The state of Florida's estuaries and future needs in estuarine research: Part 2. an academic research agenda (review draft)

As a program supporting academic research that addresses recognized societal needs, the Florida Sea Grant Program is developing a research theme area on estuaries to provide a uniquely academic product that will augment mission-oriented research undertaken by government and by the private sector. This report is not a call for proposals. It does not prescribe a specific research plan. Rather, it is a concept paper designed to focus research on two broad "organizing themes": (1) the hydrology of Florida's estuaries, and (2) the impact of cyclic environmental variability on estuarine function. (46pp.

Comparison of Indices Proposed as Criteria for Assigning Skin Notation

Objectives: Skin notations are used as a hazard identification tool to flag chemicals associated with a potential risk related to transdermal penetration. The transparency and rigorousness of the skin notation assignment process have recently been questioned. We compared different approaches proposed as criteria for these notations as a starting point for improving and systematizing current practice. Methods: In this study, skin notations, dermal acute lethal dose 50 in mammals (LD50s) and two dermal risk indices derived from previously published work were compared using the lists of Swiss maximum allowable concentrations (MACs) and threshold limit values (TLVs) from the American Conference of Governmental Industrial Hygienists (ACGIH). The indices were both based on quantitative structure-activity relationship (QSAR) estimation of transdermal fluxes. One index compared the cumulative dose received through skin given specific exposure surface and duration to that received through lungs following inhalation 8 h at the MAC or TLV. The other index estimated the blood level increase caused by adding skin exposure to the inhalation route at kinetic steady state. Dermal-to-other route ratios of LD50 were calculated as secondary indices of dermal penetrability. Results: The working data set included 364 substances. Depending on the subdataset, agreement between the Swiss and ACGIH skin notations varied between 82 and 87%. Chemicals with a skin notation were more likely to have higher dermal risk indices and lower dermal LD50 than chemicals without a notation (probabilities between 60 and 70%). The risk indices, based on cumulative dose and kinetic steady state, respectively, appeared proportional up to a constant independent of chemical-specific properties. They agreed well with dermal LD50s (Spearman correlation coefficients −0.42 to −0.43). Dermal-to-other routes LD50 ratios were moderately associated with QSAR-based transdermal fluxes (Spearman correlation coefficients −0.2 to −0.3). Conclusions: The plausible but variable relationship between current skin notations and the different approaches tested confirm the need to improve current skin notations. QSAR-based risk indices and dermal toxicity data might be successfully integrated in a systematic alternative to current skin notations for detecting chemicals associated with potential dermal risk in the workplac

Freezing of Water in Hardboard: Absence of Changes in Mechanical Properties

One-eighth-inch dry process and two species mixes of 7/16 in. wet process hardboard roofings plus 1/8 in. dry process and 7/16 in. wet process standard hardboards were examined using differential thermal analysis to ascertain the maximum moisture content that exterior hardboard could attain without exhibiting significant freezing. All samples with moisture contents greater than ≈20% exhibited high temperature freezing near - 10 C. Additionally, dry process materials with moisture contents near or above 30% had a distinct low temperature freezing event near -35 C. Integration of the area under the freezing curves indicated that ≈ 3% of the water contained in these samples froze at low temperature. During thawing, this fraction of water melted above -10 C. This type of thermal hysteresis is characteristic of the freeze/thaw behavior expected for supercooled water. Mechanical strength tests performed on dry process (4.1 and 41.1% moisture content) and wet process (4.4 and 34.3% moisture content) standard hardboard exposed to freeze/thaw cycling to -50 C revealed no consistent changes in the modulus of elasticity, modulus of rupture, tensile strength parallel to surface, or internal bond strength

Novel insights into the architecture and protein interaction network of yeast eIF3.

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3(rec)) exhibits the same size and activity as the natively purified eIF3 (eIF3(nat)). The homogeneity and stoichiometry of eIF3(rec) and eIF3(nat) were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex

Role of M. uclerans extracellular matrix

Date du colloque&nbsp;: 04/2008</p

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments—specifically, the GFP1-10 and GFP11 fragments—we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2&nbsp;pmol of polypeptide, with fluorescence plateauing after 4&nbsp;h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein

Establishment of resistance to Leishmania major infection in susceptible BALB/c mice requires parasite-specific CD8+ T cells

Although CD4+ T cells are generally accepted to be responsible for the determination of resistance to infection in experimental murine cutaneous leishmaniasis, a contribution of CD8+ lymphocytes to immunity can be demonstrated under certain well-defined conditions. Normally highly susceptible BALB/c mice can be rendered resistant to infection with Leishmania major promastigotes by a single injection of monoclonal anti-CD4 antibodies at the beginning of infection. Mice treated in such a way can heal their primary cutaneous lesions and acquire immunity to subsequent challenge infection. Both the resolution of the primary infection and the induced state of immunity to reinfection in these mice is shown to be dependent upon the anti-leishmanial effector functions of CD8+ T cells. Furthermore, in contrast to control infected BALB/c mice, which are unable to mount a delayed-type hypersensitivity (DTH) response to viable parasites, mice cured as a result of treatment with anti-CD4 antibodies in vivo exhibit a strong DTH response, which can be significantly reduced by injection of either anti-CD4 or anti-CD8 monoclonal antibodies prior to antigenic challenge with viable promastigotes. Moreover, increased numbers of specific CD8+ T cells, able to transfer Leishmania-specific DTH responses, were found in lymphoid organs of BALB/c mice rendered resistant to infection by immunointervention with anti-CD4 monoclonal antibodies at the beginning of infection. Neutralization in vivo of interleukin 4 during the course of infection in BALB/c mice also enables these otherwise susceptible mice to resolve their cutaneous lesions and to decrease the parasite burden in infected tissues. CD8+ T cells are required for both of these beneficial effects. Taken together, these results indicate that in the immune BALB/c mouse, as in the normally resistant CBA mouse, CD8+ lymphocytes are involved in the elimination of L. major and in the establishment and maintenance of immunity against infection with this parasite