Resveratrol as a Promising Nutraceutical: Implications in Gut Microbiota Modulation, Inflammatory Disorders, and Colorectal Cancer.

Natural products have been a long-standing source for exploring health-beneficial components from time immemorial. Modern science has had a renewed interest in natural-products-based drug discovery. The quest for new potential secondary metabolites or exploring enhanced activities for existing molecules remains a pertinent topic for research. Resveratrol belongs to the stilbenoid polyphenols group that encompasses two phenol rings linked by ethylene bonds. Several plant species and foods, including grape skin and seeds, are the primary source of this compound. Resveratrol is known to possess potent anti-inflammatory, antiproliferative, and immunoregulatory properties. Among the notable bioactivities associated with resveratrol, its pivotal role in safeguarding the intestinal barrier is highlighted for its capacity to prevent intestinal inflammation and regulate the gut microbiome. A better understanding of how oxidative stress can be controlled using resveratrol and its capability to protect the intestinal barrier from a gut microbiome perspective can shed more light on associated physiological conditions. Additionally, resveratrol exhibits antitumor activity, proving its potential for cancer treatment and prevention. Moreover, cardioprotective, vasorelaxant, phytoestrogenic, and neuroprotective benefits have also been reported. The pharmaceutical industry continues to encounter difficulties administering resveratrol owing to its inadequate bioavailability and poor solubility, which must be addressed simultaneously. This report summarizes the currently available literature unveiling the pharmacological effects of resveratrol.

Inferring Recombination Events in SARS-CoV-2 Variants In Silico.

Over the last 34 months, at least 10 severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) distinct variants have evolved. Among these, some were more infectious while others were not. These variants may serve as candidates for identification of the signature sequences linked to infectivity and viral transgressions. Based on our previous hijacking and transgression hypothesis, we aimed to investigate whether SARS-CoV-2 sequences associated with infectivity and trespassing of long noncoding RNAs (lncRNAs) provide a possible recombination mechanism to drive the formation of new variants. This work involved a sequence and structure-based approach to screen SARS-CoV-2 variants in silico, taking into account effects of glycosylation and links to known lncRNAs. Taken together, the findings suggest that transgressions involving lncRNAs may be linked with changes in SARS-CoV-2-host interactions driven by glycosylation events.

Zirconium copper oxide microflowers based non-enzymatic screen-printed electrochemical sensor for the detection of glucose in saliva, urine, and blood serum.

Zirconium copper oxide microflowers (Zr/CuO MF) based non-enzymatic sensor was developed for glucose detection in saliva, urine, and blood. An easy urea hydrolysis method was employed for the synthesis of the metal oxide and further calcined to improve the catalytic property. The flower-like morphology of the Zr/CuO was confirmed by SEM analysis and the presence of copper and zirconium was examined using energy dispersive X-ray analysis (EDAX). The Zr/CuO MF modified screen-printed electrodes exhibited excellent glucose sensing performance in 0.15 M NaOH medium and could quantify glucose in the range from 10 µM to 27 mM. A high sensitivity of 1.815 ± 0.003 mA mM-1 cm-2 was obtained for lower glucose concentration from 15 µM to 3 mM and 1.250 ± 0.006 mA mM-1 cm-2 for higher concentration glucose from 3 to 27 mM. The limit of detection of the fabricated sensor was found to be 0.8 µM. The sensor displayed high selectivity and stability towards glucose in different body fluids like saliva, urine, and blood serum at a working potential of 0.6 V (vs. Ag/AgCl). In saliva, urine, and serum samples, the sensor exhibited excellent recovery of 95-108, 92-108, and 93-101% in saliva, urine, and serum, respectively, with a relative standard deviation of less than 10%, demonstrating high accuracy and reliability of the sensor. The developed sensor is promising for developing an invasive and non-invasive point-of-care testing device for glucose detection.

Increased Innate Immune Susceptibility in Hyperpigmented Bacteriophage-Resistant Mutants of Pseudomonas aeruginosa.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

A novel and improved selective media for the isolation and enumeration of Klebsiella species.

Bacterial pathogens are fostered in and transmitted through wastewater. Hence, monitoring their impact on sanitation and hygiene is imperative. As part of the monitoring process, culture-based methodologies are primarily used, which centre on the use of selective and differential media. Media available today are, at best, difficult to formulate and, at worst, prohibitively expensive. To address this lacuna, the study proposes a selective and differential medium for Klebsiella spp. Klebsiella blue agar (KBA) is completely selective against selected gram-positive bacteria (Bacillus spp., Staphylococcus aureus) and a few gram-negative bacteria (Acinetobacter baumanii, Serratia marcescens). On the other hand, it supports the growth of the chosen members of the Klebsiella pneumoniae species-complex with a characteristic green colouration. Methylene blue, tryptophan, and bile salt make up the selective components of KBA. Moreover, methylene blue, 0.6% NaCl, and glycerol render it differential. KBA was more selective than HiCrome™ Klebsiella Selective Agar Base (KSA) in replica plating experiments. KBA promoted only 157 CFUs against 209 CFUs in KSA when stamped with 253 CFUs grown on LB. The colonies so isolated were predominantly Klebsiella spp., on identification through colony polymerase chain reaction. Moreover, the differential nature of KBA distinguished Klebsiella aerogenes from other species. On the contrary, KSA lodged colonies indistinguishable from each other and Klebsiella spp. Due to its ease of formulation, high selectivity, differential nature, and cost-effective composition, KBA is a viable option for the routine culture of Klebsiella spp. in environmental and clinical settings. KEY POINTS: • Formulated a novel selective and differential media for Klebsiella spp., named Klebsiella Blue agar • Facile formulation methodology • Can be employed to isolate Klebsiella spp. from complex sources such as wastewater.

Anacardic acid-mediated regulation of osteoblast differentiation involves mitigation of inflammasome activation pathways.

Disruption of the finely tuned osteoblast-osteoclast balance is the underlying basis of several inflammatory bone diseases, such as osteomyelitis, osteoporosis, and septic arthritis. Prolonged and unrestrained exposure to inflammatory environment results in reduction of bone mineral density by downregulating osteoblast differentiation. Earlier studies from our laboratory have identified that Anacardic acid (AA), a constituent of Cashew nut shell liquid that is used widely in traditional medicine, has potential inhibitory effect on gelatinases (MMP2 and MMP9) which are over-expressed in numerous inflammatory conditions (Omanakuttan et al. in Mol Pharmacol, 2012 and Nambiar et al. in Exp Cell Res, 2016). The study demonstrated for the first time that AA promotes osteoblast differentiation in lipopolysaccharide-treated osteosarcoma cells (MG63) by upregulating specific markers, like osteocalcin, receptor activator of NF-κB ligand, and alkaline phosphatase. Furthermore, expression of the negative regulators, such as nuclear factor-κB, matrix metalloproteinases (MMPs), namely MMP13, and MMP1, along with several inflammatory markers, such as Interleukin-1ß and Nod-like receptor protein 3 were downregulated by AA. Taken together, AA expounds as a novel template for development of potential pharmacological therapeutics for inflammatory bone diseases.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage.

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

Proteasomal dysfunction and ER stress triggers 2'-hydroxy-retrochalcone-induced paraptosis in cancer cells.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

A graphite pencil electrode with electrodeposited Pt-CuO for nonenzymatic amperometric sensing of glucose over a wide linear response range.

A disposable nonenzymatic glucose sensor was obtained by pulsed electrodeposition of Pt-CuO on a graphite pencil electrode (GPE). The morphology of the modified GPE was studied using SEM, and the chemical composition of the coating was examined by EDAX and XRD. The electrochemical response of the modified GPE was compared with individual copper- and platinum-modified GPEs. The electrodeposition parameters were optimized with respect to the electrocatalytic activity of the deposits towards glucose oxidation. Best operated at a working potential of 0.6 V vs. Ag/AgCl, the sensor has a sensitivity of 2035 µA mM-1 cm-2, a 0.1 µM detection limit and a wide linear response range that extends up to 25 mM. It is highly selective for glucose in the presence of various exogenous and endogenous interfering species. Eventhough the requirement of alkaline medium for sensing is a limitation, easy fabrication procedure, very high sensitivity and selectivity, wide analytical range, and disposable sensor characteristics show potential application towards blood glucose determination. Graphical abstractSchematic representation of the Pt-CuO electrodeposited pencil graphite electrode for the nonenzymatic determination of glucose.

(I-3,II-3)-Biacacetin-mediated cell death involves mitochondria.

Dysregulation of the dynamic balance between cell proliferation and cell death leads to several malignancies including cancer. Biflavones are known to possess anti-proliferative activity against numerous cancer cell lines. The current study was undertaken to understand the mechanism of action of the biflavonoid (I-3,II-3)-biacacetin on MDA-MB-231. Biacacetin induces dose-dependent cell death in MDA-MB-231 cells from concentrations as low as 0.5 µM, which was further confirmed by an increase in sub-G1 cells. Furthermore, the cell death induced by biacacetin was found to be mitochondria-dependent, since cells devoid of mitochondria were viable in the presence of biacacetin even at the highest concentration tested (25 µM). Fluorescence studies clearly indicated nuclear changes and apoptotic body formation that are characteristic of apoptosis. These results were further corroborated by studies that demonstrate biacacetin to regulate several key markers of apoptosis like Caspase 3, p53, Bax, and poly-ADP-ribose polymerase-1. Furthermore, biacacetin did not induce cell death in normal macrophage cell line, RAW at concentrations up to 15 µM. In addition to MDA-MB-231 cells, biacacetin also induces apoptotic cell death in the highly chemo-resistant cell line, OVISE, where the cells stained positive for annexin. Biacacetin also induces cell death in the highly malignant fibrosarcoma cell line HT1080. Furthermore, biacacetin also induces significant cell death (50%) in 3D tumor spheroids, at a concentration of 25 µM. Taken together, these results provide an understanding of biacacetin-mediated cell death and thereby provides a strong basis for the use of such compounds as novel templates for anti-cancer therapeutics.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

An α, ß-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of ß5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells.

Paper-based device for the colorimetric assay of bilirubin based on in-situ formation of gold nanoparticles.

A paper-based colorimetric assay for the determination of bilirubin has been developed. The method is based on the in-situ reduction of chloroauric acid to form gold nanoparticles. A chromatographic paper was patterned using a wax printer. Chloroauric acid was drop-cast onto the reagent zone. In the presence of bilirubin, gold(III) ions are reduced and form gold nanoparticles. This leads to a color change from yellow to purple. The intensity of the purple color (peak at 530 nm) increases with bilirubin concentration in the 5.0 to 1000 mg L-1 range. The detection limit is 1.0 mg L-1. For the quantification of bilirubin, images were captured using a digital camera, and data were processed with the help of machine learning-based supervised prediction using Random Forest classification. The method was applied to the determination of bilirubin in urine samples. The spiked urine samples exhibit more than 95% recovery. Graphical abstractSchematic representation of the paper-based colorimetric assay for the detection of bilirubin based on the in-situ formation of gold nanoparticles. A color band is generated for visual interpretation and used for the testing of bilirubin in urine.

Fabrication of Highly Sensitive Nonenzymatic Electrochemical H2O2 Sensor Based on Pt Nanoparticles Anchored Reduced Graphene Oxide.

A highly sensitive nonenzymatic hydrogen peroxide (H2O2) sensor was fabricated using platinum nanoparticles decorated reduced graphene oxide (Pt/rGO) nanocomposite. The Pt/rGO nanocomposite was prepared by single-step chemical reduction method. Nanocomposite was characterized by various analytical techniques including Raman spectroscopy, X-ray diffraction, field emission scanning electron microscope and high-resolution transmission electron microscopy. Screen printed electrodes (SPEs) were fabricated and the nanocomposite was cast on the working area of the SPE. Cyclic voltammetry and amperometry demonstrated that the Pt/rGO/SPE displayed much higher electrocatalytic activity towards the reduction of H2O2 than the other modified electrodes. The sensor exhibited wide linear detection range (from 10 µM to 8 mM), very high sensitivity of 1848 µA mM-1 cm-2 and a lower limit of detection of 0.06 µM. The excellent performance of Pt/rGO/SPE sensor were attributed to the reduced graphene oxide being used as an effective matrix to load a number of Pt nanoparticles and the synergistic amplification effect of the two kinds of nanomaterials. Moreover, the sensor showed remarkable features such as good reproducibility, repeatability, long-term stability, and selectivity.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

Nitric Oxide and ERK mediates regulation of cellular processes by Ecdysterone.

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2' ,7' -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway.

Anacardic acid inhibits gelatinases through the regulation of Spry2, MMP-14, EMMPRIN and RECK.

Earlier studies from our laboratory have identified Anacardic acid (AA) as a potent inhibitor of gelatinases (MMP-2 and 9), which are over-expressed in a wide variety of cancers (Omanakuttan et al., 2012). Disruption of the finely tuned matrix metalloproteinase (MMP) activator/inhibitor balance plays a decisive role in determining the fate of the cell. The present study demonstrates for the first time, that in addition to regulating the expression as well as activity of gelatinases, AA also inhibits the expression of its endogenous activators like MMP-14 and Extracellular Matrix MetalloProteinase Inducer (EMMPRIN) and induces the expression of its endogenous inhibitor, REversion-inducing Cysteine-rich protein with Kazal motifs (RECK). In addition to modulating gelatinases, AA also inhibits the expression of various components of the Epidermal Growth Factor (EGF) pathway like EGF, Protein Kinase B (Akt) and Mitogen-activated protein kinases (MAPK). Furthermore, AA also activates the expression of Sprouty 2 (Spry2), a negative regulator of EGF pathway, and silencing Spry2 results in up-regulation of expression of gelatinases as well as MMP-14. The present study thus elucidates a novel mechanism of action of AA and provides a strong basis for utilizing this molecule as a template for cancer therapeutics.

Vertically Aligned TiO2 Nanotube Arrays Decorated with CuO Mesoclusters for the Nonenzymatic Sensing of Glucose.

Highly ordered titanium dioxide (TiO2) nanotubes were modified with CuO mesoclusters through electrodeposition followed by electrooxidation. Field Emission Scanning Electron Microscopy (FESEM) revealed the presence of vertically aligned TiO2 nanotubes with a diameter of 60 nm and CuO mesoclusters of ~500 nm in diameter. Glucose oxidation on the CuO modified TiO2 electrode occurred at +0.55 V. The electrode exhibited a sensitivity of 1836 and 1416 µA mmol⁻¹ L cm−2 for glucose concentrations ranging from 0.625 to 6.25 mmol L⁻¹ and 6.87 to 12.5 mmol L⁻¹ respectively, a limit of detection of 3.4 µmol L⁻¹ (S/N = 3) and a rapid response time of ≤2 s. Physiological concentrations of ascorbic acid, uric acid and dopamine had no effect on the glucose oxidation. Interference from other sugars (lactose and galactose) was negligible. Result obtained from the estimation of blood glucose was found to be in good agreement with those obtained from commercially available glucose sensor strips.

Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor.

Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9µM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9µM to as high as 333µM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA.

Anacardic acid induces apoptosis-like cell death in the rice blast fungus Magnaporthe oryzae.

Anacardic acid (6-pentadecylsalicylic acid), extracted from cashew nut shell liquid, is a natural phenolic lipid well known for its strong antibacterial, antioxidant, and anticancer activities. Its effect has been well studied in bacterial and mammalian systems but remains largely unexplored in fungi. The present study identifies antifungal, cytotoxic, and antioxidant activities of anacardic acid in the rice blast fungus Magnaporthe oryzae. It was found that anacardic acid causes inhibition of conidial germination and mycelial growth in this ascomycetous fungus. Phosphatidylserine externalization, chromatin condensation, DNA degradation, and loss of mitochondrial membrane potential suggest that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that anacardic acid induces caspase-independent apoptosis in M. oryzae. Expression of a predicted ortholog of apoptosis-inducing factor (AIF) was upregulated during the process of apoptosis, suggesting the possibility of mitochondria dependent apoptosis via activation of apoptosis-inducing factor. Anacardic acid treatment leads to decrease in reactive oxygen species rather than increase in reactive oxygen species (ROS) accumulation normally observed during apoptosis, confirming the antioxidant properties of anacardic acid as suggested by earlier reports. Our study also shows that anacardic acid renders the fungus highly sensitive to DNA damaging agents like ethyl methanesulfonate (EMS). Treatment of rice leaves with anacardic acid prevents M. oryzae from infecting the plant without affecting the leaf, suggesting that anacardic acid can be an effective antifungal agent.

Identification and characterization of a library of surfactins and fengycins from a marine endophytic Bacillus sp.

An endophytic bacterial strain from a marine green alga, Ulva lactuca, was isolated and identified by 16S rRNA gene sequencing method. The bacterial isolate was found to secrete two major families of cyclic depsilipopeptides, surfactins, and fengycins. Sequencing of the isolated lipopeptides was carried out using the MSn data obtained from an electrospray ionization (ESI) ion trap mass spectrometer coupled to an HPLC system. The assigned sequences were confirmed by a chemical derivatization approach involving esterification followed by mass spectrometric analysis. Distinction of leucine residues from isoleucine was established through a combined electron transfer dissociation-collision-induced dissociation (ETD-CID) method. The fengycins described in this study were found to cause significant delay of growth of two plants, Vigna radiata (mung bean) and Oryza sativa (rice). To the best of our knowledge, this is the first study describing identification and characterization of cyclic peptides from an endophytic Bacillus sp. isolated from marine algae.