Storage Methods of Lactic Acid Bacteria Strains separated from DVS

Abstract: Randomly amplified polymorphic DNA (RAPD) and morphological observation and fermentation property analysis were used to evaluate the different storage methods of three lactic acid bacteria strains separated from Directed Vat Set (DVS). Methods: Evaluate the genome change of the different samples from different periods during 4℃ storage, -80℃ storage and lyophilization using RAPD. Observe the morphological features by microscopy after recovered from different time of storage, then used each strain as a starter culture of fresh milk, observe the fermentation properties. Results: The RAPD patterns and fermentation property of the three strains all changed during 4℃ and -80℃ storage, and they all showed a good stability when stored by lyophilization. Suggests: This is the first time to use RAPD technique and fermentation property observation in lactic acid bacteria storage research work, RAPD appears to be an efficient method for evaluating of storage effect of lactic acid bacteria strain

Structural Analysis of Alkaline β-Mannanase from Alkaliphilic Bacillus sp. N16-5: Implications for Adaptation to Alkaline Conditions

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)8-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)8-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pKa calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism

Immunization of Mice with Recombinant Protein CobB or AsnC Confers Protection against Brucella abortus Infection

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy

DMSP-producing bacteria are more abundant in the surface microlayer than subsurface seawater of the East China Sea

Microbial production and catabolism of dimethylsulfoniopropionate (DMSP), generating the climatically active gases dimethyl sulfide (DMS) and methanethiol (MeSH), have key roles in global carbon and sulfur cycling, chemotaxis, and atmospheric chemistry. Microorganisms in the sea surface microlayer (SML), the interface between seawater and atmosphere, likely play an important role in the generation of DMS and MeSH and their exchange to the atmosphere, but little is known about these SML microorganisms. Here, we investigated the differences between bacterial community structure and the distribution and transcription profiles of the key bacterial DMSP synthesis (dsyB and mmtN) and catabolic (dmdA and dddP) genes in East China Sea SML and subsurface seawater (SSW) samples. Per equivalent volume, bacteria were far more abundant (~ 7.5-fold) in SML than SSW, as were those genera predicted to produce DMSP. Indeed, dsyB (~ 7-fold) and mmtN (~ 4-fold), robust reporters for bacterial DMSP production, were also far more abundant in SML than SSW. In addition, the SML had higher dsyB transcripts (~ 3-fold) than SSW samples, which may contribute to the significantly higher DMSP level observed in SML compared with SSW. Furthermore, the abundance of bacteria with dmdA and their transcription were higher in SML than SSW samples. Bacteria with dddP and transcripts were also prominent, but less than dmdA and presented at similar levels in both layers. These data indicate that the SML might be an important hotspot for bacterial DMSP production as well as generating the climatically active gases DMS and MeSH, a portion of which are likely transferred to the atmosphere

Development of aptamer-based low-background assays for adenosine triphosphate detection

Aptamers are unique single-stranded oligonucleic acids that can recognize some biomolecules with high specificity and affinity. In this study, this unique property has been applied to develop two different aptamer-based fluorescence bioassays for the detection of adenosine triphosphate (ATP). The first assay made use of an aptamer-hairpin complex composed of an ATP-specific aptamer sequence and a signaling sequence. In the presence of ATP, the aptamer specifically binded to ATP to open the hairpin structure, subsequently allowing the signaling sequence to hybridize with substrate for fluorescence signal readout. The high fluorescence background of the substrate was minimized by a Zn2+-induced energy transfer mechanism from the fluorophore to the quencher so as to significantly improve the signal-to-noise ratio. This assay demonstrated a linear detection range of ATP from 1 M to 2 mM with a detection limit of 0.15 M. To further improve the assay sensitivity for the ATP detection, a Mung Bean Nuclease (MBN) assisted signal amplification scheme was adopted in the second assay. A sensing element was designed by hybridizing the ATP-specific aptamer with two complementary sequences, one modified with fluorescein amidite (FAM) and the other with black-hole quencher (BHQ). Upon the binding of the aptamer with ATP, the double-stranded structure was denatured, and MBN then digested the single-stranded sequences to release the bound ATP from aptamer-ATP complex for binding in the next cycle. Under optimal conditions, the assay achieved a low detection limit of 7.9 nM for ATP detection, with a wide dynamic range of 10 nM to 2 mM. The two assays provided convenient means for protein detection, with potential clinical applications.Master of Engineering (MSE

A highly sensitive and specific biosensor for ligation- and PCR-free detection of MicroRNAs

10.1016/j.bios.2011.02.029Biosensors and Bioelectronics2693768-377

Synthetic Life with Alternative Nucleic Acids as Genetic Materials

DNA, the fundamental genetic polymer of all living organisms on Earth, can be chemically modified to embrace novel functions that do not exist in nature. The key chemical and structural parameters for genetic information storage, heredity, and evolution have been elucidated, and many xenobiotic nucleic acids (XNAs) with non-canonical structures are developed as alternative genetic materials in vitro. However, it is still particularly challenging to replace DNAs with XNAs in living cells. This review outlines some recent studies in which the storage and propagation of genetic information are achieved in vivo by expanding genetic systems with XNAs

Systematic Chemical Analysis Approach Reveals Superior Antioxidant Capacity via the Synergistic Effect of Flavonoid Compounds in Red Vegetative Tissues

The flavonoid system comprises an abundance of compounds with multiple functions; however, their potential synergism in antioxidant function remains unclear. We established an approach using ever-red (RL) and ever-green leaves (GL) of crabapple cultivars during their development to determine interrelationships among flavonoid compounds. RL scored significantly better than GL in terms of the type, composition, and diversity of flavonoids than GL. Principal component analysis predicted flavonoids in RL to have positive interaction effects, and the total antioxidant capacity was significantly higher than the sum of antioxidant capacities of the individual compounds. This synergy was verified by the high antioxidant capacity in rat serum after feeding on red leaves. Our findings suggest that the synergistic effect is a result of the high transcription levels regulated by McMYBs in RL. In summary, individual flavonoids cooperate in a flavonoid system, thus producing a synergistic antioxidant effect, and the approach used herein can provide insights into the roles of flavonoids and other compounds in future studies