Attention Mobilization as a Modulator of Listening Effort: Evidence From Pupillometry.

Listening to speech in noise can require substantial mental effort, even among younger normal-hearing adults. The task-evoked pupil response (TEPR) has been shown to track the increased effort exerted to recognize words or sentences in increasing noise. However, few studies have examined the trajectory of listening effort across longer, more natural, stretches of speech, or the extent to which expectations about upcoming listening difficulty modulate the TEPR. Seventeen younger normal-hearing adults listened to 60-s-long audiobook passages, repeated three times in a row, at two different signal-to-noise ratios (SNRs) while pupil size was recorded. There was a significant interaction between SNR, repetition, and baseline pupil size on sustained listening effort. At lower baseline pupil sizes, potentially reflecting lower attention mobilization, TEPRs were more sustained in the harder SNR condition, particularly when attention mobilization remained low by the third presentation. At intermediate baseline pupil sizes, differences between conditions were largely absent, suggesting these listeners had optimally mobilized their attention for both SNRs. Lastly, at higher baseline pupil sizes, potentially reflecting overmobilization of attention, the effect of SNR was initially reversed for the second and third presentations: participants initially appeared to disengage in the harder SNR condition, resulting in reduced TEPRs that recovered in the second half of the story. Together, these findings suggest that the unfolding of listening effort over time depends critically on the extent to which individuals have successfully mobilized their attention in anticipation of difficult listening conditions.

Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate.

Plant biomass is a low-cost and abundant source of carbohydrates for production of fuels, "green" chemicals and materials. Currently, biochemical conversion of the biomass into sugars via enzymatic hydrolysis is the most viable technology. Here, the role of carbohydrate binding modules (CBMs) in the disruption of insoluble polysaccharide structures and their capacity to enhance cellulase-promoted lignocellulosic biomass hydrolysis was investigated. We show that CBM addition promotes generation of additional reducing ends in the insoluble substrate by cellulases. On the contrary, bovine serum albumin (BSA), widely used in prevention of a non-specific protein binding, causes an increase in soluble reducing-end production, when applied jointly with cellulases. We demonstrate that binding of CBMs to cellulose is non-homogeneous, irreversible and leads to its amorphisation. Our results also reveal effects of CBM-promoted amorphogenesis on cellulose hydrolysis by cellulases.

OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were used to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through PPI.

On the subtle tuneability of cellulose hydrogels: implications for binding of biomolecules demonstrated for CBM 1.

Cellulose-based hydrogel materials prepared by regeneration from cellulose solutions in ionic liquids, or ionic liquid containing solvent mixtures (organic electrolyte solutions), are becoming widely used in a range of applications from tissue scaffolds to membrane ionic diodes. In all such applications knowledge of the nature of the hydrogel with regards to porosity (pore size and tortuosity) and material structure and surface properties (crystallinity and hydrophobicity) is critical. Here we report significant changes in hydrogel properties, based on the choice of cellulose raw material (α- or bacterial cellulose - with differing degree of polymerization) and regeneration solvent (methanol or water). Focus is on bioaffinity applications, but the findings have wide ramifications, including in biomedical applications and cellulose saccharification. Specifically, we report that the choice of cellulose and regeneration solvent influences the surface area accessible to a family 1 carbohydrate-binding module (CBM), CBM affinity for the cellulose material, and rate of migration through the hydrogel. By regenerating bacterial cellulose in water, a maximum accessible surface area of 33 m2 g-1 was achieved. However, the highest CBM migration rate, 1.76 µm2 min-1, was attained by regenerating α-cellulose in methanol, which also resulted in the maximum affinity of the biomolecule for the material. Thus, it is clear that if regenerated cellulose hydrogels are to be used as support materials in bioaffinity (or other) applications, a balance between accessible surface area and affinity, or migration rate, must be achieved.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

Mice deficient in the candidate tumor suppressor gene Hic1 exhibit developmental defects of structures affected in the Miller-Dieker syndrome.

HIC1 is a candidate tumor suppressor gene which is frequently hypermethylated in human tumors, and its location within the Miller-Dieker syndrome's critical deletion region at chromosome 17p13.3 makes it a candidate gene for involvement in this gene deletion syndrome. To study the function of murine Hic1 in development, we have created Hic1 -deficient mice. These animals die perinatally and exhibit varying combinations of gross developmental defects throughout the second half of development, including acrania, exencephaly, cleft palate, limb abnormalities and omphalocele. These findings demonstrate a role for Hic1 in the development of structures affected in the Miller-Dieker syndrome, and provide functional evidence to strengthen its candidacy as a gene involved in this disorder.

Enzyme-linked immunosorbent assays in a chromatographic format.

This paper describes the execution of enzyme-linked immunosorbent assays (ELISA), (i) in immunosorbent columns with antibodies immobilized on porous particles, (ii) where samples and reagents are metered by valves and syringe pumps, (iii) samples, reagents, substrate, and wash buffers are transported into or through the system by high pressure liquid chromatography pumps, and (iv) enzyme reaction product is detected by absorbance. The normal protocol used for ELISA was modified in that antigen was complexed with enzyme conjugated antibody in the autosampler and an aliquot introduced into the system where it was transported to the immunosorbent and captured. Substrate was subsequently pumped into the immunosorbent bed and the product swept to an absorbance detector for quantitation.

Distribution of polysaccharide side chains of lipopolysaccharide determine resistance of Escherichia coli to the bactericidal activity of serum.

The lipopolysaccharide (LPS) of serum-sensitive strains of Escherichia coli was compared with LPS derived from serum-resistant clones. Polysaccharide O-antigen side chains (PSSC) of LPS from serum-resistant clones contained 12%-40% more of the longer carbohydrate molecules (L-PSSC) than did LPS from serum-sensitive parent strains; in contrast, 12%-27% more of the shorter PSSC (S-PSSC) were found in LPS from serum-sensitive strains. The sensitivity or resistance to the bactericidal activity of human serum correlated with the distribution and the length of PSSC fractions of LPS. This was demonstrated in a liposome model in which LPS was incorporated into simulated bacterial membranes. The incubation of serum with liposomes incorporated with various ratios of S-PSSC-to-L-PSSC concentrations resulted in liposomal lysis at S-PSSC-to-L-PSSC ratios greater than 2:1. These findings demonstrate the importance of the length of carbohydrate side chains of LPS in determining sensitivity or resistance to the bactericidal activity of human serum.

Effects of endotoxin-associated protein on hematopoiesis.

Endotoxin-associated protein (EAP), a gram-negative bacterial cell wall component, was evaluated for its effects on hematopoietic colony formation in vitro. Colony-stimulating activity, induced by EAP on circulating and bone marrow progenitor cells, was found to be partially mediated by T cells and augmented by interleukin-3. The addition of anti-human interleukin-1 (IL-1) antibodies reduced EAP activity, suggesting that EAP may induce IL-1 production. However, EAP was shown to promote the growth of mature progenitor cells independently, unlike the effects of IL-1 on the hematopoietic system. These studies demonstrate that bacterial components other than lipopolysaccharide, such as EAP, may have hematopoietic activity.

Distribution of Bs1 retrotransposons in Zea and related genera.

Thirty-eight accessions from Zea and 20 accessions from related genera were probed for the presence of Bs1, a retrotransposon originally found in maize. All maize and teosinte plants tested show the presence of Bs1 in one to five densely hybridizing bands. The mean copy numbers of Bs1 elements among the maize and teosinte accessions were similar: 2.92 and 3.25, respectively, with no large differences between any subgroups. Most exotic maize samples exhibited two common bands of 7.8 kb and 4.7 kb. Section Zea teosintes (but not teosintes of section Luxuriantes) also show the presence of a common band of the same size as the smaller common band in maize. At reduced stringency, Tripsacum dactyloides exhibited a single hybridizing band at 6.9 kb. Results argue for the evolution of maize from a mexicana or parviglumis teosinte, and the evolution of the Bs1 element within the tribe Andropogoneae. Additionally, recombinant inbred lines were probed for the presence of Bs1, in order to map the chromosomal locations of Bs1 elements in four different maize lines. Two of the recombinant inbred parental lines had an element (Bs1-F) on chromosome 5, while the other two lines had an element (Bs1-S) on chromosome 8. Restriction site polymorphisms have apparently arisen in the vicinity of Bs1-S since its insertion. Segregation analysis of other lines was also performed; the data indicate that Bs1 has the distribution expected of a transposable element, different locations in different lines, and not that of a fixed gene locus. However, the common bands in the Zea mays lines and the recombinant inbred data imply that Bs1 is not highly mobile.

An unusually compact retrotransposon in maize.

Unlike any other known plant transposon, the maize transposable element Bs1 is similar to the retrotransposons previously described in yeast and Drosophila. Bs1 is bounded by 302 bp identical long terminal repeats (LTRs), and it contains open reading frames with apparent amino acid sequence similarity to reverse transcriptase and other retroviral pol gene enzymes. Bs1 is 3203 bp long, very short for a retrotransposon, and the apparent organization of its genetic information is significantly different from any previously described element. Although transcription of Bs1 has not been observed, it is probably an active transposon, since it was observed to transpose in a maize line that contains only two sequences hybridizing to Bs1 probes. Both of these sequences share 35 restriction sites with the cloned Bs1 element, and thus must be very similar or identical with it.

Immunization with rough mutants of Salmonella minnesota: initial studies in human subjects.

Vaccines prepared from unheated and boiled, acetone-precipitated Salmonella minnesota R595 (Re chemotype mutant) were administered subcutaneously to 122 healthy volunteers. Titers of antibody to Re lipopolysaccharide, the basal core structure of endotoxin, as measured by indirect hemagglutination, rose in a dose-responsive fashion after immunization. Febrile reactions, usually mild, occurred after 7% of injections with the highest doses (2.0 and 3.0 x 10(10) organisms), and mild local soreness and tenderness were noted after approximately one-third of injections. Passive immunization of mice with sera from immunized subjects demonstrated that protective activity against challenge with both heterologous, viable gram-negative bacilli and endotoxin developed. Although measuring serum protective activity, developing a serological assay that correlates with protective activity, and potential vaccine toxicity remain problems, immunization of humans with the Re mutant results in serum protective activity against endotoxin and viable bacilli and has the potential for clinical usefulness.

Immunization with rough mutants of Salmonella minnesota: protective activity of IgM and IgG antibody to the R595 (Re chemotype) mutant.

We evaluated the immunoglobulin class responsible for the protective activity in serum obtained from humans and rabbits after immunization with the R595 (Re chemotype) mutant of Salmonella minnesota. Whole serum obtained before immunization and the IgG and IgM fractions failed to protect mice against lethal challenge with viable Klebsiella pneumoniae or Morganella morganii or with Salmonella typhi lipopolysaccharide (LPS). The protective activity of postimmunization serum resided solely in IgM antibody, whereas IgG antibody exhibited no protective activity. IgM antibody to the Re mutant was protective against bacterial challenge with both test strains of bacteria and S. typhi LPS. IgM antibody, at approximately the same concentration present in postimmunization serum, increased the LD50 of K. pneumoniae from less than 8.0 x 10(2) to greater than 2.0 x 10(4). These findings indicate that commercially prepared human IgG with high titers of antibody to antigens of the core portion of LPS would have little clinical utility.

Endotoxin-associated protein: interleukin-1-like activity on serum amyloid A synthesis and T-lymphocyte activation.

Bacterial endotoxins or lipopolysaccharides (LPS) elicit a variety of biologic activities in intact animals and various in vitro systems. LPS from most gram-negative bacteria have appeared to have similar biologic activities regardless of the species of origin or method of preparation of the LPS. More recent studies have suggested differences in the effects of protein-rich as opposed to protein-free LPS in inducing mitogenesis of lymphocytes from endotoxin-resistant C3H/HeJ mice. These studies examine other activities of endotoxin-associated protein (EAP), purified to less than 0.007% contamination with LPS, and demonstrate that this material has activity mimicking some of the effects of interleukin-1 (IL-1). EAP proved to be as potent as LPS in eliciting rises in concentrations of serum amyloid A (SAA) and was active in both endotoxin-sensitive (CF1) and endotoxin-resistant (C3H/HeJ) mice. In contrast to LPS, which mediates its SAA-inducing activity by release of an inducer (IL-1) from LPS-stimulated macrophages, EAP appeared to act directly to induce SAA production, in that incubation with macrophages failed to increase its activity. EAP also exhibited IL-1-like activity in the lymphocyte-activating factor assay when both CF1 and C3H/HeJ thymocytes and macrophages were tested. The lymphocyte-activating factor activity of EAP was not blocked by addition of polymyxin B. In addition, EAP exerted stimulatory activity on resting human T lymphocytes, costimulated with Sepharose-bound anti-CD3 monoclonal antibody 64.1, comparable to that observed with purified human monocyte IL-1. These studies indicate that proteins from procaryotic cells may act as cytokines for some eucaryotic cells.

Potential for fecundin to influence the reproductive performance of merino ewes in Western Australia.

Four experiments were conducted at different locations in Western Australia to evaluate the effectiveness of immunizing young (maiden, 1 1/2 year old) and adult Merino ewes with Fecundin to improve their reproductive performances. The ovulation rates of immunized maiden ewes was increased (0.06-0.30) above that of untreated ewes in both experiments 1 and 2. However, there were no significant improvements in the marking percentages for the immunized ewes with the differences between the untreated and immunized ewes ranging from -16.4 to 5.8%. In comparison with untreated ewes immunization 6 and 2 weeks before the start of joining depressed ewe fertility by 26.1% whereas immunization 8 and 4 weeks before the start of joining did not significantly affect fertility. The availability of oat grain ad libitum prior to parturition and during early lactation did not improve the survival of lambs born to immunized ewes. In experiments 3 and 4 immunization of adult Merino ewes increased their ovulation rates (0.41-0.63) above untreated controls and tended to increase the proportion of pregnant ewes which had multiple pregnancies (from -2.3 to 34.2%). The responses at the end of lambing were variable (from -19.8 to 37.5% lambs marked) with high lamb mortalities occurring in some experiments. There was no adverse effect on the reproductive performances following consecutive annual immunizations over 3 years and the absence of treatment for 1 year did not prevent a response in the following year.

Selective pressures and lipopolysaccharide subunits as determinants of resistance of clinical isolates of gram-negative bacilli to human serum.

Differences in molecular composition of lipopolysaccharides (LPS) between serum-sensitive (S) clinical isolates of Escherichia coli and serum-resistant (R) clones derived by serial passage in serum were demonstrated to determine sensitivity or resistance to killing by normal human serum (NHS). LPS from R clones had a greater proportion of higher-molecular-weight, more highly O-antigen-substituted subunits than LPS from their serum S parents. Utilization of a liposomal model with inserted LPS simulating bacterial cell walls established LPS as the site of serum bactericidal action. Liposomes containing S LPS were lysed, while liposomes containing R LPS were unaffected by NHS. R and S LPS were fractionated into higher (F1)- and lower (F2)-molecular-weight fractions. Liposomes containing R LPS or the F1 fraction of S and R LPS were not lysed by serum. Liposomes containing the F2 fraction of S or R LPS were lysed by serum analogous to that observed with liposomes containing intact S LPS. These findings establish LPS to be one site of serum bactericidal activity and demonstrate that the higher-molecular-weight, highly O-antigen-substituted LPS subunits mediate resistance to killing by NHS.