Bile Salt Hydrolase (Bsh) Activity Screening of Lactobacilli: In Vitro Selection of Indigenous Lactobacillus Strains with Potential Bile Salt Hydrolysing and Cholesterol-Lowering Ability

The bile salt hydrolase (Bsh) activity of probiotic bacterium residing in gastrointestinal tract has often being associated with its cholesterol-lowering effects. Hence, Bsh activity was explored in this study as the criterion for the selection of most potential Bsh-active and cholesterol-lowering indigenous Lactobacillus strains. Forty lactobacilli were adjudged Bsh active after a preliminary screening of 102 lactobacilli and occurrence of Bsh activity correlated well with their natural habitats. Of the 40 shortlisted lactobacilli, fifteen putative Lactobacillus strains were selected and further tested for their comparative Bsh activity. In the end, indigenous Lactobacillus plantarum strains Lp91 and Lp21 were emerged as the promising Bsh-active lactobacilli with their substrate preference inclined more towards glycocholate than other bile acid amino conjugates. In addition, strains Lp91 and Lp21 also exhibited significantly high bile salt deconjugation, cholesterol assimilation and cholesterol co-precipitation ability in vitro. In conclusion, indigenous L. plantarum strains Lp91 and Lp21 may be the promising candidate probiotics to elucidate the ecological significance of probiotic Bsh activity in vivo.     

Functional remineralization of dentin lesions using polymer-induced liquid-precursor process

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 μm deep artificial caries lesions were remineralized via the PILP process for 7-28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (E(R) = 0.2 GPa) region extending about 70 μm into the lesion, with a sloped region to about 140 μm where values reached normal dentin (18-20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7-14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of E(R) compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone.     

Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1

? Interactions between the Arabidopsis NitRate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity nitrate transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for nitrate transport. ? Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and nitrate uptake in Xenopus oocytes. ? All NRT2s, except for AtNRT2.7, restored growth and β-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 transporters interact strongly with AtNAR2.1. ? Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.     

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.     

Antioxidative potential of lactobacilli isolated from the gut of Indian people

Oxidative stress is one of the major causes of degenerative conditions occurring at cellular level with serious health implications. This study was aimed at investigating the antioxidative potentials of probiotic lactobacilli of Indian gut origin and their ability to augment antioxidant defense enzyme systems in the host cells under oxidative stress conditions. A total of 39 Lactobacillus cultures were assessed for their resistance against reactive oxygen species. Most of the cultures were moderately to strongly resistant towards 0.4 mM H(2)O(2). The Lactobacillus isolate CH4 was the most H(2)O(2) resistant culture with only 0.06 log cycle reduction. Majority of the cultures demonstrated high resistance towards hydroxyl ions and Lp21 was the most resistant with log count reduction of 0.20 fold only. Almost all the cultures were also quite resistant to superoxide anions. Lp21 also showed the highest superoxide dismutase content (0.8971 U). Amongst the 39 cultures, Lactobacillus spp. S3 showed the highest total antioxidative activity of 77.85 ± 0.13 % followed by Lp55 (56.1 ± 1.2 %) in terms of per cent inhibition of linolenic acid oxidation. Lp9 up-regulated the expression of superoxide dismutase 2 gene in HT-29 cells both at 0.1 mM (1.997 folds) and 1.0 mM H(2)O(2) (2.058 folds) concentrations. In case of glutathione peroxidase-1, Lp9, Lp91 and Lp55 showed significant (P < 0.001) up-regulation in the gene expression to the level of 5.451, 8.706 and 10.083 folds, respectively when HT-29 was challenged with 0.1 mM H(2)O(2). The expression of catalase gene was also significantly up-regulated by all the cultures at 0.1 mM H(2)O(2) conditions. It can be concluded that the antioxidative efficacy of the putative probiotic lactobacilli varied considerably between species and strains and the potential strains can be explored as prospective antioxidants to manage oxidative stress induced diseases.     

Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury

Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in this protection, we airway-instilled mice first with lipopolysaccharide (LPS) and then with either mouse BMSCs (mBMSCs) or human BMSCs (hBMSCs). Live optical studies revealed that the mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelia in these mice, releasing mitochondria-containing microvesicles that the epithelia engulfed. The presence of BMSC-derived mitochondria in the epithelia was evident optically, as well as by the presence of human mitochondrial DNA in mouse lungs instilled with hBMSCs. The mitochondrial transfer resulted in increased alveolar ATP concentrations. LPS-induced ALI, as indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by the instillation of wild-type mBMSCs but not of mutant, GJC-incompetent mBMSCs or mBMSCs with dysfunctional mitochondria. This is the first evidence, to our knowledge, that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.     

Natural transformation of Gallibacterium anatis

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Molecular characterization of hot spring cyanobacteria and evaluation of their photoprotective compounds

Phylogenetic analysis of 4 cyanobacterial strains isolated from hot springs in Rajgir, India, was carried out using the 16S rRNA gene (1400 bp). These strains were identified as members of Chroococcales ( Cyanothece sp. strain HKAR-1) and Nostocales ( Nostoc sp. strain HKAR-2, Scytonema sp. strain HKAR-3, and Rivularia sp. strain HKAR-4). Furthermore, we evaluated the presence of ultraviolet-screening and (or) photoprotective compounds, such as mycosporine-like amino acids (MAAs) and scytonemin, in these cyanobacteria by using high-performance liquid chromatography. Well-characterized MAAs, including the critical and highly polar compounds shinorine, porphyra-334, and mycosporine-glycine, as well as several unknown MAAs, were found in these hot-spring-inhabiting microorganisms. The presence of scytonemin was detected only in Scytonema sp. strain HKAR-3 and Rivularia sp. strain HKAR-4. The results indicate that hot spring cyanobacteria, namely Cyanothece, Nostoc, Scytonema, and Rivularia, belonging to different groups possess various photoprotective compounds to cope up with the negative impacts of damaging radiations.