Stress-induced changes in CARF expression serve as a quantitative predictive measure of cell proliferation fate.

Discovery of CARF (Collaborator of ARF)/CDKN2AIP as an ARF-interacting protein that promotes ARF-p53-p21WAF1 signaling and cellular senescence, initially established its role in genomic stress. Multiple reports further unraveled its role in regulation of senescence, growth arrest, apoptosis, or malignant transformation of cells in response to a variety of stress conditions in cultured human cells. It has been established as an essential protein. Whereas CARF-compromised cells undergo apoptosis, its enrichment has been recorded in a variety of cancer cells and has been associated with malignant transformation. We earlier demonstrated its role in stress-induced cell phenotypes that ranged from growth arrest, apoptosis, or malignant transformation. In the present study, we assessed the molecular mechanism of quantitative impact of change in CARF expression level on these cell fates. Stress-induced changes in CARF expression were assessed quantitatively with proteins involved in proteotoxicity, oxidative, genotoxic, and cytotoxic stress. These comparative quantitative analyses confirmed that (i) CARF responds to diverse stresses in a quantitative manner, (ii) its expression level serves as a reliable predictive measure of cell fates (iii) it correlates more with the DNA damage and MDA levels than the oxidative and proteotoxic signatures and (iv) CARF-expression based quantitative assay may be recruited for stress diagnostic applications.

Quadruplex and q-PCR based diagnostic assay to delineate the major quarantine and other seed-borne fungal pathogens of soybean.

Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely, Diaporthe phaseolorum (stem blight), D. longicolla (seed decay), Peronospora manshurica (downy mildew), along with Macrophomina phaseolina causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as Histone-3 for detection of D. longicolla and M. phaseolina. The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309 bp amplicons for D. longicolla and M. phaseolina, respectively. Actin gene based marker DpActF1&R2 was developed for D. phaseolorum which provided 113 bp amplicon whereas, COX2 based marker PmCoxF2&R2 was developed for P. manshurica with amplified product of 152 bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank.

Development of PCR-based assays to diagnose the major fungal pathogens infecting pulse crops, potential for germplasm health certification and quarantine processing.

Plant diseases cause serious economic losses of agriculture production worldwide. Rapid, accurate and reliable diagnostic methods are required to alleviate the detection of fungal plant pathogens to prevent their spread and achieve effective management. This study was aimed to develop fast, reliable and highly sensitive diagnostics to detect fungal plant pathogens for quarantine processing, safe exchange and conservation of germplasms of pulse crops. Multiplex and real time PCR assays were developed for detection of Rhizoctonia solani, Macrophomina phaseolina, Ascochyta rabiei, Alternaria alternata, A. tenuissima, Fusarium oxysporum f. sp. ciceris, Sclerotium (Athelia) rolfsii, Sclerotinia sclerotiorum, Pseudocercospora cruenta and Cercospora canescens causing various diseases in pulse crops. Twenty-two sets of primers from various genomic regions such as cytochrome oxidase subunit (COX 1), internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF-1α), large subunit (LSU), small subunit (SSU) and ß-tubulin as well as two SCAR primers from RAPD profile were designed. The developed markers proved to be species-specific and validated against other fungal plant pathogens associated with pulses for cross-reactivity. The markers proved highly sensitive during conventional and qPCR analysis. Duplex PCR assays for R. solani and M. phaseolina; C. canescens and P. cruenta; A. alternata and A. tenuissima; and a quadruplex PCR assay for A. rabiei, S. sclerotiorum, S. rolfsii and F. oxysporum f. sp. ciceris were developed and validated for simultaneous detection of these pathogens in a single reaction. The assays developed in the present study were able to detect and identify major fungal plant pathogens causing disease in pulse crops.

Thermal properties of different types of starch: A review.

Starch is present in high amount in various cereals, fruits and roots & tubers which finds major application in industry. Commercially, starch is rarely consumed or processed in its native form, thus modification of starch is widely used method for increasing its application and process stability. Due to the high demand for starch in industrial applications, researchers were driven to hunt for new sources of starch, including modification of starch through green processing. Thermal properties are significant reference parameters for evaluating the quality of starch when it comes to cooking and processing. Modification of starches affects the thermal properties, which are widely studied using Differential scanning calorimeter or Thermogravimetric analysis. It could lead to a better understanding of starch's thermal properties including factors influencing and expand its commercial applications as a thickener, extender, fat replacer, etc. in more depth. Therefore, the review presents the classification of starches, factors influencing the thermal properties, measurement methods and thermal properties of starch in its native and modified form. Further, this review concludes that extensive research on the thermal properties of new sources of starch, as well as modified starch, is required to boost thermal stability and extend industrial applications.

Effect of process parameters of microwave-assisted hot air drying on characteristics of fried black gram papad.

Papad is a crunchy wafer-like snack food consumed all over India and other Asian countries. Traditionally, sun-drying requires more drying time and reduces less moisture from papad, which absorbs more oil during frying. The current study was carried out using microwave-assisted hot air drying (MAHD) to reduce fried papad oil content. Box-Behnken design (BBD) was selected to perform 17 trials for drying of papad using MAHD along with different power levels (300, 600, and 900 W), exposure times (30, 60, and 90 s) and hot air temperatures (40, 50 and 60 °C). The fried papad characteristics were evaluated in terms of oil content, oil uptake ratio, porosity, expansion and texture using standard procedures. MAHD helped reduce higher than 30% of fried papad oil content compared to the traditional method. Scanning electron microscope (SEM) confirmed that the MAHD papad has fewer pore structures than sun dried-fried papad. Using response surface methodology (RSM), the optimized parameters of MAHD was found to be 653 W power level with an exposure time of 56 s at 43 °C of drying temperature, which reduced oil content (7.90 ± 0.02%), oil uptake ratio (1.50 ± 0.03), porosity (16.33 ± 0.29%) and expansion (7.97 ± 0.02%) of fried papad.

Pineapple processing waste (PPW): bioactive compounds, their extraction, and utilisation: a review.

Fruits and vegetable processing industries contribute to the largest portion of food waste. With changing diet habits, the demand for the production and processing of fruits and vegetables has increased greatly to fulfil the rising demand amongst the masses. Waste generation begins from the harvesting of raw material until it gets processed. Pineapple processing industries produce processing waste (peel, core, pomace, and crown) which are rich in various bioactive compounds. In most cases, the by-products contain larger amounts of valuable compounds which have higher nutritional and therapeutic importance than its final produce. Researchers have studied the potential of pineapple wastes primarily for the extraction of enzymes (bromelain, pectinase, xylanase and cellulase) and secondarily as a low-cost substrate to produce dietary fibre, organic acids, and phenolic antioxidants. This review describes the bioactive compounds in pineapple wastes, their extraction techniques, and their potential applications as a polymer material, bio-sorbents, bioethanol and vanillin production, etc. It focuses primarily on bioactive compounds that have functional and medicinal value and can be used independently or incorporated with other ingredients to form the valorised product.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C.

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60-80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, ß-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

Partially defatted tomato seed flour as a fat replacer: effect on physicochemical and sensory characteristics of millet-based cookies.

ABSTRACT: Fat is an essential component for cookies structure and sensory attributes, but there is a necessity to replace or mimic fat in order to prevent lifestyle diseases. Hence, this study investigated the effect of industrial by-product, i.e., Partially defatted tomato seed flour (PDTSF) as a fat replacer on physicochemical and sensory characteristics of millet-based cookies at 10, 20, 30, and 40% (w/w) fat replacement levels. The physicochemical and functional properties of PDTSF were analyzed. PDTSF exhibited excellent nutritional profile and functional properties. PDTSF supplemented cookies showed a significant increase (p ≤ 0.05) in hardness, protein, ash, and carbohydrate content and the significant decrease in spread ratio, fat, and energy value with an increment of PDTSF. Descriptive sensory evaluation of millet-based cookies at all fat replacement levels showed good acceptability. Storage analysis for 30 days revealed as acceptance of cookies is not affected due to moisture intake and hardness. Hence, the results confirmed the positive effect on PDTSF as a whole food fat replacer to produce Reduced- fat millet cookies at 10 and 20% fat replacement levels.

Differential expression and localization of TRPV channels in the mature sperm of Anas platyrhynchos.

Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca2+ -signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck (Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca2+ -selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization.

Bioinformatics and Molecular Insights to Anti-Metastasis Activity of Triethylene Glycol Derivatives.

The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1' pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial-mesenchymal transition (EMT) signaling.

Effect of particle size, concentration, temperature and pH on rheological properties of shallots flour.

Shallot flour was prepared and sieved into three different particle sizes of < 180 µm (sample A), 180 µm (sample B) and 250 µm (sample C). Effect of concentration [0.5%, 1.0%, 1.5% and 2.0% (w/w)], temperature (20, 30, 40, 60 and 80 °C), pH (4, 7 and 10) and freezing (- 20 °C) on rheological properties of shallot flour were studied at increasing shear of 0.1-100 s-1. Effect of dynamic change in temperature (15-95 °C) was also measured at constant shear rate of 50 s-1. Power law (Eq. 2) model with coefficient of determination (R2) above 0.90, well described the rheological behavior of the shallot flour as a shear thinning, non-Newtonian fluid at different concentration, temperature and pH. All the samples had n values below 1 and increase in viscosity or consistency index (k) value with increase in concentration of the sample was observed, while inverse relation was observed when temperature was increased. All samples showed increase in k value when the pH of the dispersion was varied from acidic to alkaline condition. Viscosity of samples were found unaffected even after freezing in freeze-thaw cycle. These data show sample A to be most suitable for their application as thickener, having highest k value. The obtained research provides information for utilization of shallot as a thickener in various food industries.

TRPA1 is selected as a semi-conserved channel during vertebrate evolution due to its involvement in spermatogenesis.

TRPA1 is a non-selective cation channel originated in invertebrates. The genomic locus containing TRPA1 gene remains highly conserved and retained in all vertebrates. TRPA1 gene is evolutionarily selected, yet maintained as a highly diverged protein. Throughout the vertebrate evolution, the extracellular loops of TRPA1 become most diverged indicating that TRPA1 may be involved in detecting large spectrum and uncertain stimulus which is critical for adaptive benefit. We tested the expression of TRPA1 in mature sperm from different vertebrates. This is the first report demonstrating that TRPA1 is expressed endogenously in mature spermatozoa of multiple species representing entire vertebrate phyla. However, its specific localization within sperm remains species-specific. Accordingly, we report that in rodents TRPA1 expression correlates with different stages of spermatogenesis. We propose that presence of endogenous TRPA1 in testes and in mature sperm provides reproductive benefit.

Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin.

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence.

Fucoxanthin is commonly found in marine organisms; however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin; hence, it warrants further attention for basic mechanistic studies as well as drug development.

Effect of ultrasound treatment on dehulling efficiency of blackgram.

Present study was conducted to evaluate the effect of Power ultrasound, on dehulling efficiency, dhal yield, dehulling loss and total colour difference of black gram using response surface methodology. Nine treatments were performed with variation in ultrasound power 343-525 W and treatment time 1-3.5 h. It was observed that ultrasound treatment significantly improved the dehulling efficiency and dhal yield of the black gram and reduced the dehulling loss. The optimized treatment condition obtained for optimum dehulling yield (75.71%), dhal yield (74.63%) dehulling loss (12.72%), and total colour difference (5.08) was ultrasound power of 513.39 W and exposure time of 2.12 h. Moreover the blackgram pretreated with ultrasound required lesser cooking time when compared to soaked alone sample. The SEM analysis revealed the significant effect of ultrasound on the blackgram kernel which led to uniform cavitation of the surface of the kernel compared to the soaked sample without ultrasound treatment. In food industry blackgram is preprocessed i.e. soaked and cooked to produce various soups, canned products, batter, snack foods etc. Hence ultrasonic treatment can be applied to improve and facilitate a faster dehulling efficiency, with added advantage of increased soaking rate and a decrease in the cooking time for blackgram.

Molecular dynamics-based identification of novel natural mortalin-p53 abrogators as anticancer agents.

INTRODUCTION: Cancer is one of the leading causes of mortality worldwide that requires attention in terms of extensive study and research. Eradication of mortalin-p53 interaction that leads to the inhibition of transcriptional activation or blocking of p53 from functioning as a suppressor and induction of nuclear translocation of p53 can prove to be one of the useful approaches for cancer management. RESULTS: In this study, we used structure-based approach to target the p53-binding domain of mortalin in order to prevent mortalin-p53 complex formation. We screened compounds from ZINC database against the modeled mortalin protein using Glide virtual screening. The top two compounds, DTOM (ZINC 28639308) and TTOM (ZINC 38143676) with Glide score of -12.27 and -12.16, respectively, were identified with the potential to abrogate mortalin-p53 interaction. Finally, molecular dynamics simulations were used to analyze the dynamic stability of the ligand-bound complex and it was observed that residues Tyr196, Asn198, Val264 and Thr267 were involved in intermolecular interactions in both the simulated ligand-bound complexes, and thus, these residues may have a paramount role in stabilizing the binding of the ligands with the protein. CONCLUSION: These detailed insights can further facilitate the development of potent inhibitors against mortalin-p53 complex.

2,3-Dihydro-3ß-methoxy Withaferin-A Protects Normal Cells against Stress: Molecular Evidence of Its Potent Cytoprotective Activity.

2,3-Dihydro-3ß-methoxy withaferin-A (3ßmWi-A) is a natural withanolide that is structurally close to withaferin-A (Wi-A), is cytotoxic to human cancer cells, and is a candidate anticancer natural compound. Using cell-based biochemical, molecular, and imaging assays, we report that Wi-A and 3ßmWi-A possess contrasting activities. Whereas Wi-A caused oxidative stress to normal cells, 3ßmWi-A was well tolerated at even 10-fold higher concentrations. Furthermore, it promoted survival and protected normal cells against oxidative, UV radiation, and chemical stresses. We provide molecular evidence that 3ßmWi-A induces antistress and pro-survival signaling through activation of the pAkt/MAPK pathway. We demonstrate that 3ßmWi-A (i) contrary to Wi-A is safe and possesses stress-relieving activity, (ii) when given subsequent to a variety of stress factors including Wi-A, protects normal cells against their toxicity, and (iii) is a vital compound that may guard normal cells against the toxicity associated with various targeted therapeutic regimes in clinical practice.

Functional significance of point mutations in stress chaperone mortalin and their relevance to Parkinson disease.

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.