Insights into the susceptibility of raspberries to Drosophila suzukii oviposition

The spotted wing drosophila (SWD) causes massive losses in red raspberry (Rubus idaeus L.) cultivation by direct oviposition leading to damages of the soft skin fruits. Knowledge of the fly's host preference could help farmers in managing the pest. We used a laboratory-based oviposition assay for screening the germplasm of Rubus to ascertain whether the spotted wing drosophila prefers certain cultivars to others for oviposition and if preference is based on citric acid and Brix content or firmness. Correlation analyses of evaluated characters with no-choice tests results in 3 years obtained no correlation between citric acid, Brix content and oviposition. Primocane raspberries were lower affected by SWD than floricane raspberries. The Rubus hybrid cultivar “Dorman Red” and the primocane cultivar “Pokusa” showed the lowest oviposition rate compared to the other 58 evaluated genotypes. We found that oviposition correlates to firmness of the investigated cultivars, which strongly indicates that host preference is partly connected to that character in raspberries. The results are discussed regarding the use of Rubus genetic resources in breeding and integrated pest management to control spotted wing drosophila in the field.     

Oviposition and development potential of the spotted‐wing drosophila,Drosophila suzukii (Diptera: Drosophilidae), on uninjured Campbell Early grape

The spotted‐wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), originally distributed across a few Asian countries including South Korea, has invaded North America and Europe but is absent from Australia. In order to export the South Korean grape cultivar Campbell Early to Australia, its potential to serve as an oviposition and development medium for SWD must first be determined. In this study, we determined the oviposition and development potential of SWD on Campbell Early, after elucidating the SWD life cycle and establishing an artificial diet‐based mass‐culturing system. An investigation of the life cycle under five temperature regimes (16, 19, 22, 25 and 28°C) showed that the durations of the egg, larval and adult stages were shortened when temperature was increased from 16, 19, 22, 25 and 28°C, but pupal duration was shortest at 25°C and extended again at 28°C. A test of oviposition and development potential of SWD on Campbell Early grape clusters showed oviposition of 30.8 ± 6.8 eggs per cluster of injured grapes and 157.7 ± 16.2 eggs on a culture dish of artificial diet. However, in a similar experiment using uninjured grape clusters, only a single egg was deposited on the grape skin, which soon dried. In light of these results, newly harvested grapes left at vineyards during daily harvests are unlikely to serve as an oviposition and development medium for SWD, as long as the grapes remain uninjured.     

Contrasting Patterns in Mammal–Bacteria Coevolution: Bartonella and Leptospira in Bats and Rodents

Background

Emerging bacterial zoonoses in bats and rodents remain relatively understudied. We conduct the first comparative host–pathogen coevolutionary analyses of bacterial pathogens in these hosts, using Bartonella spp. and Leptospira spp. as a model.

Methodology/Principal Findings

We used published genetic data for 51 Bartonella genotypes from 24 bat species, 129 Bartonella from 38 rodents, and 26 Leptospira from 20 bats. We generated maximum likelihood and Bayesian phylogenies for hosts and bacteria, and tested for coevoutionary congruence using programs ParaFit, PACO, and Jane. Bartonella spp. and their bat hosts had a significant coevolutionary fit (ParaFitGlobal = 1.9703, P≤0.001; m² global value = 7.3320, P≤0.0001). Bartonella spp. and rodent hosts also indicated strong overall patterns of cospeciation (ParaFitGlobal = 102.4409, P≤0.001; m² global value = 86.532, P≤0.0001). In contrast, we were unable to reject independence of speciation events in Leptospira and bats (ParaFitGlobal = 0.0042, P = 0.84; m² global value = 4.6310, P = 0.5629). Separate analyses of New World and Old World data subsets yielded results congruent with analysis from entire datasets. We also conducted event-based cophylogeny analyses to reconstruct likely evolutionary histories for each group of pathogens and hosts. Leptospira and bats had the greatest number of host switches per parasite (0.731), while Bartonella and rodents had the fewest (0.264).

Conclusions/Significance

In both bat and rodent hosts, Bartonella exhibits significant coevolution with minimal host switching, while Leptospira in bats lacks evolutionary congruence with its host and has high number of host switches. Reasons underlying these variable coevolutionary patterns in host range are likely due to differences in disease-specific transmission and host ecology. Understanding the coevolutionary patterns and frequency of host-switching events between bacterial pathogens and their hosts will allow better prediction of spillover between mammal reservoirs, and ultimately to humans.     

Abiotic and Biotic Factors Associated with Tick Population Dynamics on a Mammalian Host: Ixodes hexagonus Infesting Otters,Lutra lutra

The Eurasian otter, Lutra lutra, hosts several parasites with zoonotic potential. As this semiaquatic mammal has large ranges across terrestrial, freshwater and marine habitats, it has the capacity for wide dispersion of pathogens. Despite this, parasites of otters have received relatively little attention. Here, we examine their ectoparasite load and assess whether this is influenced by abiotic or biotic variables. Climatic phenomena such as the North Atlantic Oscillation (NAO) affect weather conditions in northern Europe. Consequently parasite distributions, particularly species with life stages exposed to the external environment, can be affected. We assessed the extent to which inter-annual variations in large-scale weather patterns (specifically the NAO and Central England (CE) temperatures) and host characteristics influenced tick prevalence and intensity. Ectoparasites consisted of a single species, the nidiculous tick Ixodes hexagonus (prevalence  = 24.3%; mean intensity  = 7.2; range  = 1–122; on n  = 820 otter hosts). The prevalence, but not intensity of infestation, was associated with high CE temperatures, while both prevalence and intensity were associated with positive phases of the NAO. Such associations indicate that I. hexagonus are most abundant when weather conditions are warmer and wetter. Ticks were more prevalent on juvenile than sub-adult or adult otters, which probably reflects the length of time the hosts spend in the holt where these ticks quest. High tick number was associated with poor host condition, so either poor condition hosts are more susceptible to ticks, or tick infestations negatively impact on host condition. Otters are clearly an important and common host for I. hexagonus, which has implications for vector-borne diseases. This work is the first to consider the impacts of long-term weather patterns on I. hexagonus and uses wild-animal cadavers to illustrate the importance of abiotic and biotic pressures impacting parasitic populations.     

Do Parasitic Trematode Cercariae Demonstrate a Preference for Susceptible Host Species?

Many parasites are motile and exhibit behavioural preferences for certain host species. Because hosts can vary in their susceptibility to infections, parasites might benefit from preferentially detecting and infecting the most susceptible host, but this mechanistic hypothesis for host-choice has rarely been tested. We evaluated whether cercariae (larval trematode parasites) prefer the most susceptible host species by simultaneously presenting cercariae with four species of tadpole hosts. Cercariae consistently preferred hosts in the following order: Anaxyrus ( = Bufo) terrestris (southern toad), Hyla squirella (squirrel tree frog), Lithobates ( = Rana) sphenocephala (southern leopard frog), and Osteopilus septentrionalis (Cuban tree frog). These host species varied in susceptibility to cercariae in an order similar to their attractiveness with a correlation that approached significance. Host attractiveness to parasites also varied consistently and significantly among individuals within a host species. If heritable, this individual-level host variation would represent the raw material upon which selection could act, which could promote a Red Queen “arms race” between host cues and parasite detection of those cues. If, in general, motile parasites prefer to infect the most susceptible host species, this phenomenon could explain aggregated distributions of parasites among hosts and contribute to parasite transmission rates and the evolution of virulence. Parasite preferences for hosts belie the common assumption of disease models that parasites seek and infect hosts at random.     

Multi Frequency Phase Fluorimetry (MFPF) for Oxygen Partial Pressure Measurement: Ex Vivo Validation by Polarographic Clark-Type Electrode

Background

Measurement of partial pressure of oxygen (P_O2) at high temporal resolution remains a technological challenge. This study introduces a novel P_O2 sensing technology based on Multi-Frequency Phase Fluorimetry (MFPF). The aim was to validate MFPF against polarographic Clark-type electrode (CTE) P_O2 measurements.

Methodology/Principal Findings

MFPF technology was first investigated in N = 8 anaesthetised pigs at F_IO2 of 0.21, 0.4, 0.6, 0.8 and 1.0. At each F_IO2 level, blood samples were withdrawn and P_O2 was measured in vitro with MFPF using two FOXY-AL300 probes immediately followed by CTE measurement. Secondly, MFPF-P_O2 readings were compared to CTE in an artificial circulatory setup (human packed red blood cells, haematocrit of 30%). The impacts of temperature (20, 30, 40°C) and blood flow (0.8, 1.6, 2.4, 3.2, 4.0 L min⁻¹) on MFPF-P_O2 measurements were assessed. MFPF response time in the gas- and blood-phase was determined. Porcine MFPF-P_O2 ranged from 63 to 749 mmHg; the corresponding CTE samples from 43 to 712 mmHg. Linear regression: CTE = 15.59+1.18*MFPF (R² = 0.93; P<0.0001). Bland Altman analysis: mean_diff 69.2 mmHg, range_diff -50.1/215.6 mmHg, 1.96-SD limits -56.3/194.8 mmHg. In artificial circulatory setup, MFPF-P_O2 ranged from 20 to 567 mmHg and CTE samples from 11 to 575 mmHg. Linear regression: CTE = −8.73+1.05*MFPF (R² = 0.99; P<0.0001). Bland-Altman analysis: mean_diff 6.6 mmHg, range_diff -9.7/20.5 mmHg, 1.96-SD limits -12.7/25.8 mmHg. Differences between MFPF and CTE-P_O2 due to variations of temperature were less than 6 mmHg (range 0–140 mmHg) and less than 35 mmHg (range 140–750 mmHg); differences due to variations in blood flow were less than 15 mmHg (all P-values>0.05). MFPF response-time (monoexponential) was 1.48±0.26 s for the gas-phase and 1.51±0.20 s for the blood-phase.

Conclusions/Significance

MFPF-derived P_O2 readings were reproducible and showed excellent correlation and good agreement with Clark-type electrode-based P_O2 measurements. There was no relevant impact of temperature and blood flow upon MFPF-P_O2 measurements. The response time of the MFPF FOXY-AL300 probe was adequate for real-time sensing in the blood phase.     

Differentiation between Primary Cerebral Lymphoma and Glioblastoma Using the Apparent Diffusion Coefficient: Comparison of Three Different ROI Methods

Objective

Apparent diffusion coefficients (ADC) can help differentiate between central nervous system (CNS) lymphoma and Glioblastoma (GBM). However, overlap between ADCs for GBM and lymphoma have been reported because of various region of interest (ROI) methods. Our aim is to explore ROI method to provide the most reproducible results for differentiation.

Materials and Methods

We studied 25 CNS lymphomas and 62 GBMs with three ROI methods: (1) ROI₁, whole tumor volume; (2) ROI₂, multiple ROIs; and (3) ROI₃, a single ROI. Interobserver variability of two readers for each method was analyzed by intraclass correlation(ICC). ADCs were compared between GBM and lymphoma, using two-sample t-test. The discriminative ability was determined by ROC analysis.

Results

ADCs from ROI₁ showed most reproducible results (ICC >0.9). For ROI₁, ADC_mean for lymphoma showed significantly lower values than GBM (p = 0.03). The optimal cut-off value was 0.98×10⁻³ mm²/s with 85% sensitivity and 90% specificity. For ROI₂, ADC_min for lymphoma was significantly lower than GBM (p = 0.02). The cut-off value was 0.69×10⁻³ mm²/s with 87% sensitivity and 88% specificity.

Conclusion

ADC values were significantly dependent on ROI method. ADCs from the whole tumor volume had the most reproducible results. ADC_mean from the whole tumor volume may aid in differentiating between lymphoma and GBM. However, multi-modal imaging approaches are recommended than ADC alone for differentiation.     

DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure

Altered DNA methylation patterns in CD4⁺ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N_patients = 8, N_controls = 8) and gene expression (N_patients = 9, N_controls = 10) profiles of CD4⁺ T-cells from SAR patients and healthy controls using Illumina''s HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (N_patients = 12, N_controls = 12), but not by gene expression (N_patients = 21, N_controls = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (N_patients = 35) and controls (N_controls = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4⁺ T cells.     

Oral Vaccination of White-Tailed Deer (Odocoileus virginianus) with Mycobacterium bovis Bacillus Calmette-Guerin (BCG)

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer, thus interfering with the intraspecies and interspecies transmission cycles. Thirty-three white-tailed deer were assigned to one of two groups; oral vaccination with 1×10⁸ colony-forming units of M. bovis BCG Danish (n = 17); and non-vaccinated (n = 16). One hundred eleven days after vaccination deer were infected intratonsilarly with 300 colony-forming units of virulent M. bovis. At examination, 150 days after challenge, BCG vaccinated deer had fewer gross and microscopic lesions, fewer tissues from which M. bovis could be isolated, and fewer late stage granulomas with extensive liquefactive necrosis. Fewer lesions, especially those of a highly necrotic nature should decrease the potential for dissemination of M. bovis within the host and transmission to other susceptible hosts.     

A Large Scale Gene-Centric Association Study of Lung Function in Newly-Hired Female Cotton Textile Workers with Endotoxin Exposure

Background

Occupational exposure to endotoxin is associated with decrements in pulmonary function, but how much variation in this association is explained by genetic variants is not well understood.

Objective

We aimed to identify single nucleotide polymorphisms (SNPs) that are associated with the rate of forced expiratory volume in one second (FEV₁) decline by a large scale genetic association study in newly-hired healthy young female cotton textile workers.

Methods

DNA samples were genotyped using the Illumina Human CVD BeadChip. Change rate in FEV₁ was modeled as a function of each SNP genotype in linear regression model with covariate adjustment. We controlled the type 1 error in study-wide level by permutation method. The false discovery rate (FDR) and the family-wise error rate (FWER) were set to be 0.10 and 0.15 respectively.

Results

Two SNPs were found to be significant (P<6.29×10⁻⁵), including rs1910047 (P = 3.07×10⁻⁵, FDR = 0.0778) and rs9469089 (P = 6.19×10⁻⁵, FDR = 0.0967), as well as other eight suggestive (P<5×10⁻⁴) associated SNPs. Gene-gene and gene-environment interactions were also observed, such as rs1910047 and rs1049970 (P = 0.0418, FDR = 0.0895); rs9469089 and age (P = 0.0161, FDR = 0.0264). Genetic risk score analysis showed that the more risk loci the subjects carried, the larger the rate of FEV₁ decline occurred (P _trend = 3.01×10⁻¹⁸). However, the association was different among age subgroups (P = 7.11×10⁻⁶) and endotoxin subgroups (P = 1.08×10⁻²). Functional network analysis illustrates potential biological connections of all interacted genes.

Conclusions

Genetic variants together with environmental factors interact to affect the rate of FEV₁ decline in cotton textile workers.     

Modeling of Environmental Effects in Genome-Wide Association Studies Identifies SLC2A2 and HP as Novel Loci Influencing Serum Cholesterol Levels

Genome-wide association studies (GWAS) have identified 38 larger genetic regions affecting classical blood lipid levels without adjusting for important environmental influences. We modeled diet and physical activity in a GWAS in order to identify novel loci affecting total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels. The Swedish (SE) EUROSPAN cohort (N _SE = 656) was screened for candidate genes and the non-Swedish (NS) EUROSPAN cohorts (N _NS = 3,282) were used for replication. In total, 3 SNPs were associated in the Swedish sample and were replicated in the non-Swedish cohorts. While SNP rs1532624 was a replication of the previously published association between CETP and HDL cholesterol, the other two were novel findings. For the latter SNPs, the p-value for association was substantially improved by inclusion of environmental covariates: SNP rs5400 (p _{SE,unadjusted} = 3.6×10⁻⁵, p _SE,adjusted = 2.2×10⁻⁶, p _{NS,unadjusted} = 0.047) in the SLC2A2 (Glucose transporter type 2) and rs2000999 (p _{SE,unadjusted} = 1.1×10⁻³, p _SE,adjusted = 3.8×10⁻⁴, p _{NS,unadjusted} = 0.035) in the HP gene (Haptoglobin-related protein precursor). Both showed evidence of association with total cholesterol. These results demonstrate that inclusion of important environmental factors in the analysis model can reveal new genetic susceptibility loci.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

A Genome-Wide Association Study Identifies Potential Susceptibility Loci for Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital and heterogeneous disorder characterized by the absence of intramural nervous plexuses along variable lengths of the hindgut. Although RET is a well-established risk factor, a recent genome-wide association study (GWAS) of HSCR has identified NRG1 as an additional susceptibility locus. To discover additional risk loci, we performed a GWAS of 123 sporadic HSCR patients and 432 unaffected controls using a large-scale platform with coverage of over 1 million polymorphic markers. The result was that our study replicated the findings of RET-CSGALNACT2-RASGEF1A genomic region (_rawP = 5.69×10⁻¹⁹ before a Bonferroni correction; _corrP = 4.31×10⁻¹³ after a Bonferroni correction) and NRG1 as susceptibility loci. In addition, this study identified SLC6A20 (_adjP = 2.71×10⁻⁶), RORA (_adjP = 1.26×10⁻⁵), and ABCC9 (_adjP = 1.86×10⁻⁵) as new potential susceptibility loci under adjusting the already known loci on the RET-CSGALNACT2-RASGEF1A and NRG1 regions, although none of the SNPs in these genes passed the Bonferroni correction. In further subgroup analysis, the RET-CSGALNACT2-RASGEF1A genomic region was observed to have different significance levels among subgroups: short-segment (S-HSCR, _corrP = 1.71×10⁻⁵), long-segment (L-HSCR, _corrP = 6.66×10⁻⁴), and total colonic aganglionosis (TCA, _corrP>0.05). This differential pattern in the significance level suggests that other genomic loci or mechanisms may affect the length of aganglionosis in HSCR subgroups during enteric nervous system (ENS) development. Although functional evaluations are needed, our findings might facilitate improved understanding of the mechanisms of HSCR pathogenesis.     

Modelling the Evolutionary Dynamics of Viruses within Their Hosts: A Case Study Using High-Throughput Sequencing

Uncovering how natural selection and genetic drift shape the evolutionary dynamics of virus populations within their hosts can pave the way to a better understanding of virus emergence. Mathematical models already play a leading role in these studies and are intended to predict future emergences. Here, using high-throughput sequencing, we analyzed the within-host population dynamics of four Potato virus Y (PVY) variants differing at most by two substitutions involved in pathogenicity properties. Model selection procedures were used to compare experimental results to six hypotheses regarding competitiveness and intensity of genetic drift experienced by viruses during host plant colonization. Results indicated that the frequencies of variants were well described using Lotka-Volterra models where the competition coefficients β_ij exerted by variant j on variant i are equal to their fitness ratio, r_j/r_i. Statistical inference allowed the estimation of the effect of each mutation on fitness, revealing slight (s = −0.45%) and high (s = −13.2%) fitness costs and a negative epistasis between them. Results also indicated that only 1 to 4 infectious units initiated the population of one apical leaf. The between-host variances of the variant frequencies were described using Dirichlet-multinomial distributions whose scale parameters, closely related to the fixation index F _ST, were shown to vary with time. The genetic differentiation of virus populations among plants increased from 0 to 10 days post-inoculation and then decreased until 35 days. Overall, this study showed that mathematical models can accurately describe both selection and genetic drift processes shaping the evolutionary dynamics of viruses within their hosts.     

Seasonal occurrence of spotted wing drosophila in various small fruits and berries in Pennsylvania and Maryland

Spotted wing drosophila (SWD) (Drosophila suzukii), a major invasive pest of small fruit crops, was first found in Pennsylvania and Maryland during the 2011 crop season, and since then, it has been established throughout the fruit growing regions of both states. A season‐long field study was conducted to find out the seasonal occurrence of SWD in several fruit crops (e.g. blueberry, tart and sweet cherry, floricane‐fruiting summer red raspberry, blackberry, primocane‐fruiting fall raspberries and table grapes) in Pennsylvania and Maryland in 2014. This is the first study determining seasonal occurrence of SWD using a standard commercial lure (Pherocon^® SWD Dual‐Lure^?)‐baited traps in this region. In both states, SWD adults were not captured prior to the month of July, and populations of SWD were found to build up in fruit crops only from mid‐July onwards. This indicates early season fruit crops or varieties are not at risk from SWD fruit injury in these two states. Such early fruit crops, for instance strawberry, sweet and tart cherry, are generally harvested before SWD populations build up in this region. In this context, implications of SWD population in various small fruit crops grown in this region and the utility of SWD Dual‐Lure ^? in season‐long monitoring of SWD population are discussed.     

Increased Circulating Th17 Cells after Transarterial Chemoembolization Correlate with Improved Survival in Stage III Hepatocellular Carcinoma: A Prospective Study

Transarterial chemoembolization (TACE) has therapeutic effects in patients with unresectable hepatocellular carcinoma (HCC), but its impact on the cellular immune response during disease progression is largely unknown. Here we conducted a prospective study to evaluate the effect of TACE on immune status and to identify prognostic immune markers governing treatment success. In this study, 51 stage III HCC patients, 28 stage I HCC patients (TNM classification) and 20 healthy donors were enrolled. Flow cytometry and cytometric bead array were used to evaluate the circulating immune cell subsets, including CD4⁺ T cells (Th1, Th17 and Treg cells), CD8⁺ T cells, NK cells, and NKT cells, and plasma cytokines before TACE and 30 days after TACE. Interestingly, among those immune parameters, the frequency of circulating Th17 cells was higher in stage III HCC patients than in stage I HCC patients (P = 0.015) and healthy donors (P<0.001). Moreover, an increased frequency of circulating Th17 cells was observed 30 days after TACE (Th17_D30) compared with the baseline level (P = 0.036). Kaplan-Meier analysis demonstrated that Th17_D30 was positively associated with overall survival (OS; P = 0.007) and time to progression (TTP; P = 0.009). Multivariate Cox analysis revealed that Th17_D30 was an independent prognostic factor for OS (HR = 0.317, P = 0.032) and TTP (HR = 0.304, P = 0.010). These results provide a potential prognostic marker for stage III HCC patients undergoing TACE and may be useful for identifying patients who can benefit from adjuvant immunotherapies.     

Strain Differences in Fitness of Escherichia coli O157:H7 to Resist Protozoan Predation and Survival in Soil

Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C⁺) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C⁻ cells remained after 7 days. The C⁺ phenotype correlated negatively with D-values of EcO157 exposed to soil (r _s = −0.683; P = 0.036), Vorticella (r _s = −0.465; P = 0.05) or Colpoda (r _s = −0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C⁺ phenotype (Vorticella, r _s = 0.730, P = 0.0004; Colpoda, r _s = 0.625, P = 0.006) suggesting a preference for consumption of C⁺ cells, although they grew on C⁻ strains also. We speculate that the C⁻ phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.