Discovery of small molecule guanylyl cyclase B receptor positive allosteric modulators.

Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3',5'-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure-activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700 days under anoxic condition. The synthetic wastewater containing 0.382 g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

Control of aflatoxin biosynthesis by sulfur containing benzimidazole derivatives: In-silico interaction, biological activity, and gene regulation of Aspergillus flavus.

Aspergillus flavus producing aflatoxins is one of the potent contaminants of raw food commodities during pre-and post-harvest crops. Aflatoxins are the group of secondary metabolites a subset of natural polyketides. Our major focus is on the inhibition of the biosynthesis pathway of aflatoxin by targeting the enzymes involved. Benzimidazoles are known antimicrobial compounds. In this study the sulfur containing benzimidazole derivatives were tested for their antifungal and antiaflatoxigenic activity. The fungal growth and aflatoxin production was analysed in culture medium as well as in the rice. Inhibition of specific genes was studied in terms of mRNA expression and the interaction of test compound with polyketide synthases by in-silico molecular docking. Substitution at the 6th position of 2-(2-thienyl) benzimidazole (2-TBD) reduced the antifungal property of benzimidazole but effectively inhibited the aflatoxin synthesis in the culture medium as well as in the rice from the toxigenic strain of A. flavus. Among the derivatives tested, the methyl group containing 2-(2-thienyl)- 6-methylbenzimidazole (6-MTBD) inhibited aflatoxin B1 most effectively followed by carboxylic group containing 2-(2-thienyl) benzimidazole-6-carboxylic acid (6-TBCA) with IC50 value of 12.36 and 18.25 µg/mL respectively. Molecular docking study shows that 2-(2-thienyl) benzimidazole-6-carbonitrile (6-CTBD) and 6-MTBD occupy same pocket on TE domain of PksA with similar range of binding energy, however the experimental data show a different effect on the biosynthesis of AFB1. 6-MTBD effectively inhibited the AFB1 synthesis (97%) while 6-CTBD could not (39.5%). Data obtained from the expression study also supports the experimental observations. These compounds are non-toxic to mammalian cells. These benzimidazole derivatives inhibit toxic secondary metabolites without affecting the growth of the fungi hence can be used during fermentation to avoid mycotoxin contamination.

Triazolothiadiazine derivative positively modulates CXCR4 signaling and improves diabetic wound healing.

Development of specific therapies that target and accelerate diabetic wound repair is an urgent need to alleviate pain and suffering and the huge socioeconomic burden of this debilitating disease. C-X-C Motif Chemokine Ligand 12 (CXCL12) also know an stromal cell-derived factor 1α (SDF-1α) is a chemokine that binds the CXC chemokine receptor type 4 (CXCR4) and activates downstream signaling resulting in recruitment of hematopoietic cells to locations of tissue injury and promotes tissue repair. In diabetes, low expression of CXCL12 correlates with impaired wound healing. Activation of CXCR4 receptor signaling with agonists or positive allosteric modulators (PAMs) provides a potential for small molecule therapeutic discovery and development. We recently reported high throughput screening and identification of the CXCR4 partial agonist UCUF-728, characterization of in vitro activity and reduced wound closure time in diabetic mice at 100 µM as a proof-of-concept study. We report here, the discovery of a second chemical scaffold demonstrating increased agonist potency and represented by thiadiazine derivative, UCUF-965. UCUF-965 is a potent partial agonist of ß-arrestin recruitment in CXCR4 receptor overexpressing cell line. Furthermore, UCUF-965 potentiates the CXCL12 maximal response in cAMP signaling pathway, activates CXCL12 stimulated migration in lymphoblast cells and modulates the levels of specific microRNA involved in the complex wound repair process, specifically in mouse fibroblasts. Our results indicate that UCUF-965 acts as a PAM agonist of the CXCR4 receptor. Furthermore, UCUF-965 enhanced angiogenesis markers and reduced wound healing time by 36% at 10.0 µM in diabetic mice models compared to untreated control.

The main Aflatoxin B1 degrading enzyme in Pseudomonas putida is thermostable lipase.

Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by Aspergillus flavus and A. parasiticus, and prevalent in food and feed. Microbial degradation is a promising strategy which can be performed in mild and environmental friendly condition. This work is a step towards identifying the enzyme responsible for biodegradation of AFB1 by P. putida. Experiments were performed with P. putida lysate and compared with commercial lipase to see the degradation efficiency and the temperature stability. The cell free lysate of P. putida efficiently degraded AFB1 in a range of temperature from 20 to 90 °C. The lysate is thermostable and could retain its activity on pre-incubation up to 90 °C. Highest rate of degradation was observed at 70 °C. These observations show that the P. putida lysate is not only stable at higher temperatures but its enzymatic activity increases after incubation. Similarly, the commercial lipase degraded AFB1 efficiently. However, both, the P. putida lysate and lipase ceased degradation in presence of a lipase inhibitor, HgCl2. The Hill function accurately predicted enzyme activity at various times and temperatures. Like lipase, the lysate also hydrolyses the p-nitrophenyl palmitate to p-nitrophenol. Kinetic parameters such as Vmax, Km and n values are good measures to characterize the lysate response with respect to changing paranitro phenyl palmitate levels. The substrate specificity test of lipase showed linear correlation between the absorbance at 410 nm vs amount of product paranitro phenol. The value of Km, Vmax and n are 0.62 mM, 355.7 µmol min-1 and 1.29, respectively. The lipase gene presence in P. putida was confirmed using PCR technique. These observations indicate that the main enzyme responsible for AFB1 degradation by P. putida is lipase. Thus, lipase as a multifunctional biocatalyst provides a promising future for a variety of industries and may also help to ensure the food safety by degrading the mycotoxins.

Rapid and sensitive detection of mycotoxins by advanced and emerging analytical methods: A review.

Quantification of mycotoxins in foodstuffs is extremely difficult as a limited amount of toxins are known to be presented in the food samples. Mycotoxins are secondary toxic metabolites, made primarily by fungal species, contaminating feeds and foods. Due to the presence in globally used grains, it is an unpreventable problem that causes various acute and chronic impacts on human and animal health. Over the previous few years, however, progress has been made in mycotoxin analysis studies. Easier techniques of sample cleanup and advanced chromatographic approaches have been developed, primarily high-performance liquid chromatography. Few extremely sophisticated and adaptable tools such as high-resolution mass spectrometry and gas chromatography-tandem MS/MS have become more important. In addition, Immunoassay, Advanced quantitative techniques are now globally accepted for mycotoxin analysis. Thus, this review summarizes these traditional and highly advance methods and their characteristics for evaluating mycotoxins.

VITPOR AI, A Coagulation Factor XIIa Inhibitor from Porphyra yezoensis: In Vivo Mode of Action and Assessment of Platelet Function Analysis.

BACKGROUND: Thrombosis represents as the prime contributor to the burden of diseases, worldwide. Conventional anticoagulants for thrombosis therapy have a common bleeding side effect. Bioactive peptides are studied to be an effective alternative for currently available therapeutic drugs. OBJECTIVE: In this study, VITPOR AI peptide, a previously reported coagulation FXIIa inhibitor from Nori (Porphyra yezoensis), was assessed for its inhibitory activity against FXIIa and its in vivo mode of action. METHODS: In vivo efficacy as well as the antithrombotic property of the peptide was evaluated in mice model by ex vivo activated Partial Thromboplastin Time assay, tail transection model and whole blood clotting time. The enzyme kinetics was studied using chromogenic substrate assay. RESULTS: The kinetic behaviour of VITPOR AI showed that the peptide is a competitive inhibitor of FXIIa. Peptide showed significant inhibition of platelet adhesion and aggregation. VITPOR AI exhibited significant antithrombotic activity. Furthermore, ex vivo activated Partial Thromboplastin Time assay revealed that VITPOR AI exhibited potent anticoagulant activity in vivo. Tail bleeding assay revealed that the peptide did not prolong bleeding time in mice even at a higher dose of 5 mg/kg. Cytotoxicity studies of the peptide against human blood leukocytes indicated the safety of the peptide. CONCLUSION: VITPOR AI could be prospected as a potent anticoagulant with Factor XIIa inhibition, antiplatelet aggregation and antithrombotic activity. It was also studied to have no bleeding side effect.

Inhibition of aflatoxin B1 biosynthesis and down regulation of aflR and aflB genes in presence of benzimidazole derivatives without impairing the growth of Aspergillus flavus.

Aflatoxins are mutagenic secondary metabolites produced by certain ubiquitous saprophytic fungi. These contaminate agricultural crops and pose a serious health threat to humans and livestock all over the world. Benzimidazole and its derivatives are biologically active heterocyclic compounds known for their fungicidal activity. In the present study, second and sixth position substituted benzimidazole derivatives are tested for their antifungal and anti-aflatoxigenic activity. Aflatoxigenic strain of Aspergillus flavus cultured in Yeast extract sucrose (YES) medium as well as in rice in the presence and absence of test compounds. 2-(2-Furyl) benzimidazole (FBD) showed complete inhibition of fungal growth at 50 µg/mL. However, the polar derivatives of FBD viz. 6-NFBD, 6-AFBD, 6-CAFBD, and 6-CFBD did not impair the fungal growth but effectively inhibited aflatoxin B1 biosynthesis. Significant down-regulation of aflR gene involved in regulation and aflB structural gene for aflatoxin B1 biosynthesis was observed in presence of 6-NFBD. These benzimidazole derivatives also showed good anti-aflatoxigenic activity in rice, though the IC50 concentrations in rice were comparatively higher than those in YES medium. This study summarizes the most notable structure-activity relationship (SAR) of 2-(2-Furyl) benzimidazoles for anti-aflatoxigenic and anti-fungal activities. These molecules can be further studied for their applications in industrial fermentation processes vulnerable to mold growth and subsequent aflatoxin B1 synthesis like koji fermentation, cheese production, etc.

Protein-mediated degradation of aflatoxin B1 by Pseudomonas putida.

Degradation or the removal of aflatoxin B1 from agriculture commodities is very important because of its acute toxicity and economic loss due to rejection of about 25% contaminated agri produce. The present study aimed at using Pseudomonas putida for the aflatoxin B1 (AFB1) degradation and to understand the mechanism involved. AFB1 degradation was studied with P. putida culture, culture supernatant, cell lysate, cell lysate in the presence of protease inhibitor, and heat-inactivated cell lysate. The remaining AFB1 was qualitatively and quantitatively measured by thin-layer chromatography and HPLC with a UV detector. P. putida culture and culture supernatant showed 80% reduction in AFB1 within 24 h of incubation. Cell lysate and the lysate in the presence of protease inhibitor showed the same reduction in 6 and 4 h respectively. The protease-inhibited lysate showed greater thermostability, broad pH range, and tolerance to some of the solvents and detergents in terms of aflatoxin B1 degrading activity. The heat-inactivated lysate showed only 20% reduction in 24 h of incubation indicating loss of activity on heating. As cell-free supernatant and cell lysate are capable of reducing AFB1 effectively, actively growing cells are not necessary for degradation. The active principle for degradation might be proteinaceous; therefore, heat-inactivated lysate is ineffective for reducing the AFB1. These results showed that degradation of aflatoxin B1 by P. putida might be an enzymatic process and could be used in a broad range of conditions.

An anticoagulant peptide from Porphyra yezoensis inhibits the activity of factor XIIa: In vitro and in silico analysis.

Thrombosis represents a major cause of morbidity and mortality around the world. Peptides isolated from natural sources have been proven to have anticoagulant and antithrombotic properties. VITPOR AI, a 16-mer peptide, isolated from Porphyra yezoensis was reported to have anticoagulant property. In this study, the coagulation factor XIIa activity in the presence of VITPOR AI was determined. Molecular modelling was performed to investigate the interaction between peptide and FXIIa. The structure of the peptide was predicted using PEP-FOLD3 server and simulated by molecular dynamics (MD) using GROMACS package. Molecular docking was carried out using peptide-protein docking software, pepATTRACT and its stability was confirmed by MD simulations. The chromogenic substrate assay revealed that the peptide inhibited the amidolytic activity of FXIIa with IC50 of 70.24 µM. The docking result showed peptide interactions through hydrogen bonds with Pro 96, Tyr 99, Glu 146, Gly 193 and Ser 195 of FXIIa. The MD simulation demonstrated that the peptide's binding with the FXIIa was stable as it did not move away from its binding region throughout the simulation period of 100 ns Moreover, MM/PBSA analysis also indicated a stable binding between the protein and peptide. These results suggest that the inhibition of the FXIIa activity might be due to binding of the peptide to oxyanion hole of the catalytic site. Thus, VITPOR AI could be explored as a potent anticoagulant which inhibits only intrinsic pathway of coagulation cascade but does not affect the extrinsic pathway.

Cancer stem cells and fibroblast niche cross talk in an in-vitro oral dysplasia model.

Understanding the cellular interactions during oral carcinogenesis has the potential to identify novel prognostic and therapeutic targets. This study aimed at investigating the cancer stem cell (CSC)-fibroblast niche interactions using in-vitro dysplastic cell line models developed from different stages of 4NQO-induced oral carcinogenic mice model. The spontaneously transformed epithelial cells (DysMSCTR6, 14 and 16) were developed from three time points (mild/moderate/severe), while two fibroblast cell lines (FibroMSCTR12, 16) were developed from moderate and severe dysplastic tissue. The epithelial (Epcam+/Ck+) and the fibroblast cell lines (Vimentin+/α-SMA+/Ck-) were authenticated and assessment of cells representing progressive grades of dysplastic severity indicated a significant increase in dysplastic marker profile (P < 0.05). Evaluation of the CSC characteristics showed that an increase in expression of Cd133, Cd44, Aldh1a1, Notch1, and Sox2 was accompanied by an increase in migratory (P > 0.05) and colony formation capacity (P > 0.005). Targeting Notch1 (GSI inhibitor PZ0187; 30 µM), showed a significant reduction in cell proliferation capacity (P < 0.05) and in the dysplastic marker profile. Further, Notch1 inhibition resulted in down regulation of Cd133 and Aldh1a 1 (P < 0.05) and a complete abrogation of colony formation ability (P < 0.0001). The effect of niche interactions evaluated using FibroMSCTR12-conditioned media studies, revealed an enrichment of ALDH1A1+ cells (P < 0.05), induction of spheroid formation ability (P < 0.0001) and increased proliferation capacity (3.7 fold; P < 0.005). Although PZ0187 reduced cell viability by ∼40%, was unable to abrogate the conditioned-media induced increase in proliferation capacity completely. This study reports a Notch-1 dependent enrichment of CSC properties during dysplastic progression and a Notch-1 independent dysplastic cell-fibroblast interaction during oral carcinogenesis.

Health Benefits and Pharmacological Effects of Porphyra Species.

Porphyra, one of the most cultured red algae has gained economic importance across the globe for its nutritional benefits. Porphyra is being cultivated, harvested, dried, processed and consumed in large quantities in south eastern countries. It contains relatively high amounts of proteins, carbohydrates, and micronutrients. Exploitation of its fundamental attributes led to the discovery of various biologically active compounds like polysaccharides, phycobiliproteins and peptides with effective pharmacological applications. In this review, a systematic account of the research accomplished in the past decade and up-to-date overview of various bioactive compounds and its pharmacological implications has been compiled. This review summarizes the bioactivities like antioxidative, immunomodulatory, antihypertensive, anticoagulant and anticancer properties of the bioactive compounds from Porphyra.

Essential oils: from prevention to treatment of skin cancer.

The increasing incidence of cutaneous malignancies signifies the need for multiple treatment options. Several available reviews have emphasized the potential role of various botanical extracts and naturally occurring compounds as anti-skin-cancer agents. Few studies relate to the role of chemoprevention and therapeutic activity of essential oils (EOs) and EO components. The present review summarizes an overview of chemopreventive, anti-melanoma and anti-nonmelanoma activities of EOs from various plants and EO components in in vitro and in vivo models with special emphasis on skin cancer. Also, the mechanisms by which EOs and EO components exert their effects to induce cell death are presented.

Induction of apoptosis by essential oil from P. missionis in skin epidermoid cancer cells.

BACKGROUND: The genus Pamburus (Rutaceae) comprises the only species, Pamburus missionis (Wight) Swingle. Pamburus missionis is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. PURPOSE: The present study investigates the cancer chemotherapeutic potential of essential oil (EO) from P. missionis. METHODS: EO was isolated by steam distillation and chemical composition was determined by GC-MS. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. RESULTS: GC-MS analysis of the essential oil revealed the presence of 51 components. The major components were ß-Caryophyllene, 4(14),11-Eudesmadiene, Aromadendrene oxide-(2) and Phytol. EO inhibited the growth and colony formation ability of A431 and HaCaT cells. EO treatment induced nuclear condensation and loss of membrane integrity, DNA fragmentation, increase in sub-G1 DNA content and increase in intracellular ROS level. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment blocked EO induced apoptosis, revealing that apoptotic activity was by ROS accumulation. EO induced apoptosis was found to be due to the loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study revealed cancer chemotherapeutic potential of EO from P. missionis. EO induces cell death through intrinsic (mitochondrial) and extrinsic apoptotic pathway in A431 and HaCaT cells. These results suggest that EO could be used as a potential therapeutic agent for the treatment of skin epidermoid cancer.

Synergistic interaction of ß-caryophyllene with aromadendrene oxide 2 and phytol induces apoptosis on skin epidermoid cancer cells.

BACKGROUND: Pamburus missionis (Wight) Swingle (Rutaceae) is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. In a previous study the authors demonstrated apoptotic activity of Pamburus missionis essential oil (EO) on A431 and HaCaT cells. The major components of EO were ß-caryophyllene (25.40%), 4(14),11- eudesmadiene (7.17%), aromadendrene oxide 2 (14.01%) (AO-(2) and phytol (6.88%). PURPOSE OF STUDY: To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells. METHODS: Isobolographic analysis and combination index methods were used to detect the type of interactions among the essential oil (EO) components. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding assay, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis associated proteins was investigated by western blot. RESULTS: Combination of P. missionis EO components: ß-caryophyllene/ aromadendrene oxide 2 (ß-C/AO-(2)), ß-caryophyllene/phytol (ß-C/P) and aromadendrene oxide 2 /phytol (AO-(2)/P) inhibited growth and colony formation ability of skin epidermoid A431 and precancerous HaCaT cells. Synergistic interaction was observed between ß-C/AO-(2) and ß-C/P combination while AO-(2)/P exhibited an additive effect. Combination of components induced chromatin condensation, phosphatidylserine externalisation, increase in sub-G1 DNA content, cell cycle arrest at G0/G1 phase and intracellular ROS accumulation. Inhibition of intracellular ROS by N-acetyl cysteine treatment blocked apoptosis induced by the combinations. The combinations induced apoptosis in A431 and HaCaT cells mediated by loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytosolic cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study demonstrates interactions among ß-C, AO-(2) and P in the induction of apoptosis on A431 and HaCaT cells. These data suggest the combination of ß-caryophyllene with aromadendrene oxide 2 and phytol could be potential therapeutics for the treatment of skin epidermoid cancer and precancerous cells.

Aromadendrene oxide 2, induces apoptosis in skin epidermoid cancer cells through ROS mediated mitochondrial pathway.

AIMS: Aromadendrene oxide 2 (AO-(2)) is an oxygenated sesquiterpene naturally found as a chemical component of essential oils. In the present study anticancer activity of AO-(2) has been investigated on A431 human epidermoid cancer and precancerous HaCaT cells. MATERIAL AND METHODS: Cell viability was used to detect cytotoxic activity. Mechanism of cell death induced by AO-(2) treatments was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. KEY FINDINGS: AO-(2) inhibited the growth and colony formation ability of A431 and HaCaT cells in concentration dependent manner. It induced cell cycle arrest at G0/G1 phase and apoptosis through intracellular ROS accumulation. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment completely blocked apoptotic effect. N-acetyl cysteine treatment significantly reversed G0/G1 arrest induced by AO-(2). AO-(2) treatment caused loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratios, cytochrome c release, activation of caspases (cleaved caspase-3 and caspase-9) and PARP cleavage. AO-(2) also significantly inhibited the growth of multicellular tumor spheroids of A431 and HaCaT cells. SIGNIFICANCE: The results of the present study reveals that AO-(2) a chemical component of essential oils induces apoptosis in A431 and HaCaT cells.

Cancer stem cell mediated acquired chemoresistance in head and neck cancer can be abrogated by aldehyde dehydrogenase 1 A1 inhibition.

Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, this study investigates the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI ≥ 3) by short-term treatment of head and neck squamous cell carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The cell lines (Cal-27 DoxR, Hep-2 DoxR, Hep-2 5FUR) that showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (P < 0.05). Cal-27 CisR and DoxR showed 12-14% enrichment of CD44+ cells, while CisR/5FUR showed 4-6% increase in ALDH1A1+ cells as compared to parental cells (P < 0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential, and increased in vivo tumor burden (P < 0.05). Inhibition of ALDH1A1 in Cal-27 CisR led to down regulation of the CSC markers, reduction in migratory, self-renewal and tumorigenic potential (P < 0.05) accompanied by an induction of sensitivity to Cisplatin (P < 0.05). Further, ex vivo treatment of explants (n = 4) from HNSCC patients with the inhibitor (NCT-501) in combination with Cisplatin showed a significant decrease in proliferating cells as compared to individual treatment (P = 0.001). This study hence suggests an ALDH1A1-driven, CSC-mediated mechanism in acquired drug resistance of HNSCC, which may have therapeutic implications. © 2016 Wiley Periodicals, Inc.

Decomposition and detoxification of aflatoxin B1 by lactic acid.

BACKGROUND: A degradation study of aflatoxin B1 (AFB1) was carried out using a combination of physical and chemical methods. AFB1 was heated at 80 °C in the presence of acetic, citric and lactic acids for various time periods. The cytotoxicity of the degraded AFB1 and its products were determined by MTT assay. RESULTS: The results showed that among the three organic acids lactic acid was most efficient in degrading AFB1. Although complete degradation was not observed, up to 85% degradation of AFB1 was obtained when heated for 120 min. Degradation of AFB1 was confirmed by the reduced toxicity on HeLa cells using MTT assay. Treatment with lactic acid resulted in the conversion of AFB1 into two degradation products. These products were observed at lower retention factors of 0.63 and 0.38, which were identified as AFB2 and AFB2a, respectively. The cytotoxicity of AFB2a exhibited much reduced toxicity on HeLa cells compared to that of AFB1. CONCLUSION: The results have shown the efficiency of lactic acid in degrading AFB1. This study suggest that lactic acid may be considered for use in the food and feed industry since it is present naturally in food and is considered safe.

Prevalence of toxigenic fungi in common medicinal herbs and spices in India.

Mycotoxins are unavoidable contaminants of food grains, feeds, medicinal herbs, and spices, posing as health threat to animals and humans. The objective of this study was to screen medicinal herbs and spices for fungi and mycotoxin contamination and evaluate their safety. Sixty-three samples were examined for fungal contamination and fungal load determined using standard microbiological method. Aflatoxin and citrinin were detected using thin layer chromatography and high-performance chromatography technique. Fifty-eight out of the 63 samples were contaminated, while five were free from fungal contamination. Analysis revealed that 47 % of the samples had a fungal load above 1 × 103 cfu/g which is the permissible limit set by World Health Organization. The samples Mesua ferrea-II and Terminalia chebula-III had the highest fungal load, i.e., 5.0 × 104 cfu/g. A total of 187 fungi were isolated, out of which 28 were toxigenic which included 19 aflatoxin-producing Aspergillus flavus and 9 citrinin-producing Penicillium citrinum. The natural contamination with aflatoxin B1 was detected only in one sample, i.e., Arachis hypogaea (groundnut) which was present beyond the permissible limit. Though toxigenic fungi were isolated, mycotoxins were not detected from any of the medicinal herbs and spices. Medicinal herbs and spices are susceptible to toxigenic fungi; however, they also possess intrinsic factors that inhibit mycotoxin contamination. This study provides a basis in assessing the degree of fungal and potential mycotoxin contamination in medicinal herbs and spices.

Occurrence, detection and detoxification of mycotoxins.

Mycotoxins have been identified as important toxins affecting animal species and humans ever since the discovery of aflatoxin B1 in 1960. Mycotoxigenic fungi are ubiquitous in nature and are held responsible for economic loss as they decrease crop yield and quality of food. The presence of fungi and their mycotoxins are reported not only in food grains but also in medicinal herbs and processed foods. Since prevention is not always possible, detoxification of mycotoxins have been attempted using several means; however, only few have been accepted for practical use, e.g. ammonia in the corn industry. Organizations such as the World Health Organization, US Food and Drug Administration and European Union have set regulations and safety limits of important mycotoxins, viz. aflatoxins, fusarium toxins, ochratoxin, patulin zearalenone, etc., to ensure the safety of the consumers. This review article is a brief and up-to-date account of the occurrence, detection and detoxification of mycotoxins for those interested in and considering research in this area.