Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

Diversity analysis of dairy and non-dairy strains of Lactococcus lactis ssp. lactis by multilocus sequence analysis (MLSA)

The genetic diversity of 31 identified strains of Lactococcus lactis ssp. lactis isolated from different dairy and non-dairy sources were investigated at gene level using multilocus sequence analysis (MLSA) and PCR-RFLP based on the differences in four selected partial protein coding gene sequences: araT, encoding aromatic amino acid-specific aminotransferase; dtpT, encoding di/tri peptide transporter; yueF, encoding non-proteolytic protein, peptidase, M16 family; and pdhA, encoding pyruvate dehydrogenase E1 component α-subunit. A set of seven test strains from different isolation sources and one reference strain, L. lactis ssp. lactis NCDC 094, were analyzed by MLSA. The strains showed distinct diversity among themselves and exhibited a greater percent similarity with reference strains L. lactis ssp. lactis CV56 (CP002365.1), IL1403 (AE005176.1), and KF147 (CP001834.1) in comparison with L. lactis ssp. cremoris NZ9000 (CP002094.1), MG1363 (AM406671.1), and SK11 (CP00425.1). The MLSA revealed one distinct genomic lineage within strains exclusively of L. lactis ssp. lactis. This analysis also revealed no source-wise genetic relationship in the test strains analyzed. Further, PCR-RFLP of araT, dtpT, yueF and pdhA also characterized the single genomic lineage exclusively of L. lactis ssp. lactis within a total of 24 test strains.     

Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

Lipofection and nucleofection of substrate plasmid can generate widely different readings of DNA end-joining efficiency in different cell lines

In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ) – a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters. To help standardize these assays, we searched for parameters underpinning these variations and identified transfection method as an important determinant. Here, we compare a lipid-based transfection method, lipofection, with an electroporation method, nucleofection, and find large, unanticipated and cell line-dependent differences in percent end-joining without recognizable trends. For example, in rodent cells, transfection using lipofection gives nearly WT end-joining in DNA-PKcs mutants and only mildly inhibited end-joining in Lig4 and Ku mutants. In contrast, transfection using nucleofection shows marked end-joining inhibition in all NHEJ mutants tested as compared to the WT. In human HCT116 cells, end-joining after nucleofection is strongly suppressed even in the WT and the differences to the mutants are small. After lipofection, in contrast, end-joining is high in WT cells and markedly suppressed in the mutants. We conclude that better understanding and control of the physicochemical/biological and analytical parameters underpinning these differences will be required to generate with plasmid assays results with quantitative power comparable to that of well-established methods of DSB analysis such as pulsed-field gel electrophoresis or γ-H2AX foci scoring. Until then, caution is needed in the interpretation of the results obtained – particularly with reference to pathway efficiency and residual damage – and confirmation of critical results with alternative transfection approaches is advisable.     

Comparative analysis of chromosome 2A molecular organization in diploid and hexaploid wheat

Molecular Biology Reports - Diploid A genome wheat species harbor immense genetic variability which has been targeted and proven useful in wheat improvement. Development and deployment of...     

Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.     

Variation in the Activity of Three Principal Detoxifying Enzymes in Major Sucking Pest of Tea, Helopeltis theivora Waterhouse (Heteroptera: Miridae) from Sub-Himalayan Tea Plantations of West Bengal, India

Helopeltis theivora Waterhouse (Heteroptera: Miridae), is a major sucking pest of tea in North East India along with other tea growing countries. In West Bengal, tea is cultivated in three sub-Himalayan regions, Terai (foothill plains western to river Teesta), the Dooars (foothill plains eastern to river Teesta) and the Darjeeling hill slopes. Most plantations, in these regions are managed conventionally i.e. by spraying different synthetic insecticides and a few by organic farming using different herbal and microbial insecticides. In conventional plantations, continuous application of insecticides may lead to the selection of more tolerant H. theivora populations making the pest difficult to control. So, there is a pressing need to know the biochemical variability in relation to the metabolic resistance in the pest populations and develop a population specific control strategy. Activity of three principal insecticide detoxifying enzymes in H. theivora populations were studied from three tea growing regions of North Bengal. Higher levels of activity of all the studied enzymes were found from conventional tea plantations. In male H. theivora, the activity of general esterases (GEs) was 6.6–11.2 and 10.5–11.4 fold higher, cytochrome P450 (CYPs) was 2.0–3.2 and 3.0–3.2 fold higher and glutathione S-transferase (GSTs) was 5.2–8.3 and 6.4–8.7 fold higher in Terai and the Dooars populations, respectively than organic populations from Darjeeling hill slopes. Similarly, in female H. theivora, activity of GEs was 6.2–10.3 and 8.3–9.6 folds higher, CYPs was 1.9–3.2 and 3.0–3.3 fold, and GSTs was 3.5–5.4 and 4.4–6.0 fold higher in Terai and the Dooars, respectively than the organic populations from Darjeeling hill slopes. The activities of all three enzymes were found to be significantly low in organic plantations from Darjeeling hill slopes. Esterase I–VI isozymes with higher level of expression were found in specimens from conventional tea plantations than organic populations of H. theivora from Darjeeling hill slopes in isozymes study.     

Evaluation of Nutritive Value of Raw and Fermented De-oiled Physic Nut, Jatropha curcas Seed Meal in the Formulated Diets for Rohu, Labeo rohita (Hamilton) Fingerlings

Rohu (Labeo rohita) fingerlings, were fed de-oiled Jatropha curcas seed meal (DJSM) supplemented diets for 60 days and the effectiveness, if any, on the growth was evaluated. Nine isonitrogenous (35 % crude protein) and isocaloric (4.0 kcal g^?1) diets were formulated of which one was reference diet (RD, fishmeal based control diet) and the other eight were experimental diets prepared by incorporating raw (D1–D4) and fermented (D5–D8) DJSM at 10, 20, 30 and 40 % levels by weight, respectively. Autoclaved DJSM was processed through solid state fermentation (SSF) for 15 days at 37 ± 1 °C by an exo-enzyme producing bacterium, Bacillus cereus Lr.H.23 isolated from the hindgut of rohu, L. rohita. Processing through SSF caused decrease in the contents of crude fibre and anti-nutritional factors, but increase in the levels of free amino acids and free fatty acids. In terms of growth performance, feed utilization efficiency and apparent protein digestibility, fish fed diet D7 containing 30 % fermented DJSM showed the best performance, which differed significantly (P < 0.05) from that of the fish fed diets containing raw DJSM. The results indicated that an inclusion level up to 30 % fermented DJSM replacing 15 % FM in the practical diet for rohu fingerlings can be proposed when compared to the RD. However, further experiments are required to recommend the ingredient for use in industry.