Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

StarD7 Knockdown Modulates ABCG2 Expression, Cell Migration, Proliferation, and Differentiation of Human Choriocarcinoma JEG-3 Cells

Background

StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.

Methodology/Principal Findings

Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and ³H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.

Conclusions/Significance

Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.     

Effects of R219K polymorphism of ATP-binding cassette transporter 1 gene on serum lipids ratios induced by a high-carbohydrate and low-fat diet in healthy youth

Background

Diets are the important players in regulating plasma lipid profiles. And the R219K polymorphism at the gene of ATP-binding cassette transporter 1(ABCA1) was reported to be associated with the profiles. However, no efforts have been made to investigate the changes of lipid profiles after a high-carbohydrate and low-fat diet in different subjects with different genotypes of this polymorphism. This study was to evaluate the effects of ABCA1 R219K polymorphism on serum lipid and apolipoprotein (apo) ratios induced by a high-carbohydrate/low-fat (high-CHO) diet. After a washout diet of 54.1% carbohydrate for 7 days, 56 healthy young subjects (22.89 ± 1.80 years old) were given a high-CHO diet of 70.1% carbohydrate for 6 days. Height, weight, waist circumference, hip circumference, glucose (Glu), triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apoA-1 and apoB-100 were measured on the 1^st, 8^th and 14^th days of this study. Body mass index (BMI), waist-to-hip ratios (WHR), log(TG/HDL-C), TC/HDL-C, LDL-C/HDL-C and apoA-1/apoB-100 were calculated. ABCA1 R219K was analyzed by a PCR-RFLP method.

Results

The results indicate that the male subjects of all the genotypes had higher WHR than their female counterparts on the 1^st, 8^th and 14^th days of this study. The male K carriers had higher log(TG/HDL-C) and TC/HDL-C than the female carriers on the 1^st and 14^th days, and higher LDL-C/HDL-C on the 14^th day. When compared with that on the 8^th day, TC/HDL-C was decreased regardless of the genotypes and genders on the 14^th day. Log(TG/HDL-C) was increased in the males with the RR genotype and the female K carriers. Lowered BMI, Glu and LDL-C/HDL-C were found in the male K carriers, but only lowered BMI in the female K carriers and only lowered LDL-C/HDL-C in the females with the RR genotype.

Conclusions

These results suggest that ABCA1 R219K polymorphism is associated differently in males and females with elevated log(TG/HDL-C) and decreased LDL-C/HDL-C induced by the high-CHO diet.     

ABC transporters B1, C1 and G2 differentially regulate neuroregeneration in mice

Background

ATP-binding cassette (ABC) transporters are essential regulators of organismic homeostasis, and are particularly important in protecting the body from potentially harmful exogenous substances. Recently, an increasing number of in vitro observations have indicated a functional role of ABC transporters in the differentiation and maintenance of stem cells. Therefore, we sought to determine brain-related phenotypic changes in animals lacking the expression of distinct ABC transporters (ABCB1, ABCG2 or ABCC1).

Methodology and Principal Findings

Analyzing adult neurogenesis in ABC transporter-deficient animals in vivo and neuronal stem/progenitor cells in vitro resulted in complex findings. In vivo, the differentiation of neuronal progenitors was hindered in ABC transporter-deficient mice (ABCB1^0/0) as evidenced by lowered numbers of doublecortin⁺ (−36%) and calretinin⁺ (−37%) cells. In vitro, we confirmed that this finding is not connected to the functional loss of single neural stem/progenitor cells (NSPCs). Furthermore, assessment of activity, exploratory behavior, and anxiety levels revealed behavioral alterations in ABCB1^0/0 and ABCC1^0/0 mice, whereas ABCG2^0/0 mice were mostly unaffected.

Conclusion and Significance

Our data show that single ABC transporter-deficiency does not necessarily impair neuronal progenitor homeostasis on the single NSPC level, as suggested by previous studies. However, loss of distinct ABC transporters impacts global brain homeostasis with far ranging consequences, leading to impaired neurogenic functions in vivo and even to distinct behavioral phenotypes. In addition to the known role of ABC transporters in proteopathies such as Parkinson''s disease and Alzheimer''s disease, our data highlight the importance of understanding the general function of ABC transporters for the brain''s homeostasis and the regeneration potential.     

A Novel Two Mode-Acting Inhibitor of ABCG2-Mediated Multidrug Transport and Resistance in Cancer Chemotherapy

Background

Multidrug resistance (MDR) is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.

Methods/Preliminary Findings

Using rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl)-2-[(6-{[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-yl)sulfanyl]acetamide), that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.

Conclusion/Significance

Unlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.     

Evidence of a louse-borne outbreak involving typhus in Douai, 1710-1712 during the war of Spanish succession

Background

The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR.

Methods

We identified a mass grave in Douai, France dating from the early XVIII^th century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples.

Results and Discussion

In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XX^th century.

Conclusion

Louse-borne outbreaks were raging during the XVIII^th century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America.     

Compositional development of Bifidobacterium and Lactobacillus microbiota is linked with crying and fussing in early infancy

Objectives

Our aim was to establish whether there is an interconnection between the compositional development of the gut microbiota and the amount of fussing and crying in early infancy.

Methods

Behavioral patterns of 89 infants during the 7^th and 12^th week of life were recorded in parental diaries. Total distress was defined as the sum of daily amounts of crying and fussing. Infants'' gut microbiota profiles were investigated by several molecular assays during the first six months of life.

Results

The median (range) duration of total distress among the infants was 106 (0–478) minutes a day during the 7^th and 58 (0–448) minutes a day during the 12^th week. The proportion of Bifidobacterium counts to total bacterial counts was inversely associated with the amount of crying and fussing during the first 3 months of life (p = 0.03), although the number of Bifidobacterium breve was positively associated with total distress (p = 0.02). The frequency of Lactobacillus spp. at the age of 3 weeks was inversely associated with total infant distress during the 7^th week of life (p = 0.02).

Conclusions

Bifidobacterium and Lactobacillus appear to protect against crying and fussing. Identification of specific strains with optimal protective properties would benefit at-risk infants.     

Influence of the ABCG2 gout risk 141?K allele on urate metabolism during a fructose challenge

Introduction

Both genetic variation in ATP-binding cassette sub-family G member 2 (ABCG2) and intake of fructose-containing beverages are major risk factors for hyperuricemia and gout. This study aimed to test the hypothesis that the ABCG2 gout risk allele 141 K promotes the hyperuricaemic response to fructose loading.

Methods

Healthy volunteers (n = 74) provided serum and urine samples immediately before and 30, 60, 120 and 180 minutes after ingesting a 64 g fructose solution. Data were analyzed based on the presence or absence of the ABCG2 141 K gout risk allele.

Results

The 141 K risk allele was present in 23 participants (31%). Overall, serum urate (SU) concentrations during the fructose load were similar in those with and without the 141 K allele (P_SNP = 0.15). However, the 141 K allele was associated with a smaller increase in SU following fructose intake (P_SNP <0.0001). Those with the 141 K allele also had a smaller increase in serum glucose following the fructose load (P_SNP = 0.002). Higher fractional excretion of uric acid (FEUA) at baseline and throughout the fructose load was observed in those with the 141 K risk allele (P_SNP <0.0001). However, the change in FEUA in response to fructose was not different in those with and without the 141 K risk allele (P_SNP = 0.39). The 141 K allele effects on serum urate and glucose were more pronounced in Polynesian participants and in those with a body mass index ≥25 kg/m².

Conclusions

In contrast to the predicted responses for a hyperuricemia/gout risk allele, the 141 K allele is associated with smaller increases in SU and higher FEUA following a fructose load. The results suggest that ABCG2 interacts with extra-renal metabolic pathways in a complex manner to regulate SU and gout risk.

Clinical Trials Registration

The study was registered by the Australian Clinical Trials Registry (ACTRN12610001036000).     

Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

Objective

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.

Methods

Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.

Results

Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.

Conclusion

Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.     

Fluorescence resonance energy transfer (FRET) analysis demonstrates dimer/oligomer formation of the human breast cancer resistance protein (BCRP/ABCG2) in intact cells

The human breast cancer resistance protein (BCRP/ABCG2) is a half ATP-binding cassette (ABC) efflux transporter that plays an important role in drug resistance and disposition. Although BCRP is believed to function as a homodimer or homooligomer, this has not been demonstrated in vivo in intact cells. Therefore, in the present study, we investigated dimer/oligmer formation of BCRP in intact cells. Wild-type BCRP and the mutant C603A were attached to cyan or yellow fluorescence protein and expressed in HEK293 cells by transient transfection. Protein levels, cell surface expression, and efflux activities of wild-type and mutant BCRP were determined by immunoblotting, 5D3 antibody binding, and flow cytometric efflux assay, respectively. Dimer/oligomer formation of BCRP in intact cells was analyzed using fluorescence resonance energy transfer (FRET) microscopy. Wild-type BCRP and C603A were expressed in HEK293 cells at comparable levels. C603A was predominantly expressed in the plasma membrane as was wild-type protein. Furthermore, C603A retained the same mitoxantrone efflux activity and the ability of dimer/oligmer formation as wild-type BCRP. Finally, cross-linking experiments yielded data consistent with the FRET analysis. In conclusion, we have, for the first time, demonstrated that BCRP can form a dimer/oligomer in vivo in intact cells using the FRET technique. We have also shown that Cys⁶⁰³ alone does not seem to be essential for dimer/oligomer formation of BCRP.     

Wired to be social: the ontogeny of human interaction

Background

Newborns come into the world wired to socially interact. Is a propensity to socially oriented action already present before birth? Twin pregnancies provide a unique opportunity to investigate the social pre-wiring hypothesis. Although various types of inter-twins contact have been demonstrated starting from the 11^th week of gestation, no study has so far investigated the critical question whether intra-pair contact is the result of motor planning rather then the accidental outcome of spatial proximity.

Methodology/Principal Findings

Kinematic profiles of movements in five pairs of twin foetuses were studied by using four-dimensional ultrasonography during two separate recording sessions carried out at the 14^th and 18^th week of gestation. We demonstrate that by the 14th week of gestation twin foetuses do not only display movements directed towards the uterine wall and self-directed movements, but also movements specifically aimed at the co-twin, the proportion of which increases between the 14^th and 18^th gestational week. Kinematic analysis revealed that movement duration was longer and deceleration time was prolonged for other-directed movements compared to movements directed towards the uterine wall. Similar kinematic profiles were observed for movements directed towards the co-twin and self-directed movements aimed at the eye-region, i.e. the most delicate region of the body.

Conclusions/Significance

We conclude that performance of movements towards the co-twin is not accidental: already starting from the 14th week of gestation twin foetuses execute movements specifically aimed at the co-twin.     

Treatment of Colorectal Cancer Using a Combination of Liposomal Irinotecan (Irinophore C?) and 5-Fluorouracil

Purpose

To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer.

Experimental Design

The effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU.

Results

The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg).

Conclusions

Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination.     

Effect of Doxorubicin/Pluronic SP1049C on Tumorigenicity,Aggressiveness, DNA Methylation and Stem Cell Markers in Murine Leukemia

Purpose

Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo.

Experimental Design

P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers.

Results

SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-β-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133⁺ P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133⁻ cells.

Conclusions

SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.     

Modeling the Cost-Effectiveness of the Integrated Disease Surveillance and Response (IDSR) System: Meningitis in Burkina Faso

Background

Effective surveillance for infectious diseases is an essential component of public health. There are few studies estimating the cost-effectiveness of starting or improving disease surveillance. We present a cost-effectiveness analysis the Integrated Disease Surveillance and Response (IDSR) strategy in Africa.

Methodology/Principal Findings

To assess the impact of the IDSR in Africa, we used pre- and post- IDSR meningococcal meningitis surveillance data from Burkina Faso (1996–2002 and 2003–2007). IDSR implementation was correlated with a median reduction of 2 weeks to peak of outbreaks (25^th percentile 1 week; 75^th percentile 4 weeks). IDSR was also correlated with a reduction of 43 meningitis cases per 100,000 (25^th–40: 75^th-129). Assuming the correlations between reductions in time to peak of outbreaks and cases are related, the cost-effectiveness of IDSR was $23 per case averted (25^th-$30; 75^th - cost saving), and $98 per meningitis-related death averted (25^th-$140: 75^th – cost saving).

Conclusions/Significance

We cannot absolutely claim that the measured differences were due to IDSR. We believe, however, that it is reasonable to claim that IDSR can improve the cost-effectiveness of public health surveillance.     

Relationship between the Autoantibody and Expression of β3-Adrenoceptor in Lung and Heart

Background

Evidences suggest that β₃ -adrenoceptor (β₃-AR) plays an important role in heart failure (HF), although no data is reported indicating how these effects may change with the increasing age. Pulmonary congestion and edema are the major life-threatening complications associated with HF. The purpose of this study is to explore the relationship between the anti-β₃-AR autoantibody and the expression of β₃-AR in the lungs and heart for both aged patients and rats with HF.

Methods

Synthetic β₃-AR peptides served as the target antigens in ELISA were used to screen the anti-β₃-AR autoantibody in aged patients and rats. Two aged rat models were constructed based on aortic banding and sham-operation. The expression of β₃-AR mRNA and protein in the lung and heart was measured in intervention and non-intervention groups by Western blot analysis at the baseline, 5^th, 7^th, 9^th and 11^th week, respectively.

Results

The frequency and titer of anti-β₃-AR autoantibody in aged patients and rats with HF were higher than those in the control group (p<0.05). The expression of β₃-AR mRNA and protein in pulmonary tissues decreased continually from the 7^th week (p<0.05), followed by HF observed during the 9^th week. The expression of β₃-AR in myocardial tissues continued to increase after the 9^th week (p<0.05), and the expression of both β₃-AR mRNA and protein in the BRL group [HF group with BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) (a β₃-AR agonist) injection] was positively correlated with BRL37344 when compared with non-BRL group (HF group without BRL37344 injection) (p<0.05).

Conclusion

Anti-β₃-AR autoantibody was detected in aged patients and rats with HF. The expression of β₃-AR mRNA and protein in pulmonary tissues decreased continually, and began earlier than in the heart, but its expression in myocardial tissues increased continually and could be further promoted by β₃-AR agonist.     

Quantifying Recent Ecological Changes in Remote Lakes of North America and Greenland Using Sediment Diatom Assemblages

Background

Although arctic lakes have responded sensitively to 20^th-century climate change, it remains uncertain how these ecological transformations compare with alpine and montane-boreal counterparts over the same interval. Furthermore, it is unclear to what degree other forcings, including atmospheric deposition of anthropogenic reactive nitrogen (Nr), have participated in recent regime shifts. Diatom-based paleolimnological syntheses offer an effective tool for retrospective assessments of past and ongoing changes in remote lake ecosystems.

Methodology/Principal Findings

We synthesized 52 dated sediment diatom records from lakes in western North America and west Greenland, spanning broad latitudinal and altitudinal gradients, and representing alpine (n = 15), arctic (n = 20), and forested boreal-montane (n = 17) ecosystems. Diatom compositional turnover (β-diversity) during the 20^th century was estimated using Detrended Canonical Correspondence Analysis (DCCA) for each site and compared, for cores with sufficiently robust chronologies, to both the 19^th century and the prior ∼250 years (Little Ice Age). For both arctic and alpine lakes, β-diversity during the 20^th century is significantly greater than the previous 350 years, and increases with both latitude and altitude. Because no correlation is apparent between 20^th-century diatom β-diversity and any single physical or limnological parameter (including lake and catchment area, maximum depth, pH, conductivity, [NO₃ ⁻], modeled Nr deposition, ambient summer and winter air temperatures, and modeled temperature trends 1948–2008), we used Principal Components Analysis (PCA) to summarize the amplitude of recent changes in relationship to lake pH, lake:catchment area ratio, modeled Nr deposition, and recent temperature trends.

Conclusions/Significance

The ecological responses of remote lakes to post-industrial environmental changes are complex. However, two regions reveal concentrations of sites with elevated 20^th-century diatom β-diversity: the Arctic where temperatures are increasing most rapidly, and mid-latitude alpine lakes impacted by high Nr deposition rates. We predict that remote lakes will continue to shift towards new ecological states in the Anthropocene, particularly in regions where these two forcings begin to intersect geographically.     

Systematic Monitoring of Male Circumcision Scale-Up in Nyanza,Kenya: Exploratory Factor Analysis of Service Quality Instrument and Performance Ranking

Background

Considerable conceptual and operational complexities related to service quality measurements and variability in delivery contexts of scaled-up medical male circumcision, pose real challenges to monitoring implementation of quality and safety. Clarifying latent factors of the quality instruments can enhance contextual applicability and the likelihood that observed service outcomes are appropriately assessed.

Objective

To explore factors underlying SYMMACS service quality assessment tool (adopted from the WHO VMMC quality toolkit) and; determine service quality performance using composite quality index derived from the latent factors.

Study design

Using a comparative process evaluation of Voluntary Medical Male Circumcision Scale-Up in Kenya site level data was collected among health facilities providing VMMC over two years. Systematic Monitoring of the Medical Male Circumcision Scale-Up quality instrument was used to assess availability of guidelines, supplies and equipment, infection control, and continuity of care services. Exploratory factor analysis was performed to clarify quality structure.

Results

Fifty four items and 246 responses were analyzed. Based on Eigenvalue >1.00 cut-off, factors 1, 2 & 3 were retained each respectively having eigenvalues of 5.78; 4.29; 2.99. These cumulatively accounted for 29.1% of the total variance (12.9%; 9.5%; 6.7%) with final communality estimates being 13.06. Using a cut-off factor loading value of ≥0.4, fifteen items loading on factor 1, five on factor 2 and one on factor 3 were retained. Factor 1closely relates to preparedness to deliver safe male circumcisions while factor two depicts skilled task performance and compliance with protocols. Of the 28 facilities, 32% attained between 90^th and 95^th percentile (excellent); 45% between 50^th and 75^th percentiles (average) and 14.3% below 25^th percentile (poor).

Conclusion

the service quality assessment instrument may be simplified to have nearly 20 items that relate more closely to service outcomes. Ranking of facilities and circumcision procedure using a composite index based on these items indicates that majority performed above average.     

Pulmonary Abnormalities in Mice with Paracoccidioidomycosis: A Sequential Study Comparing High Resolution Computed Tomography and Pathologic Findings

Background

Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters.

Methodology

Male BALB/c mice were intranasally inoculated with 3×10⁶ Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry.

Results

Three different patterns of lesions were evidenced by HRCT and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4^th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (−263±25 vs. −422±10 HU, p<0.001). After the 8^th and 12^th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16^th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology.

Conclusions/Significance

This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.     

Suppression of JAK2/STAT3 Signaling Reduces End-to-End Arterial Anastomosis Induced Cell Proliferation in Common Carotid Arteries of Rats

Background

JAK2/STAT3 pathway was reported to play an essential role in the neointima formation after vascular intima injury. However, little is known regarding this pathway to the whole layer injury after end-to-end arterial anastomosis (AA). Here, we investigated the role of JAK2/STAT3 pathway in common carotid arterial (CCA) anastomosis-induced cell proliferation, phenotypic change of vascular smooth muscle cells (VSMCs) and re-endothelialization.

Methods

CCAs of adult male Wistar rats were resected at 3, 7, 14, and 30 days after end-to-end CCA anastomosis. Activation of JAK2/STAT3 pathway was detected by Western blotting and Immunofluorescence, and expression of proliferating cell nuclear antigen (PCNA) was detected by Q-PCR and Western blotting. Under the treatment with AG490 (a JAK2 inhibitor), protein levels of JAK2, STAT3 and PCNA, morphological changes of artery, phenotypic change of VSMCs, and re-endothelialization were measured by Western blotting, H&E, Q-PCR, and Evans blue staining respectively.

Results

The protein levels of p-JAK2, p-STAT3, and PCNA were up-regulated, peaked on the 7^th day in the vessel wall after AA. AG490 down-regulated the levels of p-JAK2, p-STAT3, and PCNA on the 7^th-day-group, resulting in reduced vessel wall proliferation on the 7^th and 14^th day after AA. Besides, AG490 switched the phenotypic change of VSMCs after AA representing inhibited mRNA levels of synthetic phase markers (osteopoitin and SMemb) and up-regulated contractile phase markers (ASMA, SM2 and SM22α). Furthermore, AG490 did not affect the re-endothelialization process on all indicated time points after AA (the 3^rd, 7^th, 14^th, and 30^th day).

Conclusion

Our study indicated that JAK2/STAT3 signaling pathway played an important role on cell proliferation of the injured vessel wall, and probably a promising target for the exploration of drugs increasing the patency or reducing the vascular narrowness after AA.