure associated with advanced age, respectively. Two hundred and twenty-one patients (42%) died within one year of beginning MDT. The second month of MDT was the riskiest for newly treated patients; approximately 20% of patients succumbed to liver failure, 33% to dapsone (diaminodiphenylsulfone/DDS) allergy, and 27% to renal insufficiency during this period. Among 143 deaths related to leprosy, 37 (26%) occurred within three months of starting MDT. We recommend that newly diagnosed patients should be provided with no more than two months of MDT blister packs.

Occurrence and management of leprosy reaction in China in 2005.

BACKGROUND: Leprosy reactions are a major cause of disability before, during and after anti-bacterial treatment. Prompt diagnosis and correct management of reaction is a crucial matter for improving the quality of leprosy health services. OBJECTIVES: To describe the pattern of leprosy reaction and its management in China during 2005. METHODS: A retrospective survey using a questionnaire was carried out in all the provinces of China at the beginning of 2006. Patients included were those presenting with leprosy reaction between 1 January and 31 December 2005. RESULTS: 452 questionnaires from 25 provinces were analysed. There were 313 male and 139 female patients who had 159 Type I reactions, 273 Type II reactions and 20 Type I and II mixed reaction. 72.4% of reactions occurred in the first year of MDT and 27.6% of patients during the second year of MDT. The highest frequency of reaction was during the first 6 months of MDT; 57.3% of patients developed new nerve impairment during and after MDT. CONCLUSIONS: New nerve function impairment and disability still occurs among patients during and after MDT. The early detection and management of leprosy reaction remains important.

Current situation of leprosy colonies/leprosaria and their future in P.R. China.

OBJECTIVE: To identify the current situation of leprosy colonies/leprosaria and make some recommendations for improving the quality of life of people affected by leprosy in China. METHODS: A national survey using designed forms was carried out in 2004. The forms were filled in by local heads responsible for the management of leprosy colonies/leprosaria and sent to Provincial and National Centres for analysis. RESULTS: China had 605 leprosy colonies/leprosaria with 555 active leprosy patients (on treatment) and 18,175 ex-patients (people affected by leprosy) living in them at the end of 2004. Among 18,730 patients and people affected by leprosy, 13,430 (71.7%) had grade 2 disabilities. Among those with visible disability, 6392 (47.6%) lost the ability to take care of themselves due to serious deformity. Because of a decrease in health workers working at leprosy colonies and a shortage of medical materials, the health care quality of these people was neglected. Most colonies/leprosaria were located at remote and isolateS places with difficult transportation, and most buildings/houses were in danger of collapse because the colonies/leprosaria were built in the 1950's. Those affected by leprosy were in great need of help. CONCLUSION: The authors recommend that small, remote and isolated leprosy colonies should be closed. New leprosaria at District, Provincial or National levels should be established or some old leprosaria with good transportation should be reconstructed to house those affected by leprosy from closed leprosy colonies/leprosaria. The newly established or reconstructed leprosaria could act as centres for reference, training, rehabilitation and research on leprosy.

Risk of syphilis in STI clinic patients: a cross-sectional study of 11,500 cases in Guangxi, China.

OBJECTIVE: To measure prevalence of syphilis among the STI clinic population in Guangxi, China, and to assess the socioeconomic and behavioural characteristics associated with the infection. METHODS: We undertook a cross-sectional survey and syphilis and HIV serologic testing among 11 473 patients attending 14 community and hospital-based dermatovenereal clinics across eight cities in Guangxi between December 2004 and February 2006. RESULTS: 1297 (11.9%) patients demonstrated positive toludine red unheated serum test and Treponema pallidum particle agglutination results with serologic testing. A total of 58% (752) of seropositive subjects presented with a genital ulcer, palmar/plantar rash or inguinal lymphadenopathy. Female sex (OR = 2.23, 95% confidence intervals (CI) = 1.69 to 3.00, p<0.001), less education (middle school, OR = 1.70, 95% CI = 1.11 to 2.62, p = 0.023; primary school or less, OR = 1.98, 95% CI = 1.13 to 3.46, p = 0.017) and high annual income (OR = 1.91, 95% CI = 1.18 to 3.10, p = 0.009 for >30 000 RMB yuan) were associated with serologically positive status. Syphilis infection was significantly more prevalent in city 2 (19.5%, OR = 3.07, 95% CI = 1.83 to 5.16, p<0.001), city 4 (16.6%, OR = 1.90, 95% CI = 1.10 to 3.28, p = 0.011) and city 8 (13.8%, OR = 1.83, 95% CI = 1.13 to 2.97, p = 0.006). A total of 40.1% (532) of infected subjects engaged in commercial sex and increased rates of the infection was associated with multiple sexual partners (OR = 1.54, 95% CI = 1.16 to 2.06, p = 0.003). A total of 1.2% (133) of participants carried laboratory markers for HIV and 1.8% (23) of patients with syphilis were positive for HIV. CONCLUSIONS: Syphilis infection has reached alarming rates in China's STI clinic population, suggesting a generalised spread of the disease through commercial sex and bridging populations. Syphilis control is deserving of China's highest priority. Universal screening for syphilis and HIV testing in STI clinics should be considered as measures for control.

Relapse in MB leprosy patients treated with 24 months of MDT in south west China: a short report.

This study investigates the relapse rate among multibacillary leprosy patients treated with 24 months of MDT in south west China. A retrospective relapse survey was conducted in the southwest of China. A detailed questionnaire was designed to collect the data on relapse among MB patients who completed 2 years of the WHO/MB regimen, from 1989 to 2000. The data about 2517 multibacillary leprosy patients in 27 counties in the southwest of China were collected. Among 2517 MB patients, 235 patients died or were lost to follow-up and 2374 were followed up for more than 3 years after completion of MDT. The total duration of follow-up was 20,825 person-years, with a mean duration of 8.27 years per patient. Five patients with relapse were identified with an accumulated relapse rate of 0.21/1000 person-years. Their initial BIs ranged from 1.8 to 5. The patients with relapse occurred 48-158 months after the completion of MDT. The relapse rate of MB patients treated with 24 months of the WHO/MB regimen was observed to be very low after long-term follow-up.

Recidivas en pacientes de Lepra MB tratados con 24 meses de MDT en el suroeste de China: Un informe breve / No disponible

Este estudio evalúa el índice de recidivas entre los pacientes de lepra tratados con 24 meses de MDT en el suroeste de China. Para ello se llevó a cabo un estudio retrospectivo de recidivas en esta parte del país. Se diseñó un cuestionario muy detallado para recoger los datos sobre las recidivas entre pacientes MB que habían completado 2 años de tratamiento OMS/MB desde 1989 a 2000. Los datos se obtuvieron sobre 2.517 pacientes multibacilares en 27 países del suroeste de China. Entre los 2.517 pacientes MB, 235 pacientes fallecieron o se dieron de baja durante el seguimiento y 2.374 fueron controlados durante más de 3 años después de completar la MDT. La duración total del seguimiento fue de 20,825 personas-año, con una duración media de 8.27 años por paciente. Se identificaron cinco pacientes con recidivas con un índice acumulado de recidivas de 0-21/1.000 personas-año. Los IB iníciales variaron entre 1.8 a 5. Las recidivas se presentaron entre 48-158 mese después de completar la MDT. El índice de recidivas de los pacientes MB tratados con 24 meses con una pauta OMS/MB se consideró muy bajo después de un largo período de seguimiento

This study investigates the relapse rate among multibacillary leprosy patients treated with 24 months of MDT in south west China. A retrospective relapse survey was conducted in the southwest of China. A detailed questionnaire was designed to collect the data on relapse among MB patients who completed 2 years of the WHO/MB regimen, from 1989 to 2000. The data about 2.517 multibacillary leprosy patients in 27 counties in the southwest of China were collected. Among 2.517 MB patients, 235 patients died or were lost to follow-up and 2.374 were followed up for more than 3 years after completion of MDT. The total duration of follow-up was 20,825 person-years, with a mean duration of 8.27 years per patient. Five patients with relapse were identified with an accumulated relapse rate of 0.21-/1.000 person-years. Their initial Bls ranged from 1.8 to 5. The patients with relapse occurred 48-158 months after the completion of MDT. The relapse rate of MB patients treated with 24 months of the WHO/MB regimen was observed to be very low after long-term follow-up

Recidivas en pacientes de Lepra MB tratados con 24 meses de MDT en el suroeste de China: Un informe breve / No disponible

Este estudio evalúa el índice de recidivas entre los pacientes de lepra tratados con 24 meses de MDT en el suroeste de China. Para ello se llevó a cabo un estudio retrospectivo de recidivas en esta parte del país. Se diseñó un cuestionario muy detallado para recoger los datos sobre las recidivas entre pacientes MB que habían completado 2 años de tratamiento OMS/MB desde 1989 a 2000. Los datos se obtuvieron sobre 2.517 pacientes multibacilares en 27 países del suroeste de China. Entre los 2.517 pacientes MB, 235 pacientes fallecieron o se dieron de baja durante el seguimiento y 2.374 fueron controlados durante más de 3 años después de completar la MDT. La duración total del seguimiento fue de 20,825 personas-año, con una duración media de 8.27 años por paciente. Se identificaron cinco pacientes con recidivas con un índice acumulado de recidivas de 0-21/1.000 personas-año. Los IB iníciales variaron entre 1.8 a 5. Las recidivas se presentaron entre 48-158 mese después de completar la MDT. El índice de recidivas de los pacientes MB tratados con 24 meses con una pauta OMS/MB se consideró muy bajo después de un largo período de seguimiento (AU)

This study investigates the relapse rate among multibacillary leprosy patients treated with 24 months of MDT in south west China. A retrospective relapse survey was conducted in the southwest of China. A detailed questionnaire was designed to collect the data on relapse among MB patients who completed 2 years of the WHO/MB regimen, from 1989 to 2000. The data about 2.517 multibacillary leprosy patients in 27 counties in the southwest of China were collected. Among 2.517 MB patients, 235 patients died or were lost to follow-up and 2.374 were followed up for more than 3 years after completion of MDT. The total duration of follow-up was 20,825 person-years, with a mean duration of 8.27 years per patient. Five patients with relapse were identified with an accumulated relapse rate of 0.21-/1.000 person-years. Their initial Bls ranged from 1.8 to 5. The patients with relapse occurred 48-158 months after the completion of MDT. The relapse rate of MB patients treated with 24 months of the WHO/MB regimen was observed to be very low after long-term follow-up (AU)