<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pineapple var. Queen using a novel encapsulation-based antibiotic selection technique

A protocol for Agrobacterium-mediated transformation of a local ‘elite’ Indian variety (Queen) of pineapple [Ananus comosus (L.) Merr, family Bromeliaceae] has been established using a standard transformation vector (pCAMBIA 1304). High transformation efficiency, expressed as the mean percentage of transgenic micro-shoots regenerated from initial callus explants (20.6%) was achieved using a novel encapsulation-based, antibiotic selection procedure. The Agrobacterium-infected micro-shoots derived from callus explants survived selection in high concentration of hygromycin (60 mg l⁻¹ and beyond) in encapsulated alginate beads. The integration of transgene in hygromycin-resistant shoots and plants was confirmed by histochemical GUS assay, PCR amplification and Southern hybridization. It is possible to eliminate false antibiotic positives in pineapple transformation program to a large extent following this procedure.     

Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions

High-affinity antibodies are critical for numerous diagnostic and therapeutic applications, yet their utility is limited by their variable propensity to aggregate either at low concentrations for antibody fragments or high concentrations for full-length antibodies. Therefore, determining the sequence and structural features that differentiate aggregation-resistant antibodies from aggregation-prone ones is critical to improving their activity. We have investigated the molecular origins of antibody aggregation for human V(H) domain antibodies that differ only in the sequence of the loops containing their complementarity determining regions (CDRs), yet such antibodies possess dramatically different aggregation propensities in a manner not correlated with their conformational stabilities. We find the propensity of these antibodies to aggregate after being transiently unfolded is not a distributed property of the CDR loops, but can be localized to aggregation hotspots within and near the first CDR (CDR1). Moreover, we have identified a triad of charged mutations within CDR1 and a single charged mutation adjacent to CDR1 that endow the poorly soluble variant with the desirable biophysical properties of the aggregation-resistant antibody. Importantly, we find that several other charged mutations in CDR1, non-CDR loops and the antibody scaffold are incapable of preventing aggregation. We expect that our identification of aggregation hotspots that govern antibody aggregation within and proximal to CDR loops will guide the design and selection of antibodies that not only possess high affinity and conformational stability, but also extreme resistance to aggregation.     

Root associated iron oxidizing bacteria increase phosphate nutrition and influence root to shoot partitioning of iron in tolerant plant Typha angustifolia

Aims

Typha angustifolia is a heavy metal tolerant plant that grows in a uranium mine tailings highly contaminated with iron. In this study three iron oxidizing microbes (FeOBs) isolated from Typha rhizoplane were investigated for their role in plant growth promotion (PGP). Their effect on iron nutrition in Typha under iron replete and excess condition was also evaluated.

Methods

The PGP activities of the FeOBs were studied by measuring their influence on plant growth. To investigate the mechanism of growth promotion their ability to solubilize phosphate, and to produce Indole acetic acid and siderophores were studied. The influence of the FeOBs on root to shoot partitioning of iron was tested by measuring total iron content in roots and shoots treated with microbes.

Results

The FeOBs were named as Paenibacillus cookii JGR8, (MTCC12002), Pseudomonas jaduguda JGR2 (LMG25820) and Bacillus megaterium JGR9 (MTCC12001). The siderophore producers, influenced iron accumulation in the plant root. Additionally P. pseudoalcaligenes JGR2 increased shoot iron content overcoming the root- shoot barrier that allows Typha to exclude metals from its shoot. Among the PGP mechanisms tested, ability to solubilize phosphate appeared to be most significant for increasing the plant biomass.

Conclusion

FeOBs that produce siderophore increased iron content in plant and therefore can be of immense biotechnological importance. However Biomass increase was directly correlated with increased phosphate acquisition and not with enhanced iron accumulation in Typha.     

Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes

In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.     

Regulating the discriminatory response to antigen by T-cell receptor

The cell-mediated immune response constitutes a robust host defense mechanism to eliminate pathogens and oncogenic cells. T cells play a central role in such a defense mechanism and creating memories to prevent any potential infection. T cell recognizes foreign antigen by its surface receptors when presented through antigen-presenting cells (APCs) and calibrates its cellular response by a network of intracellular signaling events. Activation of T-cell receptor (TCR) leads to changes in gene expression and metabolic networks regulating cell development, proliferation, and migration. TCR does not possess any catalytic activity, and the signaling initiates with the colocalization of several enzymes and scaffold proteins. Deregulation of T cell signaling is often linked to autoimmune disorders like severe combined immunodeficiency (SCID), rheumatoid arthritis, and multiple sclerosis. The TCR remarkably distinguishes the minor difference between self and non-self antigen through a kinetic proofreading mechanism. The output of TCR signaling is determined by the half-life of the receptor antigen complex and the time taken to recruit and activate the downstream enzymes. A longer half-life of a non-self antigen receptor complex could initiate downstream signaling by activating associated enzymes. Whereas, the short-lived, self-peptide receptor complex disassembles before the downstream enzymes are activated. Activation of TCR rewires the cellular metabolic response to aerobic glycolysis from oxidative phosphorylation. How does the early event in the TCR signaling cross-talk with the cellular metabolism is an open question. In this review, we have discussed the recent developments in understanding the regulation of TCR signaling, and then we reviewed the emerging role of metabolism in regulating T cell function.     

Synthesis and reactivity of azobenzene-based bispropargyl sulfones: interesting comparison between cyclic and acyclic systems

Azobenzene-based bispropargyl bissulfone 3 containing stable E-azo moiety has been synthesized. Upon irradiation with long wavelength UV it isomerized to the Z-form 4, which can be thermally reisomerized to the E-isomer. Reactivity towards isomerization to the allenic system as well as DNA-cleaving efficiency under basic conditions was found to be significantly lower as compared to the previously synthesized cyclic sulfones 1 and 2. This lowering of reactivity can be explained in terms of low conversion to the allenic form and hence the lower extent of alkylation of DNA-bases, the only possible DNA-cleavage pathway for 3 and 4.     

U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.     

Blood pressure and waist circumference: an empirical study of the effects of waist circumference on blood pressure among Bengalee male jute mill workers of Belur,West Bengal,India

An investigation of 150 adult Bengalee Hindu male jute mill workers in Belur, a suburb of Kolkata, West Bengal, India, was conducted to study the relationship between central obesity and blood pressure. In accordance with their waist circumference measurement, the subjects were divided into two categories: centrally non-obese (CNO) and centrally obese (CO). The participants were classified as the CO group if they had a WC of 80 cm or more. Results showed that none of the CNO subjects was mild hypertensive (SBP>/=140 mmHg and/or DBP>/=90 mmHg) while 85 of the CO subjects (82.5%) were mild hypertensives, the difference being statistically significant (chi-square=9.33; p<0.0025). Moreover, the data also revealed that the CO subjects had much (p<0.001) greater mean weight, body mass index (BMI), systolic (SBP), diastolic (DBP) and mean arterial (MAP) blood pressure than the CNO group members. The significant difference in blood pressure was found even after correcting the confounding effects of age and BMI variables. The results of this study showed that, the Bengalee male jute mill workers in the CO group had significantly higher blood pressure irrespective of age and overall adiposity (BMI). Therefore, the presence of central obesity is deemed a risk factor, for hypertension regardless of age and BMI. Thus, a WC cut-off point of 80 cm could be employed for health promotion among Bengalee men so as to prevent and manage hypertension effectively.     

Rorippa indica HSPRO2 expression in transgenic Brassica juncea induces tolerance against mustard aphid Lipaphis erysimi

We have previously established that RiHSPRO2, a nematode resistance protein-like homolog from wild crucifer Rorippa indica (L.) Hiern is a potent candidate to control mustard aphid Lipaphis erysimi. The present study further exploits this protein through structure prediction, biosafety assessment and transgenic study. The RiHSPRO2 protein showed an abundance of alpha helices intervened with loops in the homology-based three-dimensional model. No allergenic moiety was found in its amino acid sequence based on homology search in Protein Data Base. The secondary structure of RiHSPRO2 was unstable at temperatures above 50 °C. In vitro pepsin digestion assay revealed the protein to be digested in pepsin supplemented Simulated Gastric Fluid (SGF) within 2 minutes. The protein is proved to be biologically safe as per the FAO/WHO guidelines. An efficient Agrobacterium-mediated Brassica juncea transformation involving direct organogenesis with mean transformation frequency of 5.06?±?0.28% is reported. RiHSPRO2, under the influence of constitutive promoter CaMV35S, was transformed into susceptible B. juncea cv. B85. Southern hybridization confirmed stable integration of the transgene and Western blotting confirmed consistent expression of RiHSPRO2 in the genetically modified Brassica lines. The transgene segregated following Mendelian 3:1 ratio in the successive generation. Detached leaf aphid bioassay and in planta aphid bioassay in transgenic B. juncea lines revealed a reduction in aphid survivability by 45% and a decrease in aphid fecundity by 45.6%.