Site-selective sulfur anchoring produces sintering-resistant intermetallic ORR electrocatalysts for membrane electrode assemblies.

Intermetallic compounds are emerging as promising oxygen reduction reaction (ORR) catalysts for fuel cells due to their typically higher activity and durability compared to disordered alloys. However, the preparation of intermetallic catalysts often requires high-temperature annealing, which unfortunately leads to adverse sintering of the metal nanoparticles. Herein, we develop a scalable site-selective sulfur anchoring strategy that effectively suppresses alloy sintering, ensuring the formation of efficient intermetallic electrocatalysts with small sizes and high ordering degrees. The alloy-support interactions are precisely modulated by selectively modifying the alloy-support interfaces with oxidized sulfur species, thus simultaneously blocking both the nanoparticle migration and Oswald ripening pathways for sintering. Using this strategy, sub-5 nm PtCo intermetallic electrocatalysts enclosed by two atomic layers of Pt shells have been successfully prepared even at a metal loading higher than 30 wt%. The intermetallic catalysts exhibit excellent ORR performances in both rotating disk electrode and membrane electrode assembly conditions with a mass activity of 1.28 A mgPt-1 at 0.9 V (vs. RHE) and a power density of 1.0 W cm-2 at a current density of 1.5 A cm-2. The improved performances result from the enhanced Pt-Co electronic interactions and compressive surface strain generated by the highly ordering structure, while the atomic Pt shells prevent the dissolution of Co under highly acidic conditions. This work provides new insights to inhibit the sintering of nanoalloys and would promote the scalable synthesis and applications of platinum-based intermetallic catalysts.

Sintilimab-related diabetes mellitus and psoriasis: A case report and literature review.

RATIONALE: With the popularity of ICIs in different oncology treatments, immune-related adverse events have raised concerns, mostly occurring in skin and endocrine gland injury. This disease involves different organ systems and presents with a variety of clinical manifestations. Most patients with immune checkpoint inhibitor-induced type 1 diabetes are reported to have no combination of autoimmune disease. We report a case of Sintilimab-related diabetes mellitus and psoriasis. PATIENT CONCERNS: We report a case of a 65-year-old female with Sintilimab-related diabetes mellitus and psoriasis. DIAGNOSIS: The patient treated with anti-programmed cell death protein 1 (Sintilimab) for 4 cycles. The patient presented with inexplicable bouts of nausea and vomiting, accompanied by chest discomfort and a feeling of breathlessness, prompting their admission to the local hospital. The initial assessment upon admission revealed an abrupt elevation in blood glucose levels, alongside normal ketone levels, lactic acidosis, and hyperuricemia. A comprehensive regimen was provided to regulate glucose levels and address the symptoms, resulting in notable improvement and subsequent discharge. Regrettably, the patient's personal decision to discontinue medication for a single day led to the emergence of acute ketoacidosis, coupled with a recurrence of psoriasis vulgaris. Consequently, readmission became necessary. Based on the patient's medical history and diabetes antibody testing, the diagnosis of immune checkpoint inhibitor induced diabetes mellitus has been confidently established. INTERVENTIONS: The patient ceased treatment with Sintilimab and was initiated on insulin therapy for glycemic control, alongside symptomatic management for psoriasis. Upon stabilization of the condition, long-term administration of exogenous insulin was implemented as a substitute treatment. OUTCOME: Outside of the hospital, insulin therapy effectively maintained stable blood glucose levels, and there were no further episodes of psoriasis flare-ups. LESSON: The clinical manifestations of immune checkpoint inhibitor induced diabetes mellitus are variable, and in this case the patient presented with unique primary symptoms. Therefore, it is crucial to accumulate relevant cases, understand the different clinical presentations and identify the underlying mechanisms of the disease. This will provide further evidence for early therapeutic intervention in similar patients in the future.

Engineering Cf /ZrB2 -SiC-Y2 O3 for Thermal Structures of Hypersonic Vehicles with Excellent Long-Term Ultrahigh Temperature Ablation Resistance.

Ultrahigh temperature ceramic matrix composites (UHTCMCs) are critical for the development of high Mach reusable hypersonic vehicles. Although various materials are utilized as the thermal components of hypersonic vehicles, it is still challenging to meet the ultrahigh temperature ablation-resistant and reusability. Herein, the Y2 O3 reinforced Cf /ZrB2 -SiC composites are designed, which demonstrates near-zero damage under long-term ablation at temperatures up to 2500 °C for ten cycles. Notably, the linear ablation rate of the composites (0.33 µm s-1 ) is over 24 times better than that of the conventional Cf /C-ZrC at 2500 °C (8.0 µm s-1 ). Moreover, the long-term multi-cycle ablation mechanisms of the composites are investigated with the assistance of DFT calculations. Especially, the size effect and the content of the Zr-based crystals in the oxide layer fundamentally affect the stability of the oxide layer and the ablation properties. The ideal component and structure of the oxide layer for multi-cycle ablation condition are put forward, which can be obtained by controlling the Y2 O3 /ZrB2 mole ratio and establishing Y-Si-O - t-Zr0.9 Y0.1 O1.95 core-shell nano structure. This work proposes a new strategy for improving the long-term multi-cycle ablation resistance of UHTCMCs.

Coexistence of Superhardness and Metal-Like Electrical Conductivity in High-Entropy Dodecaboride Composite with Atomic-Scale Interlocks.

High electrical conductivity and super high hardness are two sought-after material properties, but both are contradictory because the effective suppression of dislocation movement generally increases the scattering of conducting electrons. Here we synthesized a high-entropy dodecaboride composite (HEDC) with a large number of atomic-scale interlocking layers. It shows a Vickers hardness of 51.2 ± 3.6 GPa under an applied load of 0.49 N and an electrical resistivity of 44.5 µΩ·cm at room temperature. Such HEDC achieves superhardness by inheriting the high intrinsic hardness of its constituent phases and restricting the dislocation motion to further enhance the extrinsic hardness through forming numerous atom-scale interlocks between different slip systems. Moreover, the HEDC maintains the excellent electrical conductivity of the constituent borides, and the competition between two correlating structures produces the special kind of coherent boundary that minimizes the scattering of conducting electrons and does not largely deteriorate the electrical conductivity.

Destroying pathogen-tumor symbionts synergizing with catalytic therapy of colorectal cancer by biomimetic protein-supported single-atom nanozyme.

The crucial role of intratumoral bacteria in the progression of cancer has been gradually recognized with the development of sequencing technology. Several intratumoral bacteria which have been identified as pathogens of cancer that induce progression, metastasis, and poor outcome of cancer, while tumor vascular networks and immunosuppressive microenvironment provide shelters for pathogens localization. Thus, the mutually-beneficial interplay between pathogens and tumors, named "pathogen-tumor symbionts", is probably a potential therapeutic site for tumor treatment. Herein, we proposed a destroying pathogen-tumor symbionts strategy that kills intratumoral pathogens, F. nucleatum, to break the symbiont and synergize to kill colorectal cancer (CRC) cells. This strategy was achieved by a groundbreaking protein-supported copper single-atom nanozyme (BSA-Cu SAN) which was inspired by the structures of native enzymes that are based on protein, with metal elements as the active center. BSA-Cu SAN can exert catalytic therapy by generating reactive oxygen species (ROS) and depleting GSH. The in vitro and in vivo experiments demonstrate that BSA-Cu SAN passively targets tumor sites and efficiently scavenges F. nucleatum in situ to destroy pathogen-tumor symbionts. As a result, ROS resistance of CRC through elevated autophagy mediated by F. nucleatum was relieved, contributing to apoptosis of cancer cells induced by intracellular redox imbalance generated by BSA-Cu SAN. Particularly, BSA-Cu SAN experiences renal clearance, avoiding long-term systemic toxicity. This work provides a feasible paradigm for destroying pathogen-tumor symbionts to block intratumoral pathogens interplay with CRC for antitumor therapy and an optimized trail for the SAN catalytic therapy by the clearable protein-supported SAN.

Quantifying Electrochemical Driving Force for Exsolution in Perovskite Oxides by Designing Graded Oxygen Chemical Potential.

Metal nanoparticles exsolved and anchored at the parent perovskite oxide surfaces can greatly enhance the activity and antisintering stability for high-temperature (electro-) chemical catalytic reactions. While exsolution of nanoparticles triggered by using conventional high-temperature thermal reduction suffers from slow kinetics, using an electrochemical driving force can promote the exsolution rate. However, a quantitative correlation between the applied electrochemical driving force and the spatial density of exsolved nanoparticles remains unknown. In this work, we use a specially designed electrochemical device to induce a spatially graded voltage in a La0.43Ca0.37Ti0.94Ni0.06O3-δ electrode, in order to systematically investigate the effect of electrochemical switching on exsolution. With increasing driving force, which leads to decreasing oxygen chemical potential, the density of nanoparticles was observed to increase dramatically, while the average particle size remained roughly constant. We further identified oxygen vacancy pairs or clusters as the preferential nucleation sites for exsolution. Our work provided a high-throughput platform for the systematic study of exsolution of perovskite oxides targeted for fuel electrode materials with improved electrocatalytic performance and stability.

FORT-1: Phase II/III Study of Rogaratinib Versus Chemotherapy in Patients With Locally Advanced or Metastatic Urothelial Carcinoma Selected Based on FGFR1/3 mRNA Expression.

PURPOSE: Rogaratinib, an oral pan-fibroblast growth factor receptor (FGFR1-4) inhibitor, showed promising phase I efficacy and safety in patients with advanced urothelial carcinoma (UC) with FGFR1-3 mRNA overexpression. We assessed rogaratinib efficacy and safety versus chemotherapy in patients with FGFR mRNA-positive advanced/metastatic UC previously treated with platinum chemotherapy. METHODS: FORT-1 (ClinicalTrials.gov identifier: NCT03410693) was a phase II/III, randomized, open-label trial. Patients with FGFR1/3 mRNA-positive locally advanced or metastatic UC with ≥ 1 prior platinum-containing regimen were randomly assigned (1:1) to rogaratinib (800 mg orally twice daily, 3-week cycles; n = 87) or chemotherapy (docetaxel 75 mg/m2, paclitaxel 175 mg/m2, or vinflunine 320 mg/m2 intravenously once every 3 weeks; n = 88). The primary end point was overall survival, with objective response rate (ORR) analysis planned following phase II accrual. Because of comparable efficacy between treatments, enrollment was stopped before progression to phase III; a full interim analysis of phase II was completed. RESULTS: ORRs were 20.7% (rogaratinib, 18/87; 95% CI, 12.7 to 30.7) and 19.3% (chemotherapy, 17/88; 95% CI, 11.7 to 29.1). Median overall survival was 8.3 months (95% CI, 6.5 to not estimable) and 9.8 months (95% CI, 6.8 to not estimable; hazard ratio, 1.11; 95% CI, 0.71 to 1.72; P = .67). Grade 3/4 events occurred in 37 (43.0%)/4 (4.7%) patients and 32 (39.0%)/15 (18.3%), respectively. No rogaratinib-related deaths occurred. Exploratory analysis of patients with FGFR3 DNA alterations showed ORRs of 52.4% (11/21; 95% CI, 29.8 to 74.3) for rogaratinib and 26.7% (4/15; 95% CI, 7.8 to 55.1) for chemotherapy. CONCLUSION: To our knowledge, these are the first data to compare FGFR-directed therapy with chemotherapy in patients with FGFR-altered UC, showing comparable efficacy and manageable safety. Exploratory testing suggested FGFR3 DNA alterations in association with FGFR1/3 mRNA overexpression may be better predictors of rogaratinib response.

Novel BaTiO3-Based, Ag/Pd-Compatible Lead-Free Relaxors with Superior Energy Storage Performance.

Ceramic dielectrics are reported with superior energy storage performance for applications, such as power electronics in electrical vehicles. A recoverable energy density (Wrec) of ∼4.55 J cm-3 with η ∼ 90% is achieved in lead-free relaxor BaTiO3-0.06Bi2/3(Mg1/3Nb2/3)O3 ceramics at ∼520 kV cm-1. These ceramics may be co-fired with Ag/Pd, which constitutes a major step forward toward their potential use in the fabrication of commercial multilayer ceramic capacitors. Compared to stoichiometric Bi(Mg2/3Nb1/3)O3-doped BaTiO3 (BT), A-site deficient Bi2/3(Mg1/3Nb2/3)O3 reduces the electrical heterogeneity of BT. Bulk conductivity differs from the grain boundary only by 1 order of magnitude which, coupled with a smaller volume fraction of conducting cores due to enhanced diffusion of the dopant via A-site vacancies in the A-site sublattice, results in higher breakdown strength under an electric field. This strategy can be employed to develop new dielectrics with improved energy storage performance.

Bioinspired Copper Single-Atom Catalysts for Tumor Parallel Catalytic Therapy.

The oxidation of intracellular biomolecules by reactive oxygen species (ROS) forms the basis for ROS-based tumor therapy. However, the current therapeutic modalities cannot catalyze H2 O2 and O2 concurrently for ROS generation, thereby leading to unsatisfactory therapeutic efficacy. Herein, it is reported a bioinspired hollow N-doped carbon sphere doped with a single-atom copper species (Cu-HNCS) that can directly catalyze the decomposition of both oxygen and hydrogen peroxide to ROS, namely superoxide ion (O2 •- ) and the hydroxyl radical (•OH), respectively, in an acidic tumor microenvironment for the oxidation of intracellular biomolecules without external energy input, thus resulting in an enhanced tumor growth inhibitory effect. Notably, the Fenton reaction turnover frequency of Cu species in Cu-HNCS is ≈5000 times higher than that of Fe in commercial Fe3 O4 nanoparticles. Experimental results and density functional theory calculations reveal that the high catalytic activity of Cu-HNCS originates from the single-atom copper, and the calculation predicts a next-generation Fenton catalyst. This work provides an effective paradigm of tumor parallel catalytic therapy for considerably enhanced therapeutic efficacy.

Targeted Therapeutic-Immunomodulatory Nanoplatform Based on Noncrystalline Selenium.

Developing versatile nanomaterials has offered a myriad of opportunities to surmount cancer. In particular, the combination of therapy and immunomodulatory effect to further enhance immune response provides a new idea for effective tumor treatment. Herein, for the first time, an in situ growth strategy is developed to construct highly dispersed noncrystalline selenium nanoparticles (Se NPs) with thiolated cyclo(Arg-Gly-Asp-Phe-Lys-(mpa)) (RGD) peptide modification (R-Se@DMSND) for targeted cancer treatment. Se NPs could be homogeneously grown into the pore channels of dendritic mesoporous silica nanoparticles (DMSNs) since the DMSNs could stabilize Se NPs to prevent their aggregations. Moreover, Se NPs could not only act as a therapeutic agent, inducing ROS overproduction, to effectively suppress primary tumor but also as an immunomodulatory agent to simultaneously inhibit the growth of secondary tumors by enhancement of the immune response, as confirmed by the in vivo results. Such the therapeutic-immunomodulatory strategy for tumorous therapy combining with immunomodulation using one simple nanoplatform may pave a new avenue in the biomedical field.

Randomized Phase III Trial of Adjuvant Pazopanib Versus Placebo After Nephrectomy in Patients With Localized or Locally Advanced Renal Cell Carcinoma.

Purpose This phase III trial evaluated the efficacy and safety of pazopanib versus placebo in patients with locally advanced renal cell carcinoma (RCC) at high risk for relapse after nephrectomy. Patients and Methods A total of 1,538 patients with resected pT2 (high grade) or ≥ pT3, including N1, clear cell RCC were randomly assigned to pazopanib or placebo for 1 year; 403 patients received a starting dose of 800 mg or placebo. To address toxicity attrition, the 800-mg starting dose was lowered to 600 mg, and the primary end point analysis was changed to disease-free survival (DFS) for pazopanib 600 mg versus placebo (n = 1,135). Primary analysis was performed after 350 DFS events in the intent-to-treat (ITT) pazopanib 600 mg group (ITT600mg), and DFS follow-up analysis was performed 12 months later. Secondary end point analyses included DFS with ITT pazopanib 800 mg (ITT800mg) and safety. Results The primary analysis results of DFS ITT600mg favored pazopanib but did not show a significant improvement over placebo (hazard ratio [HR], 0.86; 95% CI, 0.70 to 1.06; P = .165). The secondary analysis of DFS in ITT800mg (n = 403) yielded an HR of 0.69 (95% CI, 0.51 to 0.94). Follow-up analysis in ITT600mg yielded an HR of 0.94 (95% CI, 0.77 to 1.14). Increased ALT and AST were common adverse events leading to treatment discontinuation in the pazopanib 600 mg (ALT, 16%; AST, 5%) and 800 mg (ALT, 18%; AST, 7%) groups. Conclusion The results of the primary DFS analysis of pazopanib 600 mg showed no benefit over placebo in the adjuvant setting.

Are different soil metals near the homes of pregnant women associated with mild and severe intellectual disability in children?

AIM: We explored the association of relatively low concentrations of metals in the soil proximal to maternal residence during pregnancy, with intellectual disability. We hypothesized different metals would be associated with mild versus severe intellectual disability. METHOD: We used a mixed methods design, starting with a retrospective cohort from 1996 to 2002, of 10,051 pregnant mothers, soil sampling in the areas where these mothers resided during pregnancy, and follow-up of their children to determine if there was an intellectual disability outcome. We tested the soil and then predicted the soil concentration at the maternal homes, and modeled the association with the severity of the child's intellectual disability. RESULTS: We found a significant positive association between mild intellectual disability and soil mercury (p=0.007). For severe intellectual disability, there was a significant positive association with the soil arsenic and lead (p=0.025). INTERPRETATION: This is the first report of the differential impact of metals in soil and severity of intellectual disability in children. Soil mercury concentration in the area the mother lived during pregnancy is associated with significantly increased odds of mild intellectual disability; a combination of arsenic and lead is associated with significantly increased odds of severe intellectual disability. These associations are present when controlling for maternal, child, and neighborhood characteristics.

Does the metal content in soil around a pregnant woman's home increase the risk of low birth weight for her infant?

Low birth weight (LBW) is associated with a number of maternal environmental exposures during pregnancy. This study explored the association between soil metal concentrations around the home where the mother lived during pregnancy and the outcome of LBW. We used a retrospective cohort of 9,920 mother-child pairs who were insured by Medicaid during pregnancy and lived in ten residential areas, where we conducted soil sampling. We used a grid that overlaid the residential areas and collected soil samples at the grid intersections. The soil was analyzed for the concentration of eight metals [arsenic (As), barium (Ba), chromium (Cr), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), and mercury (Hg)], and we then used Bayesian Kriging to estimate the concentration at the actual maternal addresses, since we had the GIS coordinates of the homes. We used generalized additive modeling, because the metal concentrations had nonlinear associations with LBW, to develop the best fitting multivariable model for estimating the risk of LBW. The final model showed significant associations for female infants, maternal smoking during pregnancy, non-white mothers, Cu, and As with LBW. The As variable was nonlinear in relation to LBW, and the association between higher concentrations of As with LBW was strong (p = 0.002). We identified a statistically significant association between soil concentrations of arsenic around the home of pregnant women and an increased risk of LBW for her infant.

Respiratory syncytial virus morbidity and outpatient palivizumab dosing in South Carolina, 2004-2009.

OBJECTIVE: Respiratory syncytial virus (RSV) has been identified as an important cause of lower respiratory tract disease in infants. In patients at high risk, prevention is attempted through immunoprophylaxis with palivizumab. In 2008, as a result of revisions to the American Academy of Pediatrics' guidelines, South Carolina Medicaid reduced the number of approved palivizumab doses from six to five. This study attempted to determine whether the reduction of approved doses would affect hospitalization and emergency department visits and to characterize dose administration. METHODS: We obtained data for all South Carolina Medicaid reimbursed births from November 2004 through March 2009. For each RSV season, infants who should have received palivizumab were identified. Rates of outpatient palivizumab dosing and hospitalizations and emergency department visits because of RSV also were identified. RESULTS: In the seasons sampled, 1956 infants met eligibility criteria for our study. Infants younger than 29 weeks' gestation received 34% to 48% of their total eligible palivizumab doses, whereas infants 29 to 31 weeks' gestation received 36% to 46% of their doses. The rate of emergency department visits and inpatient admissions because of RSV did not differ significantly across years. DISCUSSION: In evaluating our primary outcome, there was no increase in hospitalizations or emergency department visits. Overall, we did note a poor dosing rate in all of the groups. A statistically significant decline in dosing per eligible month was noted following the dose reductions. Despite solid evidence of the benefits of palivizumab in high-risk groups, we are doing an inadequate job of dosing these patients. CONCLUSIONS: We believe adherence to current recommendations for palivizumab dosing is suboptimal in preterm infants insured by the South Carolina Medicaid program. Healthcare professionals must work harder to identify and follow-up with patients who qualify for palivizumab dosing, including infants who meet criteria for a second season.

When are fetuses and young children most susceptible to soil metal concentrations of arsenic, lead and mercury?

This study was designed to analyze when, during pregnancy and early childhood, the association between soil metal concentrations of arsenic (As), lead (Pb) and mercury (Hg) and the outcome of intellectual disability (ID) is statistically significant. Using cluster analysis, we identified ten areas of land that contained a cluster of ID and areas of average risk for ID. We analyzed soil for As, Pb, and Hg and estimated the soil metal concentration at the residential sites where the woman and children lived during pregnancy and early childhood using a Bayesian Kriging model. Arsenic concentrations were associated with ID during the first trimester of pregnancy and Hg was associated with ID early in pregnancy and the first two years of childhood. The covariates that remained in the final models were also temporally associated with ID.

Ehrlichia type IV secretion effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by upregulating host MnSOD.

Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two-hybrid screening of E. chaffeensis hypothetical proteins with positively charged C-terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host-cell cytoplasm and localized to mitochondria. Delivery of anti-ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax-induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis-infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825-transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS-induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.

Adjusting wheal size measures to correct atopy misclassification.

PURPOSE: Skin prick testing (SPT) is fundamental to the practice of clinical allergy identifying relevant allergens and predicting the clinical expression of disease. Wheal sizes on SPT are used to identify atopic cases, and the cut-off value for a positive test is commonly set at 3 mm. However, the measured wheal sizes do not solely reflect the magnitude of skin reaction to allergens, but also skin reactivity (reflected in the size of histamine reaction) and other random or non-random factors. We sought to estimate wheal sizes exclusively due to skin response to allergens and propose gender-specific cutoff points of atopy. METHODS: We developed a Bayesian method to adjust observed wheal sizes by excluding histamine and other factor effects, based on which revised cutoff points are proposed for males and females, respectively. The method is then applied to and intensively evaluated using a study population aged 18, at a location on the Isle of Wight in the United Kingdom. To evaluate the proposed approach, two sample t-tests for population means and proportion tests are applied. RESULTS: Four common aeroallergens, house dust mite (HDM), grass pollen, dog dander, and alternaria are considered in the study. Based on 3 mm cutoff, males tend to be more atopic than females (P-values are between 0.00087 and 0.062). After applying the proposed methods to adjust wheal sizes, our findings suggest that misclassifications of atopy occur more often in males. Revised allergen-specific cutoff values are proposed for each gender. CONCLUSION: To reduce the gender discrepancy, we may have two potentially convenient solutions. One way is to apply allergen-specific and gender-specific cutoff values following the proposed method. Alternatively, we can revise the concentration of allergens in the SPT solutions but keep the cutoff values unchanged, which may be more convenient to clinicians.

Four VirB6 paralogs and VirB9 are expressed and interact in Ehrlichia chaffeensis-containing vacuoles.

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

Immunoprotection of chickens against Eimeria acervulina by recombinant alpha-tubulin protein.

Tubulins are heterodimeric molecules responsible for the polymerization of microtubules in apicomplexan parasites. The alpha-tubulin, a subcellular structural protein of Eimeria acervulina, was cloned and expressed in Escherichia coli as an alpha-tubulin-GST fusion protein. Immunogenicity of the recombinant protein was studied in chickens by subcutaneous injection of 50, 100, or 150 microg of the protein with or without Freund's adjuvant. Immunization with 150 microg alpha-tubulin-GST protein in combination with Freund's adjuvant conferred partial protection against E. acervulina oocyst challenge, as shown by a 36% reduction in oocyst shedding, a marked decrease in intestinal lesion score and a significant increase in body weight gain in comparison with the nonimmunized controls. The results suggest that alpha-tubulin protein may be used as an effective vaccine antigen for the control of Eimeria infection.

Ehrlichia ewingii infection delays spontaneous neutrophil apoptosis through stabilization of mitochondria.

The uncultivable obligate intracellular bacterium Ehrlichia ewingii, previously known only as a canine pathogen, is the most recently recognized agent of human ehrlichiosis. E. ewingii is the only Ehrlichia species known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. The inhibitory effect on neutrophil apoptosis by E. ewingii was reversible on clearance of the organism. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.