Transcriptomes of the interaction between Fusarium verticillioides and a Streptomyces strain reveal the fungal defense strategy under the pressure of a potential biocontrol agent.

The actinobacteria Streptomyces sp. AV05 appears to be a potential biocontrol agent (BCA) against mycotoxigenic fungi. It was found to significantly inhibit F. verticillioides growth and mycotoxin production during their co-cultivation. F. verticillioides growth was durably affected while the decrease of the toxin production levels was reversible, suggesting different BCA actions. The study of both transcriptomes brought useful information on the microbial interaction. RNA-seq data indicated that the dual interaction modified genetic expression of both microorganisms as 18.5 % of the genes were differentially expressed for the fungus against 3.8 % for the actinobacteria. Fungal differentially expressed genes (DEGs) were equally up and down regulated while bacterial ones were mainly upregulated. We especially focused the analysis of DEGs on fungal defense reaction to bacterial attack. For example, if this potential BCA implements a strategy of antibiosis with the over expression of 'siderophore-interacting protein' linked to the production of bacteriocins, the fungus in a state of stress is able to adapt its metabolism by up-regulation of amidase. It could correspond to the induction of resistance gene clusters and suggest a detoxification process. Moreover fumonisins-related pathway appears underexpressed in the presence of Streptomyces that explain the reduction of fumonisin accumulation observed.

Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters.

The cleavage furrow in Xenopus zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the midplane, they assemble a disk-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin that instructs the cleavage furrow. Here we investigate the mechanism that keeps the two asters separate and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg orthologue of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in Xenopus egg extracts. Prc1E was required for Kif4A recruitment but not vice versa. Microtubule plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote "anti-parallel pruning" that enforces radial organization within asters and generates boundaries to microtubule growth between asters.

Co-occurrence of second primary malignancy in patients with thyroid cancer.

BACKGROUND: The discovery of asynchronous or synchronous double or multiple malignancies in patients is not uncommon. The co-occurrence of second primary malignancy (SPM) could be randomly occurring or association with risk factors such as environmental, genetic predisposition and therapy-related. MATERIALS AND METHODS: We retrieved ∼782 million claim records consisting of 10.8 million males and 10.6 million females from Taiwan's National Health Insurance, which were collected for a period of 3 years (January 2000-December 2002). All the patient records were stratified by gender and ages at a 20-year interval with SPMs and specific groups. Interestingness or Q-value was used to measure strength of the disease-disease associations. RESULTS: A total of 9423 thyroid cancer (female: 7483, male: 1940), 276 184 SPM (female: 141 023, male: 135 161) and 861 co-occurrence cases (female: 583, male: 278) were recorded. The co-occurrence incidence rate of head and neck, breast, digestive system and lung was 1.93%, 1.59%, 1.44% and 1.18%, respectively. Malignancy of salivary glands, laryngx, sarcoma, lymphoid tissue, mouth, central nervous system and lungs found Q-value >10. Malignancies with intermediate Q-values (5.0-9.9) were observed in nasopharynx, kidney and ureter, breast, stomach and skin. Prostate, leukemia, urinary bladder, ovary, colon, liver and uterine cervix cancer have lower Q-values (1.0-4.9). CONCLUSION: Co-occurrence ratio of thyroid cancer and SPM was high, occurred in all organ systems. We postulated that the aggressive use of modern diagnostic modalities, aggressive radioiodine treatment, pre-existing molecular oncogen mutations, and thyroid hormone for simultaneously supple-mentary and suppressive therapies were responsible.

Challenges of assuring crew safety in space shuttle missions with international cargoes.

The top priority in America's manned space flight program is the assurance of crew and vehicle safety. This priority gained greater focus during and after the Space Shuttle return-to-flight mission (STS-26). One of the interesting challenges has been to assure crew safety and adequate protection of the Space Shuttle, as a national resource, from increasingly diverse cargoes and operations. The control of hazards associated with the deployment of complex payloads and cargoes has involved many international participants. These challenges are examined in some detail along with examples of how crew safety has evolved in the manned space program and how the international partners have addressed various scenarios involving control and mitigation of potential hazards to crew and vehicle safety.

Plasma lipids and cardiovascular risk: a POSCH report. Program on the Surgical Control of the Hyperlipidemias.

Quantifying the relationship between changes in lipid variables and clinical endpoints has been difficult. We studied the predictive value of various lipid variables on three endpoints in the Program on the Surgical Control of the Hyperlipidemias (POSCH): overall mortality, coronary heart disease (CHD) mortality, and CHD mortality and confirmed nonfatal myocardial infarction (MI) combined. We measured lipid variables for the annual visits from baseline to 5 years for actual follow-up values, actual and percentage differences between baseline and follow-up values, as well as the parameters comparing baseline only to 5 years for actual differences, percentage differences, and the ratio of baseline to 5 years. The lipid variables included were total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, very low density lipoprotein (VLDL) cholesterol, triglycerides, and the LDL cholesterol/HDL cholesterol ratio. The analytic method used was that of Cox regression, with age and sex as secondary covariates, and each lipid or ratio of lipids as the primary (univariate) covariate. As a result, 108 univariate Cox regressions were conducted. The combined findings for the control and the intervention groups are presented. The number of events for the combined group were: overall mortality, 190; CHD mortality, 119; and CHD mortality and confirmed nonfatal MI, 262. The highest hazard ratios were found for the lipid variable of the LDL cholesterol/HDL cholesterol ratio (e.g. 1.196 for a 1-unit increase). Only for the combined endpoint of CHD mortality and confirmed nonfatal MI was there a substantial number of statistically significant relationships (P<0.01) of lipid variables and parameters of assessment.

The effect of formulation excipients on protein stability and aerosol performance of spray-dried powders of a recombinant humanized anti-IgE monoclonal antibody.

PURPOSE: To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. METHODS: Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. RESULTS: Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300: 1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. CONCLUSIONS: Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.

Protein inhalation powders: spray drying vs spray freeze drying.

PURPOSE: To develop a new technique, spray freeze drying, for preparing protein aerosol powders. Also, to compare the spray freeze-dried powders with spray-dried powders in terms of physical properties and aerosol performance. METHODS: Protein powders were characterized using particle size analysis, thermogravimetric analysis, scanning electron microscopy, X-ray powder diffractometry, and specific surface area measurement. Aerosol performance of the powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger or an Anderson cascade impactor. Two recombinant therapeutic proteins currently used for treating respiratory tract-related diseases, deoxyribonuclase (rhDNase) and anti-IgE monoclonal antibody (anti-IgE MAb), were employed and formulated with different carbohydrate excipients. RESULTS: Through the same atomization but the different drying process, spray drying (SD) produced small (approximately 3 microns), dense particles, but SFD resulted in large (approximately 8-10 microns), porous particles. The fine particle fraction (FPF) of the spray freeze-dried powder was significantly better than that of the spray-dried powder, attributed to better aerodynamic properties. Powders collected from different stages of the cascade impactor were characterized, which confirmed the concept of aerodynamic particle size. Protein formulation played a major role in affecting the powder's aerosol performance, especially for the carbohydrate excipient of a high crystallization tendency. CONCLUSIONS: Spray freeze drying, as opposed to spray drying, produced protein particles with light and porous characteristics, which offered powders with superior aerosol performance due to favorable aerodynamic properties.

Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody.

We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.

Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders.

PURPOSE: To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. METHODS: Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). RESULTS: Spray-dried powders with a moisture level (approximately 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. CONCLUSIONS: Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.

Spray-drying of air-liquid interface sensitive recombinant human growth hormone.

Spray-drying is an attractive method for preparing fine recombinant human growth hormone (rhGH) powders if the detrimental effect of protein degradation at the air-liquid interface on the protein can be minimized. In this study, we demonstrated that rhGH degradation (insoluble and soluble aggregate formation), as the consequence of air-liquid interfacial degradation, could be prevented using the appropriate formulation. Adding polysorbate-20 surfactant into the liquid feed (with no presence of sugar protectant) significantly reduced the formation of insoluble protein aggregates, while adding the divalent metal zinc ion effectively suppressed the formation of soluble protein aggregates. The combination of the two yielded a spray-dried rhGH powder having insignificant protein degradation. Our data suggest that the two components might protect the protein through different mechanisms. Polysorbate molecules occupy the air-liquid interface of spray droplets, thereby reducing the chance for rhGH to form insoluble aggregates by surface denaturation. Two zinc ions associate with two rhGH molecules to form a dimer complex that can resist the formation of soluble protein aggregates. Characterization of spray-dried powders by scanning electron microscopy suggests that both formulation and drying conditions have a strong influence on particle morphology and shape. Overall, spherical rhGH powders of smooth surface and good biochemical quality can be prepared by spray-drying using this formulation with no addition of sugar protectant.

Spray-drying performance of a bench-top spray dryer for protein aerosol powder preparation.

The objective of this work was to improve a bench-top spray dryer's efficiency in both production recovery and throughput for preparing protein aerosol powders. A Büchi mini-spray dryer was used to prepare the powders of recombinant humanized anti-IgE antibody. The resulting powder's physical properties such as particle size, residual moisture, and morphology, along with its recovery and production rate was the basis of this development work. Mass balance suggests that approximately 10-20% of powder was lost in the exhaust air, consisting primarily of particles less than 2 micrometer. Also, significant loss (20-30%) occurred in the cyclone. Attempts were made to improve product recovery in the receiving vessel using dual-cyclone configurations, different cyclone designs, cyclones with anti-static treatment, and different receiver designs. System modifications such as replacing the original bag-filter unit with a vacuum system effectively reduced drying air flow resistance, allowing the protein to be dried at a lower inlet air temperature and the production scale to be increased. We concluded that the modified spray-drying system is advantageous over the original bench-top spray dryer. This improvement will be beneficial to early-stage research and development involving high-valued protein powders.

The effect of operating and formulation variables on the morphology of spray-dried protein particles.

The purpose of this research was to investigate the shape and morphology of various spray-dried protein powders as a function of spray-drying conditions and protein formulations. A benchtop spray dryer was used to spray dry three model proteins in formulation with a sugar or a surfactant. Physical characterizations of the powder included morphology (scanning electron microscopy), particle size, residual moisture, and X-ray powder diffraction analyses. A significant change in particle shape from irregular (e.g., "donut") to spherical was observed as the outlet temperature of the dryer was decreased. The drying air outlet temperature was shown to depend on various operating parameters and was found to correlate with the drying rate of atomized droplets in the drying chamber. The morphology of spray-dried protein particles was also affected by formulation. In protein:sugar formulations, spray-dried particles exhibited a smooth surface regardless of the protein-to-lactose ratio, whereas roughness was observed when mannitol was present at > 30% of total solids, due to recrystallization. Protein particles containing trehalose at concentrations > 50% were highly agglomerated. The presence of surfactant resulted in noticeably smoother, more spherical particles. The shape and the morphology of spray-dried powders are affected by spray drying conditions and protein formulation. This study provides information useful for development of dry proteins for fine powder (e.g., aerosol) applications.

Disease-free intervals after partial ileal bypass in patients with coronary heart disease and hypercholesterolemia: report from the Program on the Surgical Control of the Hyperlipidemias (POSCH).

OBJECTIVES: We sought to analyze the disease-free intervals and calculate the freedom from atherosclerosis events in the Program on the Surgical Control of the Hyperlipidemias (POSCH). BACKGROUND: The POSCH study was a randomized, secondary lipid/atherosclerosis intervention trial that provided strong evidence for reduction in atherosclerosis progression as demonstrated by clinical and arteriographic end points. The 417 control group patients received American Heart Association phase II diet instruction, and the 421 intervention group patients received identical dietary instruction and underwent a partial ileal bypass operation. METHODS: Four outcome measures were determined: 1) overall mortality, 2) coronary heart disease mortality, 3) coronary heart disease mortality and confirmed nonfatal myocardial infarction, and 4) coronary/cardiac interventions. RESULTS: An overall mortality rate of 10% occurred at 6.7 years in the control group and 9.4 years in the intervention group, for a gain in disease-free interval of 2.7 years in the intervention group (p = 0.032). A coronary heart disease mortality rate of 8% occurred at 7.2 years in the control group and 11 years in the intervention group, for a gain of 3.8 years (p = 0.046). Twenty percent of patients demonstrated the combined end point of coronary heart disease mortality and confirmed nonfatal myocardial infarction at 5.9 years in the control group and 11.4 years in the intervention group, for a gain of 5.5 years (p < 0.001). Twenty-five percent of patients underwent either coronary artery bypass graft surgery, percutaneous transluminal coronary angioplasty or heart transplantation at 5.4 years in the control group and 12.4 years in the intervention group, for a gain of 7 years (p < 0.001). CONCLUSIONS: The marked lipid modification achieved by partial ileal bypass in the POSCH trial led to demonstrable increases in the disease-free intervals for overall mortality, coronary heart disease mortality, coronary heart disease mortality and confirmed nonfatal myocardial infarction, and coronary intervention procedures. For the clinician and the patient, estimation of disease-free intervals may be more relevant than assessment of differences in incidence rates and risk ratios.