Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics

Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis.

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor's gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future

Fresh Water Cyanobacteria Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as an Anticancer Drug Resource.

An increasing number of cancer patients worldwide, especially in third world countries, have raised concern to explore natural drug resources, such as the less explored fresh water filamentous cyanobacteria. Six strains of cyanobacteria (Phormidium sp. CCC727, Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Phormidium sp. CCC731, Phormidium sp. CCC730, and Leptolyngbya sp. CCC732) were isolated (paddy fields and ponds in the Banaras Hindu University, campus) and five strains screened for anticancer potential using human colon adenocarcinoma (HT29) and human kidney adenocarcinoma (A498) cancer cell lines. Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 were the most potent as determined by examination of morphological features and by inhibition of growth by graded concentrations of crude extracts and thin-layer chromatography (TLC) eluates. Cell cycle analysis and multiplex assays using cancer biomarkers also confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as cancer drug resources. Apoptotic studies in the cells of A498 (cancer) and MCF-10A (normal human epithelial) exposed to crude extracts and TLC fractions revealed no significant impact on MCF-10A cells emphasizing its importance in the development of anticancer drug. Identification of biomolecules from these extracts are in progress

Status, Alert System, and Prediction of Cyanobacterial Bloom in South Korea

Bloom-forming freshwater cyanobacterial genera pose a major ecological problem due to their ability to produce toxins and other bioactive compounds, which can have important implications in illnesses of humans and livestock. Cyanobacteria such as Microcystis, Anabaena, Oscillatoria, Phormidium, and Aphanizomenon species producing microcystins and anatoxin-a have been predominantly documented from most South Korean lakes and reservoirs. With the increase in frequency of such blooms, various monitoring approaches, treatment processes, and prediction models have been developed in due course. In this paper we review the field studies and current knowledge on toxin producing cyanobacterial species and ecological variables that regulate toxin production and bloom formation in major rivers (Han, Geum, Nakdong, and Yeongsan) and reservoirs in South Korea. In addition, development of new, fast, and high-throughput techniques for effective monitoring is also discussed with cyanobacterial bloom advisory practices, current management strategies, and their implications in South Korean freshwater bodies

Screening for anticancer activity on cancer cell line.

<p>Calcein assay using a renal cell carcinoma (A498) cell line. Significant difference (p<0.001) between pairs, control with <i>Geitlerinema</i> CCC728 and <i>Arthrospira</i> CCC729 <b>(a),</b> Control, C2 and C3 with C4 and C5 <b>(b)</b> and Control and D1 with D3 and D4, D2 with D4 <b>(c)</b>. Control was lacking with crude extracts or TLC fractions.</p

Phylogenetic tree of 16S rRNA gene in cyanobacteria generated through BLASTN search in NCBI cyanobacterial database using the neighbor joining algorithm provided by MEGA 5.

<p>Phylogenetic tree of 16S rRNA gene in cyanobacteria generated through BLASTN search in NCBI cyanobacterial database using the neighbor joining algorithm provided by MEGA 5.</p

Effect of <i>Geitlerinema</i> sp. CCC728 crude extract on selected cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells showing impact on selected biomarkers.</p

Cell cycle analysis of A498 cell lines with PI staining using Flow cytometry.

<p>Interaction of cells with most potent fractions from the second TLC purification: control cells without treatment (a), cells <i>vs</i> D3 <b>(b)</b> and D4 <b>(c) from</b><i>Arthrospira</i>, C4 <b>(c)</b> and C5 <b>(d)</b> from <i>Geitlerinema</i>.</p

Apoptotic analysis by Flow cytometry using Annexin V-PI.

<p>A498 as well as MCF-10A cells were exposed to TLC fractions C4 and C5 (<i>Geitlerinema</i> sp. CCC728), D3 and D4 (<i>Arthrospira</i> sp. CCC729). Statistical analysis of A498 cell lines under treatment showed significant impact on cancer cells (F_4,10 = 106.582, p<0.001) <b>(a).</b> As far as MCF-10A cells were concerned they were not significantly affected by fractions selected in experiment (F_4,10 = 2.588, p> 0.05) <b>(b).</b></p

Effect of <i>Arthrospira</i> sp. CCC729 crude extract on selected cancer biomarkers.

<p>Multiplex assay showing levels of expression of cancer biomarkers in A498 cancer cells after treatment with crude extract at 100 μg·mL^-1 in the cells.</p