Bacteriocinogenic Potential of Bacillus amyloliquefaciens Isolated from Kimchi,a Traditional Korean Fermented Cabbage

Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.

     

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.     

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

Physiological and anatomical responses of wheat to induced dehydration and rehydration

Hydroponically grown wheat seedlings of two prominent Bulgarian cultivars (Katya and Prelom) were subjected to 48 h osmotic stress with PEG 8000 and were then rehydrated. The degree of stress was evaluated by monitoring relative water content, lipid peroxidation level, and accumulation of free proline and hydrogen peroxide in the leaves. Anatomy and ultrastructure of leaf tissue were observed under light microscopy. After imposition of stress, drought tolerant cultivar Katya displayed higher free proline content and significantly lower malondialdehyde and peroxide concentration in leaves than in the leaves of susceptible cultivar Prelom. After 24 h of rehydration Katya showed better ability to restore leaf water status and an apparent tendency towards recovery, whereas Prelom sustained higher levels of hydrogen peroxide, lipid peroxidation products and free proline and markedly low relative water content. Here, we have uncovered some of the characteristics displayed by cultivar Katya that enable it to survive and recover from severe osmotic stress. Interestingly, there was congruence between our results and the high level of cultivar Katya drought tolerance observed in the field.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

Polymer gene delivery vectors encapsulated in thermally sensitive bioreducible shell

Stable, nanosized polyelectrolyte complexes between rationally designed thermally sensitive block copolymers and plasmid DNA (polyplexes) were formed and their in vitro transfection efficiency was tested. The polyplexes were further stabilized through encapsulation into a biodegradable polymer shell. Although reduced as compared to that of the corresponding polyplexes, the encapsulated systems still show acceptable transfection efficiency. That opens the possibility to tune the balance between the safe transport and efficient delivery of DNA into the cells.     

Twist and Degrade—Impact of Molecular Structure on the Photostability of Nonfullerene Acceptors and Their Photovoltaic Blends

Nonfullerene acceptors (NFAs) dominate organic photovoltaic (OPV) research due to their promising efficiencies and stabilities. However, there is very little investigation into the molecular processes of degradation, which is critical to guiding design of novel NFAs for long‐lived, commercially viable OPVs. Here, the important role of molecular structure and conformation in NFA photostability in air is investigated by comparing structurally similar but conformationally different promising NFAs: planar O‐IDTBR and nonplanar O‐IDFBR. A three‐phase degradation process is identified: i) initial photoinduced conformational change (i.e., torsion about the core–benzothiadiazole dihedral), induced by noncovalent interactions with environmental molecules, ii) followed by photo‐oxidation and fragmentation, leading to chromophore bleaching and degradation product formation, and iii) finally complete chromophore bleaching. Initial conformational change is a critical prerequisite for further degradation, providing fundamental understanding of the relative stability of IDTBR and IDFBR, where the already twisted IDFBR is more prone to degradation. When blended with the donor polymer poly(3‐hexylthiophene), both NFAs exhibit improved photostability while the photostability of the polymer itself is significantly reduced by the more miscible twisted NFA. The findings elucidate the important role of NFA molecular structure in photostability of OPV systems, and provide vital insights into molecular design rules for intrinsically photostable NFAs.     

Characterization and potential antitumor effect of a heteropolysaccharide produced by the red alga Porphyridium sordidum

Taking into account the rising trend of the incidence of cancers of various organs, effective therapies are urgently needed to control human malignancies. However, almost all chemotherapy drugs currently on the market cause serious side effects. Fortunately, several studies have shown that some non‐toxic biological macromolecules, including algal polysaccharides, possess anti‐cancer activities or can increase the efficacy of conventional chemotherapy drugs. Polysaccharides are characteristic secondary metabolites of many algae. The efficacy of polysaccharides on the normal and cancer cells is not well investigated, but our investigations proved a cell specific effect of a newly isolated extracellular polysaccharide from the red microalga Porphyridium sordidum. The investigated substance was composed of xylose:glucose and galactose:manose:rhamnose in a molar ratio of 1:0.52:0.44:0.31. Reversible electroporation has been exploited to increase the transport through the plasma membrane into the tested breast cancer tumor cells MCF‐7 and MDA‐MB231. Application of 75 µg/mL polysaccharide in combination with 200 V/cm electroporation induced 40% decrease in viability of MDA‐MB231 cells and changes in cell morphology while control cells (MCF10A) remained with normal morphology and kept vitality.