The Effect of Maturity Status on Biochemical Composition,Antioxidant Activity,and Anthocyanin Biosynthesis Gene Expression in a Pomegranate (Punica granatum L.) Cultivar with Red Flowers,Yellow Peel,and Pinkish Arils

Pomegranate (Punica granatum L.) has widely been used as a fruit and in folk medicine since ancient civilizations in the world. It is now known that bioactive compounds present in pomegranates attribute to its therapeutic potential. Harvesting at the correct maturity stage is one of the key factors deciding the quality of harvest for consumption in fresh or value-added forms. Identification of the correct maturity stage (harvesting index) is particularly difficult for cultivars having yellowish peel and pinkish arils (sarcotesta). We studied the changes in total phenolic content (TPC), antioxidant activity (AOX), punicalagin α and β contents, color indices, total soluble solids (TSS), and expression of anthocyanin biosynthetic pathway genes from flowering to harvesting in the pomegranate cultivar Nimali, having red flowers, yellow peels, and pinkish arils at maturity over two growing seasons. Interestingly, there was no seasonal variation observed in any of the parameters over two cultivation seasons. Although the β punicalagin content did not change, the TPC, AOX, punicalagin α contents in both peel and arils gradually decreased from flowering to maturity. Though the TPC of both peels and arils, AOX and total punicalagin content of peel did not change significantly 140 days after flower initiation, the TPC and the total punicalagin of arils reached a stable level at 160 days. The TSS in both peels and arils increased significantly with the maturity having the highest values at 180 days. The peel color changed from green to yellow with maturity with a significant increase in l* and b* values and significant decrease in a* value. Nevertheless, the aril color changed from pale white to pink with the maturity with significant reduction of l* and b* values and significant increase of a* value. Changes in pomegranate dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), and leucoanthocyanidin dioxygenase (anthocyanidin synthase-ANS) gene expression in Nimali arils correlated with its color changes during maturity. These findings support to identify the harvesting index of Nimali ensuring the maximum nutritional and health benefits of pomegranate flower, arils, and peels for different downstream uses.

     

Nature and bioactivities of endolichenic fungi in Pseudocyphellaria sp., Parmotrema sp. and Usnea sp. at Hakgala montane forest in Sri Lanka

Aim:  The aim of this study was to investigate the nature and bioactivities of endolichenic fungi in three abundant lichens, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp. in the lower elevation of Hakgala montane forest in Sri Lanka.
Methods and Results:  Endolichenic fungal strains, fungi that live asymptomatically in the lichen thallus, much the same way as endophytic fungi live within healthy plant tissues, were isolated from three abundant lichen species, Pseudocyphellaria sp., Usnea sp. and Parmotrema sp., at Hakgala montane forest in Sri Lanka, using the surface sterilization method. Nine endolichenic fungal strains were isolated from Parmotrema sp. and Usnea sp. separately, while 11 endolichenic fungi were recovered from the lichen Pseudocyphellaria sp. Isolation of endolichenic fungus Chrysosporium sp. 2 was common to all three lichen species. Substrate utilization patterns and antifungal activities of eight endolichenic fungal species were evaluated and the results revealed that all the test fungi were able to produce at least one enzyme to utilize the test substrates. Nigrospora sp., Chrysosporium sp. 1 and 2 and Cladosporium sp. showed antifungal activities on growth of some selected plant pathogenic fungi.
Conclusions:  Endolichenic fungal strains (29) were isolated from the lichens Parmotrema sp., Usnea sp. and Pseudocyphellaria sp. in Sri Lanka. Chrysosporium sp. 2 was common in all three lichens. Some of these endolichenic fungal strains showed antifungal activities against common plant pathogenic fungi and they are capable of utilizing the substrates by producing specific enzymes.
Significance and Impact of the Study:  The diversity and prevalence of the endolichenic fungi have not been studied extensively and this is the first report of isolation and identification of endolichenic fungi in lichens available in Sri Lanka.     

Novel splice variants of rat CaV2.1 that lack much of the synaptic protein interaction site are expressed in neuroendocrine cells

Voltage-gated Ca(2+) channels are responsible for the activation of the Ca(2+) influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the II-III loop of Ca(V)2.1 and Ca(V)2.2 mediates a physical association with synaptic proteins that may be crucial for fast neurotransmission and axonal targeting. We report here the use of nested PCR to identify two novel splice variants of rat Ca(V)2.1 that lack much of the synprint site. Furthermore, we compare immunofluorescence data derived from antibodies directed against sequences in the Ca(V)2.1 synprint site and carboxyl terminus to show that channel variants lacking a portion of the synprint site are expressed in two types of neuroendocrine cells. Immunofluorescence data also suggest that such variants are properly targeted to neuroendocrine terminals. When expressed in a mammalian cell line, both splice variants yielded Ca(2+) currents, but the variant containing the larger of the two deletions displayed a reduced current density and a marked shift in the voltage dependence of inactivation. These results have important implications for Ca(V)2.1 function and for the mechanisms of Ca(V)2.1 targeting in neurons and neuroendocrine cells.     

Photoactivated 2,3-distyrylindoles kill multi-drug resistant bacteria

Compounds based on the 2,3-distyrylindole scaffold were found to exhibit bactericidal properties upon irradiation with white light. At the concentration of 1?μM, the lead compound 1 completely (ca. 10⁹?CFU/mL) eradicated such Gram-positive organisms as S. aureus (MRSA, MSSA), E. faecalis (VRE), S. pyogenes and S. mutans when irradiated with white light for 2?min. At the concentration of 5?μM and in the presence of polymyxin E at non-bactericidal 1.25?μg/mL concentration, 1 also showed a 7-log to 9-log reductions in bacterial counts of such Gram-negative organisms as multi-drug resistant (MDR) A. baumannii, MDR P. aeruginosa, E. coli and Klebsiella pneumoniae (CRE: KPC and NDM-1), also when irradiated with white light for 2?min. The structure-activity relationship studies revealed that unsubstituted at benzene rings 2,3-distyrylindole 2 was most potent and gave a 5-order of magnitude eradication of a MRSA strain at the concentration of 30?nM upon irradiation with white light. Initial mechanistic experiments revealed the disruption of bacterial cell membrane, but indicated that singlet oxygen production, which is commonly associated with photodynamic therapy, may not play a role in the bactericidal effects of the 2,3-distyrylindoles.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional ¹³C‐edited ¹H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.     

In Silico Structural Homology Modelling and Docking for Assessment of Pandemic Potential of a Novel H7N9 Influenza Virus and Its Ability to Be Neutralized by Existing Anti-Hemagglutinin Antibodies

The unpredictable nature of pandemic influenza and difficulties in early prediction of pandemic potential of new isolates present a major challenge for health planners. Vaccine manufacturers, in particular, are reluctant to commit resources to development of a new vaccine until after a pandemic is declared. We hypothesized that a structural bioinformatics approach utilising homology-based molecular modelling and docking approaches would assist prediction of pandemic potential of new influenza strains alongside more traditional laboratory and sequence-based methods. The newly emerged Chinese A/Hangzhou/1/2013 (H7N9) influenza virus provided a real-life opportunity to test this hypothesis. We used sequence data and a homology-based approach to construct a 3D-structural model of H7-Hangzhou hemagglutinin (HA) protein. This model was then used to perform docking to human and avian sialic acid receptors to assess respective binding affinities. The model was also used to perform docking simulations with known neutralizing antibodies to assess their ability to neutralize the newly emerged virus. The model predicted H7N9 could bind to human sialic acid receptors thereby indicating pandemic potential. The model also confirmed that existing antibodies against the HA head region are unable to neutralise H7N9 whereas antibodies, e.g. Cr9114, targeting the HA stalk region should bind with high affinity to H7N9. This indicates that existing stalk antibodies initially raised against H5N1 or other influenza A viruses could be therapeutically beneficial in prevention and/or treatment of H7N9 infections. The subsequent publication of the H7N9 HA crystal structure confirmed the accuracy of our in-silico structural model. Antibody docking studies performed using the H7N9 HA crystal structure supported the model''s prediction that existing stalk antibodies could cross-neutralise the H7N9 virus. This study demonstrates the value of using in-silico structural modelling approaches to complement physical studies in characterization of new influenza viruses.     

β-Site Amyloid Precursor Protein (APP)-cleaving Enzyme 1 (BACE1)-deficient Mice Exhibit a Close Homolog of L1 (CHL1) Loss-of-function Phenotype Involving Axon Guidance Defects

BACE1 is the β-secretase enzyme that initiates production of the β-amyloid peptide involved in Alzheimer disease. However, little is known about the functions of BACE1. BACE1-deficient mice exhibit mild but complex neurological phenotypes suggesting therapeutic BACE1 inhibition may not be completely free of mechanism-based side effects. Recently, we have reported that BACE1 null mice have axon guidance defects in olfactory sensory neuron projections to glomeruli in the olfactory bulb. Here, we show that BACE1 deficiency also causes an axon guidance defect in the hippocampus, a shortened and disorganized infrapyramidal bundle of the mossy fiber projection from the dentate gyrus to CA3. Although we observed that a classical axon guidance molecule, EphA4, was cleaved by BACE1 when co-expressed with BACE1 in HEK293 cells, we could find no evidence of BACE1 processing of EphA4 in the brain. Remarkably, we discovered that the axon guidance defects of BACE1^−/− mice were strikingly similar to those of mice deficient in a recently identified BACE1 substrate, the neural cell adhesion molecule close homolog of L1 (CHL1) that is involved in neurite outgrowth. CHL1 undergoes BACE1-dependent processing in BACE1^+/+, but not BACE1^−/−, hippocampus, and olfactory bulb, indicating that CHL1 is a BACE1 substrate in vivo. Finally, BACE1 and CHL1 co-localize in the terminals of hippocampal mossy fibers, olfactory sensory neuron axons, and growth cones of primary hippocampal neurons. We conclude that BACE1^−/− axon guidance defects are likely the result of abrogated BACE1 processing of CHL1 and that BACE1 deficiency produces a CHL1 loss-of-function phenotype. Our results imply the possibility that axon mis-targeting may occur in adult neurogenic and/or regenerating neurons as a result of chronic BACE1 inhibition and add a note of caution to BACE1 inhibitor development.