Influence of Etherel and Gibberellic Acid on Carbon Metabolism,Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Controlled environment chamber and glasshouse studies were conducted on six herbaceous annual species grown at 350 (AC) and 700 (EC) mol(CO₂) mol^-1 to determine whether growth at EC resulted in acclimation of the apparent quantum yield of photosynthesis (QY) measured at limiting photosynthetic photon flux density (PPFD), or in acclimation of net photosynthetic rate (P _N) measured at saturating PPFD. It was also determined whether acclimation in P _N at limiting PPFD was correlated with acclimation of carboxylation efficiency or ribulose-1,5-bisphosphate (RuBP) regeneration rate measured at saturating PPFD. Growth at EC reduced both the QY and P _N at limiting PPFD in three of the six species. The occurrence of photosynthetic acclimation measured at a rate limiting PPFD was independent of whether photosynthetic acclimation was apparent at saturating measurement PPFD. At saturating measurement PPFD, acclimation to EC in the apparent carboxylation efficiency and RuBP regeneration capacity also occurred independently. Thus at least three components of the photosynthetic system may adjust independently when leaves are grown at EC. Estimates of photosynthetic acclimation at both high and low PPFD are necessary to accurately predict photosynthesis at the whole plant or canopy level as [CO₂] increases.     

Phylogenetic analysis of polyomavirus BK sequences

Polyomavirus BK (BKV) has emerged as an important pathogen in kidney transplant patients. Existing taxonomic classifications of BKV come from conventional DNA sequence alignments based on limited data derived from the VP1 gene. We have used a phylogenetic whole-genome approach to examine the pattern of diversity and evolutionary relationships between 45 BKV strains isolated from multiple clinical settings. This analysis supports the classification of BKV into six genotypes, of which types V and VI have not been previously recognized. BKV strains hitherto classified as type I are, in fact, quite heterogeneous, and several cluster with our newly defined genotypes V and VI. The sequence information needed for assigning genotypes can be captured by VP1, VP2, VP3, or large T-gene sequencing. The most polymorphic coding region in the viral genome is VP1, but significant variation is also present in the large T-antigen gene, wherein polymorphisms are found in 11.39% of all nucleotide sites, 46.22% of which are cluster specific. Type-specific amino acid changes within the VP1 region are predicted to map to the BC and DE loops. The number of taxonomically informative amino acid changes in the large T antigen exceeds even that of the VP1 region. Viral strains isolated from healthy subjects and from patients with human immunodeficiency virus infection, Wiskott-Aldrich syndrome, and vasculopathy with capillary leak syndrome formed distinct subclusters. However, within the kidney transplant population, BKV strains derived from patients with asymptomatic viruria did not show complete separation from strains associated with allograft nephropathy.     

Biomarker-guided sequential targeted therapies to overcome therapy resistance in rapidly evolving highly aggressive mammary tumors

Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.     

Large catalase based bioelectrode for biosensor application

A large catalase (CAT) (Mr ~ 90 kDa), immobilized on multiwalled carbon nanotubes—Nafion^® (MWCNT-NF) matrix and encapsulated with polyethylenimine (PEI) on glassy carbon electrode (GCE), showed a pair of nearly reversible cyclic voltammetric peaks for Fe^(III)/Fe^(II) couple with formal potential of about −0.45 V (vs. Ag/AgCl electrode at pH 7.5). PEI significantly reduced the charge transfer resistance and stabilized the bioelectrode through electrostatic interaction. The electron transfer rate constant and surface coverage of the immobilized CAT were 1.05 ± 0.2 s⁻¹ and 2.1 × 10⁻¹⁰ mol cm⁻², respectively. Studies on electrocatalytic activity and kinetics of GCE/MWCNT-NF/CAT/PEI for hydrogen peroxide (H₂O₂) showed the apparent Michaelis-Menten constant of 3 mM, linear response in the range of 10 μM to 5 mM, response time of ~ 2 s for steady state current, and detection limit of ~ 1 μM. A high operational and storage stability was also demonstrated for the bioelectrode. Hence, the direct electrochemistry of the large catalase and its potential biosensor application have been established through this investigation.     

Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers

Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young''s moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers.     

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (<Emphasis Type="Italic">Eleusine coracana)</Emphasis>

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05_504, NBS-09₇₁₁, NBS-07₆₈₈, NBS-03₅₀₉ and EST-SSR-04₂₄₁) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.     

Osteogenic constituents from Pterospermum acerifolium Willd. flowers

Phytochemical investigation of ethanol extracts of the Pterospermumacerifolium flowers led to the isolation and identification of two new flavones, 4′-(2-methoxy-4-(1,2,3-trihydroxypropyl) phenoxy luteolin (1) and 5,7,3′-trihydroxy-6-O-β-d-glucopyranosyl flavone (2), and one new lactone, 3,5-dihydroxyfuran-2(5H)-one (3) along with 14 known compounds (4-17). The structure of compounds 1-17 was established based on MS, 1D and 2D NMR, spectroscopic analysis. Eight of these compounds (1-6, 8 and 9) were assessed for osteogenic activity by using primary cultures of rat osteoblast. The compounds 1, 3 and 4 significantly stimulated osteoblast differentiation and mineralization as evident from a marked increase in expression of alkaline phosphatase and alizarin red-S staining of osteoblasts.     

Use of SSR,RAPD markers and protein profiles based analysis to differentiate <Emphasis Type="Italic">Eleusine coracana</Emphasis> genotypes differing in their protein content

Fifty-two genotypes of Eleusine coracana collected from Uttarakhand hills were subjected to simple sequence repeat (SSR), random amplified polymorphic DNA (RAPD)-PCR and protein profiling analysis to investigate the variation in protein content. The main objective of the present study was to detect variability among E. coracana and also assess the discriminating ability of these three molecular methods. A total of 21 RAPD and 24 SSR primers were assayed for their specificity in detecting genetic variability in E. coracana, of which 20 RAPD and 21 SSR primers were highly reproducible and were found suitable for use in PCR analysis. Assessing genetic diversity among E. coracana genotypes by RAPD-PCR using 20 polymorphic primers yielded 56 different RAPD markers which clustered the genotypes into different groups on the basis of protein content. Similarly, SSR-PCR with 21 polymorphic primers clustered the genotypes into different groups. On the other hand, biochemical typing of E. coracana using whole seed proteins generated profiles that showed no major difference indicating the technique to be not useful in typing genotypes of this crop. However, a few of the genotypes showed the presence of a unique band of 32 kDa that needs to be further investigated to understand the role of the protein from nutritional point of view, if any. In the present study, significant negative correlation (r = −0.69*) was found between the protein and calcium content of finger millet genotypes. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis based seed storage proteins generated profiles showed no major differences in banding pattern among 52 finger millet genotypes while quantitative estimation of seed storage protein fractions using Lowry method revealed that glutelin was highest followed by prolamin, globulin and albumin.