A Seed-Endophytic Bacillus safensis Strain With Antimicrobial Activity Has Genes for Novel Bacteriocin-Like Antimicrobial Peptides.

Bacteriocins are a highly diverse group of antimicrobial peptides that have been identified in a wide range of commensal and probiotic organisms, especially those resident in host microbiomes. Rising antibiotic resistance have fueled renewed research into new drug scaffolds such as antimicrobial peptides for use in therapeutics. In this investigation, we examined mung bean seeds for endophytes possessing activity against human and plant pathogens. We isolated a novel strain of Bacillus safensis, from the contents of surface-sterilized mung bean seed, which we termed B. safensis C3. Genome sequencing of C3 identified three distinct biosynthetic systems that produce bacteriocin-based peptides. C3 exhibited antibacterial activity against Escherichia coli, Xanthomonas axonopodis, and Pseudomonas syringae. Robust antimicrobial activity of B. safensis C3 was observed when C3 was co-cultured with Bacillus subtilis. Using the cell-free supernatant of C3 and cation exchange chromatography, we enriched a product that retained antimicrobial activity against B. subtilis. The peptide was found to be approximately 3.3 kDa in size by mass spectrometry, and resistant to proteolysis by Carboxypeptidase Y and Endoproteinase GluC, suggesting that it is a modified variant of an AS-48 like bacteriocin. Our findings open new avenues into further development of novel bacteriocin-based scaffolds for therapeutic development, as well as further investigations into how our discoveries of bacteriocin-producing plant commensal microorganisms may have the potential for an immediate impact on the safety of food supplies.

Scan Parameter Optimization for Histotripsy Treatment of S. Aureus Biofilms on Surgical Mesh.

There is a critical need to develop new noninvasive therapies to treat bacteria biofilms. Previous studies have demonstrated the effectiveness of cavitation-based ultrasound histotripsy to destroy these biofilms. In this study, the dependence of biofilm destruction on multiple scan parameters was assessed by conducting exposures at different scan speeds (0.3-1.4 beamwidths/s), step sizes (0.25-0.5 beamwidths), and the number of passes of the focus across the mesh (2-6). For each of the exposure conditions, the number of colony-forming units (CFUs) remaining on the mesh was quantified. A regression analysis was then conducted, revealing that the scan speed was the most critical parameter for biofilm destruction. Reducing the number of passes and the scan speed should allow for more efficient biofilm destruction in the future, reducing the treatment time.

Impact of High-Intensity Ultrasound on Strength of Surgical Mesh When Treating Biofilm Infections.

The use of cavitation-based ultrasound histotripsy to treat infections on surgical mesh has shown great potential. However, any impact of the therapy on the mesh must be assessed before the therapy can be applied in the clinic. The goal of this study was to determine if the cavitation-based therapy would reduce the strength of the mesh thus compromising the functionality of the mesh. First, Staphylococcus aureus biofilms were grown on the surgical mesh samples and exposed to high-intensity ultrasound pulses. For each exposure, the effectiveness of the therapy was confirmed by counting the number of colony forming units (CFUs) on the mesh. Most of the exposed meshes had no CFUs with an average reduction of 5.4-log10 relative to the sham exposures. To quantify the impact of the exposure on mesh strength, the force required to tear the mesh and the maximum mesh expansion before damage were quantified for control, sham, and exposed mesh samples. There was no statistical difference between the exposed and sham/control mesh samples in terms of ultimate tensile strength and corresponding mesh expansion. The only statistical difference was with respect to mesh orientation relative to the applied load. The tensile strength increased by 1.36 N while the expansion was reduced by 1.33 mm between different mesh orientations.

Histotripsy Treatment of S. Aureus Biofilms on Surgical Mesh Samples Under Varying Scan Parameters.

Cavitation-based ultrasound histotripsy has shown potential for treating infections on surgical mesh. The goal of this paper was to explore a new scan strategy while assessing the impact of scan speed, scan step size, and the number of cycles in the tone burst on the destruction of S. aureus biofilms grown on surgical mesh samples using ultrasound histotripsy pulses (150 MPa/-17 MPa). For each exposure, the number of colony forming units (CFUs) on the mesh and released onto the surrounding gel was quantified. Most of the exposed mesh samples had no CFUs, and there was a statistically significant reduction in CFUs on the mesh for each of the exposures, with an average reduction of 3.8 log10 relative to the sham. Compared with the sham, there was also a statistically significant reduction in CFUs on the gel with the highest exposures.

Histotripsy Treatment of S. Aureus Biofilms on Surgical Mesh Samples Under Varying Pulse Durations.

Prior studies demonstrated that histotripsy generated by high-intensity tone bursts to excite a bubble cloud adjacent to a medical implant can destroy the bacteria biofilm responsible for the infection. The goal of this paper was to treat Staphylococcus aureus (S. aureus) biofilms on surgical mesh samples while varying the number of cycles in the tone burst to minimize collateral tissue damage while maximizing therapy effectiveness. S. aureus biofilms were grown on 1-cm square surgical mesh samples. The biofilms were then treated in vitro using a spherically focused transducer (1.1 MHz, 12.9-cm focal length, 12.7-cm diameter) using either a sham exposure or histotripsy pulses with tone burst durations of 3, 5, or 10 cycles (pulse repetition frequency of 333 Hz, peak compressional pressure of 150 MPa, peak rarefactional pressure of 17 MPa). After treatment, the number of colony forming units (CFUs) on the mesh and the surrounding gel was independently determined. The number of CFUs remaining on the mesh for the sham exposure (4.8 ± 0.9-log10) (sample mean ± sample standard deviation-log10 from 15 observations) was statistically significantly different from the 3-cycle (1.9 ± 1.5-log10), 5-cycle (2.2 ± 1.1-log10), and 10-cycle exposures (1 ± 1.5-log10) with an average reduction in the number of CFUs of 3.1-log10. The numbers of CFUs released into the gel for both the sham and exposure groups were the same within a bound of 0.86-log10, but this interval was too large to deduce the fate of the bacteria in the biofilm following the treatment.

Design and evaluation of a peptide-based immunotoxin for breast cancer therapeutics.

Immunotoxins are chimeric proteins comprising a specific cellular targeting domain linked to a cytotoxic factor. Here we describe the design and use of a novel, peptide-based immunotoxin that can initiate selective cytotoxicity on ErbB2-positive cells. ErbB2 is a receptor tyrosine kinase that is overexpressed in the tumor cells of approximately 30% of breast cancer patients. Immunotoxin candidates were designed to incorporate a targeting ligand with affinity for ErbB2 along with a membrane lysin-based toxin domain. One particular peptide candidate, NL1.1-PSA, demonstrated selective cytotoxicity towards ErbB2-overexpressing cell lines. We utilized a bioengineering strategy to show that recombinant NL1.1-PSA immunotoxin expression by Escherichia coli also conferred selective cytotoxicity towards ErbB2-overexpressing cells. Our findings hold significant promise for the use of effective immunotoxins in cancer therapeutics.

A multiparametric computational algorithm for comprehensive assessment of genetic mutations in mucopolysaccharidosis type IIIA (Sanfilippo syndrome).

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome) is a Lysosomal Storage Disease caused by cellular deficiency of N-sulfoglucosamine sulfohydrolase (SGSH). Given the large heterogeneity of genetic mutations responsible for the disease, a comprehensive understanding of the mechanisms by which these mutations affect enzyme function is needed to guide effective therapies. We developed a multiparametric computational algorithm to assess how patient genetic mutations in SGSH affect overall enzyme biogenesis, stability, and function. 107 patient mutations for the SGSH gene were obtained from the Human Gene Mutation Database representing all of the clinical mutations documented for Sanfilippo syndrome. We assessed each mutation individually using ten distinct parameters to give a comprehensive predictive score of the stability and misfolding capacity of the SGSH enzyme resulting from each of these mutations. The predictive score generated by our multiparametric algorithm yielded a standardized quantitative assessment of the severity of a given SGSH genetic mutation toward overall enzyme activity. Application of our algorithm has identified SGSH mutations in which enzymatic malfunction of the gene product is specifically due to impairments in protein folding. These scores provide an assessment of the degree to which a particular mutation could be treated using approaches such as chaperone therapies. Our multiparametric protein biogenesis algorithm advances a key understanding in the overall biochemical mechanism underlying Sanfilippo syndrome. Importantly, the design of our multiparametric algorithm can be tailored to many other diseases of genetic heterogeneity for which protein misfolding phenotypes may constitute a major component of disease manifestation.

Knowing is half the battle: targeting virulence factors of group A Streptococcus for vaccine and therapeutics.

Group A Streptococcus (GAS) is a leading human pathogen that causes a multitude of diseases from pharyngitis, and impetigo, to more severe outcomes such as rheumatoid arthritis and necrotizing fasciitis. GAS remains a global burden as currently no vaccine exists that is completely effective. In this review we highlight recent studies on the virulence of GAS and present several approaches that have extended those findings into aims at combating GAS disease. These and other studies such as recent genome-wide efforts into host-pathogen relationships of GAS disease will likely reveal new targets of intervention. Given the recent rise in GAS strains that have acquired resistance to several types of antibiotics, it is crucial that we continue to increase our knowledge of the mechanisms underlying GAS disease.

Influence of urban landscapes on population dynamics in a short-distance migrant mosquito: evidence for the dengue vector Aedes aegypti.

BACKGROUND: Dengue viruses are endemic across most tropical and subtropical regions. Because no proven vaccines are available, dengue prevention is primarily accomplished through controlling the mosquito vector Aedes aegypti. While dispersal distance is generally believed to be approximately 100 m, patterns of dispersion may vary in urban areas due to landscape features acting as barriers or corridors to dispersal. Anthropogenic features ultimately affect the flow of genes affecting vector competence and insecticide resistance. Therefore, a thorough understanding of what parameters impact dispersal is essential for efficient implementation of any mosquito population suppression program. Population replacement and genetic control strategies currently under consideration are also dependent upon a thorough understanding of mosquito dispersal in urban settings. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the effect of a major highway on dispersal patterns over a 2 year period. A. aegypti larvae were collected on the east and west sides of Uriah Butler Highway (UBH) to examine any effect UBH may have on the observed population structure in the Charlieville neighborhood in Trinidad, West Indies. A panel of nine microsatellites, two SNPs and a 710 bp sequence of mtDNA cytochrome oxidase subunit 1 (CO1) were used for the molecular analyses of the samples. Three CO1 haplotypes were identified, one of which was only found on the east side of the road in 2006 and 2007. AMOVA using mtCO1 and nuclear markers revealed significant differentiation between the east- and west-side collections. CONCLUSION AND SIGNIFICANCE: Our results indicate that anthropogenic barriers to A. aegypti dispersal exist in urban environments and should be considered when implementing control programs during dengue outbreaks and population suppression or replacement programs.