Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307^T. MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml⁻¹ inhibited the growth of both Gram-positive and Gram-negative bacteria by 58–89% and a dose of 500 µg ml⁻¹ scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml⁻¹ of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.     

Akt contributes to activation of the TRIF-dependent signaling pathways of TLRs by interacting with TANK-binding kinase 1

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.     

Genomic and metabolic profiling of nonulosonic acids in Vibrionaceae reveal biochemical phenotypes of allelic divergence in Vibrio vulnificus

Nonulosonic acids (NulOs) encompass a large group of structurally diverse nine-carbon backbone α-keto sugars widely distributed among the three domains of life. Mammals express a specialized version of NulOs called sialic acids, which are displayed in prominent terminal positions of cell surface and secreted glycoconjugates. Within bacteria, the ability to synthesize NulOs has been demonstrated in a number of human pathogens and is phylogenetically widespread. Here we examine the distribution, diversity, evolution, and function of NulO biosynthesis pathways in members of the family Vibrionaceae. Among 27 species of Vibrionaceae examined at the genomic level, 12 species contained nab gene clusters. We document examples of duplication, divergence, horizontal transfer, and recombination of nab gene clusters in different Vibrionaceae lineages. Biochemical analyses, including mass spectrometry, confirmed that many species do, in fact, produce di-N-acetylated NulOs. A library of clinical and environmental isolates of Vibrio vulnificus served as a model for further investigation of nab allele genotypes and levels of NulO expression. The data show that lineage I isolates produce about 20-fold higher levels of NulOs than lineage II isolates. Moreover, nab gene alleles found in a subset of V. vulnificus clinical isolates express 40-fold higher levels of NulOs than nab alleles associated with environmental isolates. Taken together, the data implicate the family Vibrionaceae as a "hot spot" of NulO evolution and suggest that these molecules may have diverse roles in environmental persistence and/or animal virulence.     

Telomere replication: poised but puzzling

Faithful replication of chromosomes is essential for maintaining genome stability. Telomeres, the chromosomal termini, pose quite a challenge to replication machinery due to the complexity in their structures and sequences. Efficient and complete replication of chromosomes is critical to prevent aberrant telomeres as well as to avoid unnecessary loss of telomere DNA. Compelling evidence supports the emerging picture of synergistic actions between DNA replication proteins and telomere protective components in telomere synthesis. This review discusses the actions of various replication and telomere-specific binding proteins that ensure accurate telomere replication and their roles in telomere maintenance and protection.     

Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning

Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.