Dietary supplementation of recombinant antimicrobial peptide Epinephelus lanceolatus piscidin improves growth performance and immune response in Gallus gallus domesticus.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.

Revisiting the relationship of three-dimensional fluid attenuation inversion recovery imaging and hearing outcomes in adults with idiopathic unilateral sudden sensorineural hearing loss.

BACKGROUND AND PURPOSE: Three-dimensional fluid attenuation inversion recovery (3D FLAIR) may demonstrate high signal in the inner ears of patients with idiopathic sudden sensorineural hearing loss (ISSNHL), but the correlations of this finding with outcomes are still controversial. Here we compared 4 3D MRI sequences with the outcomes of patients with ISSNHL. MATERIALS AND METHODS: 77 adult patients with ISSNHL underwent MRI with pre contrast FLAIR, fast imaging employing steady-state acquisition images (FIESTA-C), post contrast T1WI and post contrast FLAIR. The extent and degree of high signal in both cochleas were evaluated in all patients, and asymmetry ratios between the affected ears and the normal ones were calculated. The relationships among MRI findings, including extent and asymmetry of abnormal cochlear high signals, degree of FLAIR enhancement, and clinical information, including age, vestibular symptoms, baseline hearing loss, and final hearing outcomes were analyzed. RESULTS: 54 patients (28 men; age, 52.1±15.5years) were included in our study. Asymmetric cochlear signal intensities were more frequently observed in pre contrast and post contrast FLAIR (79.6% and 68.5%) than in FIESTA-C (61.1%) and T1WI (51.9%) (p<0.001). Age, baseline hearing loss, extent of high signal and asymmetry ratios of pre contrast and post contrast FLAIR were all correlated with final hearing outcomes. In multivariate analysis, age and the extent of high signals were the most significant predictors of final hearing outcomes. CONCLUSION: 3D FLAIR provides a higher sensitivity in detecting the asymmetric cochlear signal abnormality. The more asymmetric FLAIR signals and presence of high signals beyond cochlea indicated a poorer prognosis.

Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency.

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2-) and tumorigenesis on tomato stems (Vir+ or Vir-), the mutants could be classified into three groups: ExB2-/Vir-, ExB2-/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2-/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus-/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.

Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.