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BACKGROUND: Green synthesized nanoparticles have been earmarked for use in nanomedicine including for the development of better anticancer drugs. OBJECTIVE: The aim of this study was to undertake biochemical evaluation of anticancer activities of green synthesized silver nanoparticles (AgNPs) from ethanolic extracts of fruits (AgNPs-F) and leaves (AgNPs-L) of Annona muricata. METHODS: Previously synthesized silver nanoparticles were used for the study. The effects of the AgNPs and 5-Fluorouracil were studied on PC3, HeLa and PNT1A cells. The resazurin, migration and colonogenic assays as well as qRT-PCR were employed. RESULTS: The AgNPs-F displayed significant antiproliferative effects against HeLa cells with an IC50 of 38.58μg/ml and PC3 cells with an IC50 of 48.17μg/ml but selectively spared normal PNT1A cells (selectivity index of 7.8), in comparison with first line drug 5FU and AgNPs-L whose selectivity index were 3.56 and 2.26 respectively. The migration assay revealed potential inhibition of the metastatic activity of the cells by the AgNPs-F while the colonogenic assay indicated the permanent effect of the AgNPs-F on the cancer cells yet being reversible on the normal cells in contrast with 5FU and AgNPs-L. CASP9 was significantly over expressed in all HeLa cells treated with the AgNPs-F (1.53-fold), AgNPs-L (1.52-fold) and 5FU (4.30-fold). CXCL1 was under expressed in HeLa cells treated with AgNPs-F (0.69-fold) and AgNPs-L (0.58-fold) and over expressed in cells treated with 5FU (4.95-fold), but the difference was not statistically significant. CXCR2 was significantly over expressed in HeLa cells treated with 5FU (8.66-fold) and AgNPs-F (1.12-fold) but under expressed in cells treated with AgNPs-L (0.76-fold). CONCLUSIONS: Here we show that biosynthesized AgNPs especially AgNPs-F can be used in the development of novel and better anticancer drugs. The mechanism of action of the AgNPs involves activation of the intrinsic apoptosis pathway through upregulation of CASP9 and concerted down regulation of the CXCL1/ CXCR2 gene axis.

Animal mummies from ancient Egypt are not extensively studied in comparison with human mummies. Dogs mummies were very rare in ancient Egypt, and at the same time were not adequately studied by researchers from conservation point of view. Dog mummy in the present study is dated back to late period (664−333 BC.) and is preserved in the agriculture museum (Egypt). It suffers from different aspects of deterioration such as dirt, holes, wrinkles, missing tissue, accumulated dust, etc. This study aims to evaluate the preservation state of the mummy and explain the deterioration mechanisms to help the conservators later to choose the appropriate materials and methods for the conservation treatment. The analytical techniques used were Auto-Cad, scanning electron microscope (SEM), determination of moisture content, pH measurement, amino acid analysis (AAA), fourier transform infrared (FTIR), portable X-ray fluorescence (XRF), inductively coupled plasma mass spectrometry (ICP-MS) and X-ray diffracion analysis. The results obtained showed different forms of deterioration. Moisture content measurement proved that the mummy was dehydrated. The pH measurement of the mummy components is in acid level. FTIR and amino acid analysis proved that the mummy have been exposed to oxidation process. The analysis by XRF, ICP-MS and XRD proved that bone of the mummy suffered from deterioration.

Introduction: The biological green synthesis of nanoparticles via nanobiotechnology processes have a significant potential to boost nanoparticles production without the use of harsh, toxic, and expensive chemicals commonly used in conventional physical and chemical processes. Annona muricata, a tropical plant belonging to family Annonaceae is one of the most used plants in folk medicine because of its many medicinal uses and therefore presents a strong candidate for use in green synthesis. Aims: The aim of this study was to optimize a method for the synthesis of Silver Nanoparticles (AgNPs) from ethanolic extracts of leaves of Annona muricata as well as to characterize the green synthesized AgNPs. Methodology: AgNPs were synthesized from Annona muricata leaves using AgNO3 solution. The AgNPs were characterized using spectroscopy and microscopy techniques. Results: The formed AgNPs had an absorption maximum at 429 nm using UV–Visible spectroscopy and were stable under different pH, temperature, and storage conditions. Fourier transform infrared analysis revealed the different functional groups responsible for the synthesis and stabilization of the AgNPs. Scanning electron microscopy analysis revealed a spherical nature of the synthesized AgNPs. Energy dispersive x-ray spectroscopy analysis showed presence of Ag, O, and Cl with Ag having the highest composition at 60%. X-Ray Diffraction and Dynamic Light Scattering revealed a crystalline nature of AgNPs with an average size of 87.36 nm and a polydispersity index of 0.16 respectively. Transmission Electron Microscopy analysis further confirmed the crystalline and spherical nature of the AgNPs. Conclusion: In this article, an efficient, eco-friendly and low-cost method for the synthesis and recovery of stable AgNPs using Annona muricata leaves ethanolic extracts as both a reducing and capping agent has been reported for the first time. The synthesized AgNPs could be promising candidates for many biomedical, clinical, engineering, and polymer applications.

Green synthesis of nanoparticles from plant materials opens a new scope in nanobiotechnology and discourages the use of expensive toxic chemicals. The aim of this study was to develop and optimize a method for the synthesis of Silver Nanoparticles (AgNPs) from ethanolic extracts of fruits of Annona muricata as well as to characterize the green synthesized AgNPs. AgNPs were synthesized via AgNO3 solution. The AgNPs were characterized using spectroscopy and microscopy techniques. The formed AgNPs had an absorption maximum of 427 nm and were stable under different temperature, pH and storage conditions. Fourier Transform Infrared Resorption spectroscopy revealed the functional groups responsible for the synthesis and stabilization of the AgNPs. Scanning Electron Microscopy analysis revealed a spherical nature of the AgNPs. Energy Dispersive X-Ray spectroscopy showed presence of Ag, Cl, Ca, and Si with Ag having the highest composition at 80%. X-ray diffraction and dynamic light scattering revealed a crystalline nature of AgNPs with an average size of 60.12 nm and a polydispersity index of 0.1235 respectively. Transmission Electron Microscopy analysis further confirmed the crystalline and spherical nature of the AgNPs. In this article, an efficient, eco-friendly and low-cost method for the synthesis and recovery of stable AgNPs using ethanolic extracts of Annona muricata fruits as both reducing and capping agents has been reported. The synthesized AgNPs could have many biomedical and clinical applications.

Dioecism has always been an issue in many plant species with its numerous disadvantages, especially in woody trees such as date palms. As one of the most important crops in the Middle Eastern countries, researchers are having problems identifying of sex of the plant in its early stages of development. Hence, proper population stands in the male: female ratio for maintenance is almost impossible in the field for better production. In this study, sex determination of date palm (Phoenix dactilyfera L.) were identified in regions of the Y chromosome (Date-SRY) gene, the pivotal gene that initiates sex determination, using a new technique and thus an economically desirable objective, which will significantly impact profits in seed based cultivations. Partial sequences of the Date-SRY were taken and amplified by nested polymerase chain reaction (PCR). According to the results, the exact sex of date palm was identified in all the tested plants, while amplified regions of the Date-SRY gene closely matched with the human and papaya sequences. In addition, a primer pair was designed to amplify the sequences of the SRY-date gene with confidence that it will identify male date palms. These primer sequences include SRY-date Forward 5′- cggccctctaagtatctgtgcgcaacg-3′ (SRY-date F) and the SRY-date Reverse 5′- gtttgcacttcgaagcagag-3′ (SRY-date R). The complete sequence of the DNA has been registered and deposited in GenBank (BankIt1598036 DPSRY1 KC577225 thenKJ873056). Keywords: Date palm, Dioecious plants, Female, Male, Sex determination, SRY gene sequence

This study aims to highlight the antioxidant and cytotoxic effects of Cichorium intybus L.. The cytotoxic activity for cancer cell lines was evaluated by MTT assay. Total phenol and flavonoid conte...

Mummification was considered one of the most important processes used for the preservation of the body through ages in ancient Egypt for long times. Some natural materials were used to achieve the preservation goal. Some of these materials are natural resins which can be used alone as separate or as a mixture with others such as fat, wax, and oils. Some environmental factors (interior or exterior) were affecting these mixture components and lead to its degradation. This study aims to identify the chemical composition by gas chromatography mass spectrometry (GC/MS) and explain the deterioration mechanism of some mixture materials used in the preservation process of Late Period mummy. The results revealed that the preservative mixture samples contained resins pine pitch and mastic mixed with some biochemical substances such as fat and wax. The results also showed that the packing of the mummy’s remains in plastic bags led to the presence of phthalate compounds as contaminants. Moreover, the degradation compounds of acetonitrile and 2-amino-n-isopropylbenzamide in the ancient tissue have been demonstrated with the sample taken from the cranial cavity. Additionally, phenanthrene compounds and its derivatives were identified, and this may be due to the use of pine pitch in the embalming mixture.

Liver is the key organ (detoxifier) that gets injured by reactive oxygen species (ROS) which deplete the body's stores of anti-oxidants. ROS is present in all chronic liver diseases leading to oxidative stress. Consumption of foods high in anti-oxidants replenishes and supplies the body with important anti-oxidants which break down ROS lowering inflammatory responses and preventing liver damage. In this work, clove and honey, which are known to be rich in anti-oxidants extracts were evaluated for their potential to breakdown ROS, Reactive nitrogen species (RNS) and lower oxidative stress in rats. Purified extracts were tested in vitro for ROS scavenging by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and in vivo using CCl4-induced liver injury model. A total of 50 experimental rats were assigned to 5 groups each consisting of 10 animals and treated as follows; 1. CCl4 group: fed on CCl4 in olive oil 2. honey group: fed CCl4 then honey extract, 3. clove group: CCl4 then clove extract, 4. double negative control group: rats fed on standard diet and 5 Negative control: fed olive oil, At 10 weeks, liver histopathology was done and sera collected for liver and kidney function tests. CCl4 -only treated rats showed fibroblastic cells proliferation and inflammatory cells infiltration which was absent in the control animals. Clove extract did not exhibit fibroblastic cells proliferation at all. Both clove and honey significantly reduced cytokines (TGF-β, TNF-α and EGF) and liver function enzymes'(ALT, AST and GGT) activity (58.5,56; 119,121 and 6.1; 6.3) compared to the CCl4 group (97; 191 and 10.1). Clove highly inhibited DPPH (77.2%) compared to honey (6.5%) and conferred better liver protection against CCl4. These findings indicate that clove and honey contain compounds that have anti-oxidant activity with the ability to reverse liver fibrosis. Frequent consumption of these foods may lower liver damage and prevent development of some liver diseases. Public health education on nutritional benefits of these foods is strongly encouraged. Keywords: Clove, Honey, Liver, CCl4, Antioxidant, ROS

The resins and other biochemical substances in mixtures have an important role in achieving the preservation goal for mummification process. The identification of organic mixtures is vital in the conservation field of the unwrapped mummies, but their identification is sometimes difficult because of improper conditions, which can affect their structure and need good investigation techniques for their identification. Some endogenous or exogenous factors can cause some aspects of deterioration to organic mixtures such as cracks, color change, and chemical alteration. This study aims to evaluate the materials used for the preservation of the body in order for them to be re-used in the near future for the preservation processes on the unwrapped mummies in Egyptian museums and storehouses. Samples of organic mixtures were taken from an ancient Egyptian mummy dated to the Late Period (664–333 B.C.). A digital USB microscope and a scanning electron microscope (SEM) were used to identify the application method of the preservative mixtures used during the mummification process and to demonstrate the morphology of surface alteration on the body. Fourier transform infrared (FTIR) was used to detect the chemical changes. A portable X-ray fluorescence (XRF) was used to identify elements. The results revealed that the investigation by microscopes showed cracks in the mixtures poured inside the cranial cavity, but the application of these mixtures was by brush on the outer surface layer of the body. FTIR analysis revealed that a mixture of oil, animal fat, and resinous materials such as mastic and pine were present in the studied samples. Moreover, these samples were prone to oxidation processes through time. XRF analysis showed that the main chemical elements of the samples were calcium, potassium, phosphorus, chlorine, sulfur, and silicon and trace chemical elements such as iron, zinc, copper, and lead, which are in the chemical composition of pine and mastic resins.

The genus Nostoc is a valuable source of a wide spectrum of natural products, which have many different biological activities such as antioxidant, anticancer, anti-HIV, antimalarial, antifungal and antimicrobial drugs. The current work was aimed to evaluate the biological activities of Nostoc muscorum microalga cultivated on different waste water samples (domestic, agricultural and industrial waste water). The algae was cultivated in different kinds of waste water; sterilized BG11 media, non-sterilized treated sewage waste water media (NS-TSW), sterilized treated sewage waste water media (S-TSW), 50% BG11 + 50% sterilized TSW media (50% S-TSW), non-sterilized industrial waste water media (NS-IW), sterilized industrial waste water media (S-IW), 50% BG11 + 50% sterilized industrial waste water media (50% S-IW), non-sterilized agriculture waste water media (NS-AW), sterilized agriculture waste water media (S-AW), and 50% BG11 + 50% sterilized AW media (50% S-AW). The experiment was conducted in triplicate and cultures were incubated at 25 ± 1 °C under continuous shaking (150 rpm) and illumination (2000 lx) for 15 days. The pigments content and biological activity include antioxidant (using diphenyl picryl hydrazyl and 2,2-azino-bis ethylbenzthiazoline-6-sulfonic acid radical methods), antiviral activity against Herpes simplex virus and the cytotoxicity for aqueous extracts in vivo and in vitro assays were determined. The given results reported that use of waste water as media for cultivation of microalgae is a suitable and inexpensive method when compared with ordinary cultivation methods. Also We can conclude that the algal species has high ability to produce active ingredients used as antioxidants and antivirals especially from algae cultivated in S-TSW, 50% S-TSW, NS-AW and 50% S-AW without any toxicity in mice.

Annona muricata (A. muricata) is a tropical plant species belonging to family Annonaceae and known for its many medicinal uses. This review focuses on the research history of its traditional uses, phytochemicals, pharmacological activities, toxicological aspects of the extracts and isolated compounds, as well as the in vitro propagation studies with the objective of stimulating further studies on this plant for human consumption and treatment. A. muricata extracts have been identified in tropical regions to traditionally treat diverse conditions ranging from fever to diabetes and cancer. More than 200 chemical compounds have been identified and isolated from this plant, the most important being alkaloids, phenols and acetogenins. Using in vitro studies, its extracts and phytochemicals have been characterized as antioxidant, anti-microbial, anti-inflammatory, insecticidal, larvicidal, and cytotoxic to cancer cells. In vivo studies have revealed anxiolytic, anti-stress, anti-inflammatory, immunomodulatory, antimalarial, antidepressant, gastro protective, wound healing, hepato-protective, hypoglycemic, anticancer and anti-tumoral activities. In silico studies have also been reported. In addition, clinical studies support the hypoglycemic as well as some anticancer activities. Mechanisms of action of some pharmacological activities have been elucidated. However, some phytochemical compounds isolated from A. muricata have shown a neurotoxic effect in vitro and in vivo, and therefore, these crude extracts and isolated compounds need to be further investigated to define the magnitude of the effects, optimal dosage, and mechanisms of action, long-term safety, and potential side effects. Additionally, more clinical studies are necessary to support the therapeutic potential of this plant. Some studies were also found to have successfully regenerated the plant in vitro, but with limited success. The reported toxicity notwithstanding, A. muricata extracts seem to be some of the safest and promising therapeutic agents of the 21st century and beyond that need to be studied further for better medicinal formulations and diseases management.

Sodic-alkalinity may be more deleterious to plant growth than salinity. The objectives of this study were to determine whether 5-aminolevulinic acid (ALA: an essential precursor for chlorophyll biosynthesis) foliar application could improve the sodic-alkaline resistance of Swiss chard (Beta vulgaris L. subsp. cicla) by regulating water uptake, ionic homeostasis, photosynthetic capacity, and antioxidant metabolism. Eight-week-old uniform plants were grown in nutrient medium without and with a sodic-alkaline regime generated by a mixture of NaHCO3 and Na2CO3 (NaHCO3 : Na2CO3 = 9:1 molar ratio) for 12 d, and leaves were sprayed daily with water or ALA. The Na+ and ALA concentrations were gradually increased to 60 mM and 120 μM, respectively. ALA foliar application alleviated the physiological damage from sodic-alkalinity, as reflected by the increases in plant dry weight, relative growth rate, chlorophyll, Mg2+ concentration, and the decrease in Na+ concentration. However, ALA foliar application did not change the water uptake capacity or the concentration of K+, Fe3+, and endogenous ALA in leaf tissues under sodic-alkaline conditions. ALA foliar application effectively mitigated damage from sodic-alkalinity because of the increased activity of antioxidant enzymes (catalase and guaiacol peroxidase), particularly superoxide dismutase activity, which was maintained at the same level as for control plants. These results suggest that ALA foliar application alleviated sodic-alkaline stress mainly owing to its antioxidant capacity, and superoxide dismutase has the main responsibility for reducing oxidative stress in Swiss chard.

The genes underlying rhg1 lie at a sometimes dominant sometimes co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines). Genomic research identified; nucleotide changes within a candidate gene encoding a receptor like kinase (RLK) that were capable of altering root development and thereby part of the resistance to Hg types 0 (race 3); changes in a laccase that are capable of altering cyst development; and genes underlying changes in membrane biology. This set of three genes are subject to co-selection with a modifier locus on another linkage block. Root development is slowed in the resistant seedling and results in end of season yield loss when SCN is not present. However, in the presence of SCN resistant seedling roots grow just as vigorously as the now slower growing parasitized susceptible roots and therefore show little loss to SCN parasitism. In some genotypes but not others the RLK can act alone to confer resistance. Functional paralogs of the three gene cluster have been found on other linkage groups including A1, B1, G, and O and these can be functional in different sources of resistance like G. soja, PI 437654 and PI438489B. At rhg1 the allele differences change the structure, interacting partners and activity of the LRR protein and the laccase. The changes between the alleles result in about 30 other proteins (judged by 2 Dgels), 112 metabolites (by FTICRMS) and 8 metabolites (by GCMS) to increase in abundance in roots during SCN infection in the resistant NILs. Understanding the basis of root stunting by resistance alleles will be used to improve methods for developing new nematode resistant soybean cultivars that do not suffer from the yield suppression and low seed germination rates of existing cultivars.

In this data article, data obtained from an efficient, eco-friendly and low-cost method for the synthesis and recovery of Silver nanoparticles (AgNPs) using ethanolic extracts of Annona muricata fruits and leaves as reducing, stabilizing and capping agents has been reported. 99.7% pure silver nitrate was used as the inorganic ion source. The data was obtained using different spectroscopic and microscopic techniques. The data is presented in form of images, Microsoft excel sheets, graphs,.raw files,.dpt files, PDF files, among others. Methods of analysis and interpretation of the data have also been presented. The data can be most useful to researchers, research students, industrialists and academicians to acquire knowledge on the green synthesis of AgNPs and related applications. The data is deposited in the Mendeley Data Repository as two independent datasets accessible at https://doi.org/10.17632/jkj2x782wh.1 Gavamukulya et al., 2019 and https://doi.org/10.17632/f4mb6b488n.1 Gavamukulya et al., 2019. Keywords: Annona muricata, Green synthesis, Silver nanoparticles (AgNPs), UV/VIS, FTIR, XRD

The aim of the present study was to assess in vitro the antiradical and antioxidant activities of successive extracts and semi-purified fractions from Rumex vesicarius L. In the present work, three extracts (n-Hexane, ethyl acetate and methanol) and 22 column fractions of methanolic extract (as promising extract) were evaluated against 2,2-diphenyl- 1-picrylhydrazyl (DPPH•) and 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) radical scavenging methods as antiradical and antioxidant activities compared with Butylated hydroxytoluene (BHT) as synthetic standard and silver nanoparticles of methanolic extract (Ag-NPs-Me), in addition to analysis of chemical constituents of extract and fraction using Gas chromatography–mass spectrometry (GC-MS). The obtained results revealed that, both methods go parallel showing that the concentration of extract and incubation time are dependent and proportional with phenolic compounds concentration. Absolute methanol extract recorded the highest antioxidant activity when compared with the other crude extracts with 79.3 and 78.8% against DPPH and ABTS respectively when compared with BHT as synthetic standard (89.4 and 89.9%) against DPPH and ABTS respectively. Calculation of the antiradical activity units showed the highest values of methanolic extract and its promising fraction (No. 12) after 300 seconds (5 minutes) comparing with antioxidant activity (30 min). Also, the antioxidant activity increased with synthetic Ag-NPs-Me when compared with methanolic extract by (IC50= 53.9 and 74.6 µg/ml respectively). Thus, the GC-MS analysis of successive extracts of R. vesicarius L showed a highly complex profile, containing approximately 24 different components. One pure compound was identified from fraction No. 12. The identified compound was l-(+)- ascorbic acid 2, 6-dihexadecanoate. The data also revealed presence of closely similar antioxidant activities in methanolic extract or its pure compounds with BHT when mixed at different proportions. From the obtained results it could be concluded that R. vesicarius methanolic extracts and fractions can be extensively used in the production of potential antioxidant, antiradical and AgNPs-Me for biomedical application on the consumer’s health.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.

The merging of high-throughput gene expression techniques, such as microarray, in the screening of natural products as anticancer agents, is considered the optimal solution for gaining a better understanding of the intervention mechanism. Red yeast rice (RYR), a Chinese dietary product, contains a mixture of hypocholesterolemia agents such as statins. Typically, statins have this effect via the inhibition of HMG‑CoA reductase, the key enzyme in the biosynthesis of cholesterol. Recently, statins have been shown to exhibit various beneficial antineoplastic properties through the disruption of tumor angiogenesis and metastatic processes. Mevinolin (MVN) is a member of statins and is abundantly present in RYR. Early experimental trials suggested that the mixed apoptotic/necrotic cell death pathway is activated in response to MVN exposure. In the current study, the cytotoxic profile of MVN was evaluated against MCF‑7, a breast cancer‑derived cell line. The obtained results indicated that MVN‑induced cytotoxicity is multi‑factorial involving several regulatory pathways in the cytotoxic effects of MVN on breast cancer cell lines. In addition, MVN‑induced transcript abundance profiles inferred from microarrays showed significant changes in some key cell processes. The changes were predicted to induce cell cycle arrest and reactive oxygen species generation but inhibit DNA repair and cell proliferation. This MVN‑mediated multi‑factorial stress triggered specific programmed cell death (apoptosis) and DNA degradation responses in breast cancer cells. Taken together, the observed MVN‑induced effects underscore the potential of this ubiquitous natural compound as a selective anticancer activity, with broad safety margins and low cost compared to benchmarked traditional synthetic chemotherapeutic agents. Additionally, the data support further pre‑clinical and clinical evaluations of MVN as a novel strategy to combat breast cancer and overcome drug resistance.

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive&trade, HF Hybrid Quadrupole-Orbitrap&trade, coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of GlyoxalaseI (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1, Brassica juncea ID: Y13239.1, Arachis hypogaea, ID: DQ989209.2, and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

Pan African University Institute for Basic Sciences, Technol¬ogy and Innovation, Cairo University, Makerere University, Uganda Directorate of Government Analytical Laboratory

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.

Objectives: Resveratrol, with its robust antioxidant activity, has frequently been suggested as potentially having activity in cancer prevention and some recent reports have indicated that it has cancer treatment potential for several types of neoplasia. It has been found to block p-glycoprotein and to protect against several chemotherapeutic agents’ side effects. In this study, we assessed interactive characteristics of resveratrol with docetaxel and doxorubicin and further investigated molecular bases of this interaction in cells of three different solid tumour lines (MCF-7, HeLa and HepG2). Materials and methods and results: Resveratrol per se was found to have anti-cancer properties, but with relatively low potency in all tested cell lines (IC50 ranged from 35.1 to 83.8 μM). Doxorubicin and docetaxel showed IC50 ranging from 0.48 to 0.72 μM and from 25.9 to 77.8 nM, respectively. Resveratrol in combination with doxorubicin and docetaxel significantly increased potencies of both chemotherapeutic agents showing IC50 ranging from 0.12 to 0.34 μM and from 7.2 to 53.02 nM, respectively. The combination index showed synergistic interaction between resveratrol and doxorubicin or docetaxel on MCF-7 cells, and additive interactions on HeLa and HepG2 cells. Real time PCR revealed that expression of Bax and Bcl-2 was simultaneously elevated on combination of resveratrol with doxorubicin or docetaxel in all tested cell lines, whereas p53 exhibited marginal elevation in MCF-7 and HepG2 cells. In addition, p-glycoprotein efflux activity was significantly inhibited, with subsequent accumulation of p-glycoprotein substrate in intracellular compartments. Expression level of mdr1 gene was downregulated after resveratrol combined with doxorubicin or docetaxel in all tested cell lines. Conclusion: Resveratrol potentiates cytotoxic properties of both cancer drugs used in the study through increasing their intracellular level due to p-glycoprotein inhibition and downregulation of mdr1 gene.

Inflammatory reactions that result from microbial infections, both localized and systemic, are reported to cause transient or permanent male infertility. The cellular mechanisms underlying the inhibitory effect of microbial infection on spermatogenesis is not fully understood. However, there is evidence that spermatogenesis is affected by bacterial lipopolysaccharides (LPS) that induce acute inflammatory responses. The aim here was to use LPS treatments to investigate the potential oxidative stress and toxicity in primary cultures of adult rat Sertoli cells. The Sertoli cells were established and incubated with different concentrations of LPS (5, 10 or 20 microg/ml) for 6, 12 and 24h. Lipid peroxidation (LPO) and hydrogen peroxide (H(2)O(2)) production, along with superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), reduced glutathione (GSH), lactate, lactic acid dehydrogenase (LDH), gamma-glutamyl transpeptidase (gamma-GT) and beta-glucuronidase were measured in these cells. LPO as well as H(2)O(2) production were significantly increased while antioxidant enzyme activities and GSH concentration were significantly depressed. Effects were dose and time-dependent at all incubation periods with 10 and 20 microg/ml LPS. Moreover, markers of Sertoli cell function such as lactate production, LDH, gamma-GT and beta-glucuronidase activities were decreased in a time and dose-dependent manner. Incubation of Sertoli cells with 5 microg/ml LPS for 12 and 24h significantly increased oxidative status but significantly decreased the antioxidant enzyme activities, GSH concentration and Sertoli cell markers. In contrast, the oxidative and antioxidant status and markers of Sertoli cell function did not show any significant change in treated Sertoli cells with 5 microg/ml LPS for 6h. Therefore, it may be concluded that LPS induces oxidative stress in Sertoli cells and adversely affects Sertoli cell functions.

The aim of this study was to evaluate the potential anticancer properties and modulatory effect of selected Aloe vera (A. vera) active principles on antioxidant enzyme activities. Thus, three anthraquinones (Namely: aloesin, aloeemodin and barbaloin) were extracted from A. vera leaves by supercritical fluid extraction and subsequently purified by high performance liquid chromatography. Additionally, the N-terminal octapeptide derived from verectin, a biologically active 14 kDa glycoprotein present in A. vera, was also tested. In vivo, active principles exhibited significant prolongation of the life span of tumor-transplanted animals in the following order: barbaloin > octapeptide > aloesin > aloe-emodin. A. vera active principles exhibited significant inhibition on Ehrlich ascite carcinoma cell (EACC) number, when compared to positive control group, in the following order: barbaloin > aloe-emodin > octapeptide > aloesin. Moreover, in trypan blue cell viability assay, active principles showed a significant concentration-dependent cytotoxicity against acute myeloid leukemia (AML) and acute lymphocytes leukemia (ALL) cancerous cells. Furthermore, in MTT cell viability test, aloeemodin was found to be active against two human colon cancer cell lines (i.e. DLD-1 and HT2), with IC50 values of 8.94 and 10.78 μM, respectively. Treatments of human AML leukemic cells with active principles (100 μg ml-1) resulted in varying intensities of internucleosomal DNA fragmentation, hallmark of cells undergoing apoptosis, in the following order: aloe-emodin > aloesin > barbaloin > octapeptide. Intererstingly, treatment of EACC tumors with active principles resulted in a significant elevation activity of key antioxidant enzymes (SOD, GST, tGPx, and LDH). Our data suggest that the tested A. vera compounds may exert their chemo-preventive effect through modulating antioxidant and detoxification enzyme activity levels, as they are one of the indicators of tumorigenesis. These findings are discussed in the light of the potential of A. vera plant extracts for developing efficient, specific and non-toxic anticancer drugs that are affordable for developing countries.

Physiological processes that modulate photosynthetic acclimation to rising atmospheric CO 2 concentration are subjects of intense discussion recently. Apparently, the down-regulation of photosynthesis under elevated CO 2 is not understood clearly. In the present study, the response of soybean ( Glycine max L.) to CO 2 enrichment was examined in terms of nitrogen partitioning and water relation. The plants grown under potted conditions without combined N application were exposed to either ambient air (38 Pa CO 2 ) or CO 2 enrichment (100 Pa CO 2 ) for short (6 days) and long (27 days). Plant biomass, apparent photosynthetic rate, transpiration rate and 15 N uptake and partitioning were measured consecutively after elevated CO 2 treatment. Long-term exposure reduced photosynthetic rate, stomatal conductance and transpiration rate. In contrast, short-term exposure increased biomass production of soybean due to increase in dry weight of leaves. Leaf N concentration tended to decrease with CO 2 enrichment, however such difference was not true for stem and roots. A close correlation was observed between transpiration rate and 15 N partitioned into leaves, suggesting that transpiration plays an important role on nitrogen partitioning to leaves. In conclusion existence of a feed back mechanism for photosynthetic acclimation has been proposed. Down-regulation of photosynthetic activity under CO 2 enrichment is caused by decreasing leaf N concentration, and reduced rate of transpiration owing to decreased stomatal conductance is partially responsible for poor N translocation.

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.

The key step in ethylene (C2H4) signalling during tomato fruit ripening is initialized via the direct interaction between C2H4 and specialized membrane-bound receptors, including Never-Ripe (NR), which is strongly induced during ripening. In order to identify novel ripening-related C2H4-dependent components, a yeast two-hybrid interaction screen has previously been employed, in which NR cDNA has been used as bait. This screen has identified a clone corresponding to interacting protein 7 (SI;INT7), through its specific and strong interaction with the NR receptor (L. Alexander, Z. Lin, R. Hackett, I. Wilson and D. Grierson, unpublished work). In this work, our objective was to identify the corresponding NR-interacting gene and subsequently characterize its expression response to various stress treatments, as well as unravelling its subcellular location in the cell. By sequencing and plant database interrogation, SI;INT7 was found to be a small gene with an open reading frame (ORF) of ∼243 bb encoding a protein composed of 77 aa that shares no sequence homology with any known gene. Notably, northern analyses demonstrated that SI;INT7 gene expression is up-regulated in response to various stress signalling molecules such as salicylic acid (SA), abscissic acid, jasmonic acid, nitric oxide (NO) and salt, implicating SI;INT7 in biotic and abiotic stress signalling transduction responses. To gain more insight into the possible function of SI;INT7, a construct in which SI;INT7 is C-terminally fused to the green fluorescent protein (GFP) was generated. Subsequently, 35S::SI;INT7::GFP-containing constructs were transiently expressed in both tobacco leaves and onion peels via microprojectiles bombardment. Subsequently, confocal laser microscopic examination of bombarded tobacco and onion tissues revealed that the expression of GFP-SI;INT7 was observed predominantly in the plasma membrane, compared to the location throughout the cell observed with the control GFP construct alone. These results are discussed in the light of our present knowledge of C2H4-mediated control over fruit ripening and degree of cross-talk with other stress signalling pathways.

Tomato [Solanum lycopersicum (formerly Lycopersicon esculentum) L. cv. Momotarou] plants were grown under potted conditions inside the greenhouse of Hiroshima University, Japan. The effects of salinity stress under either ambient or elevated CO2 atmospheric conditions during the fruit-growth period were compared. Stem was the major sink organ for the carbon assimilates from the source leaves. A simple nondestructive micromorphometric technique was used to measure growth of these organs. The effect of salt stress under ambient air or elevated CO2 concentration was studied on the apparent photosynthesis (source activity), partitioning 13C, Na content, and antioxidative enzymes. Compared with the control (without salt), salt-stress treatment severely decreased whole-plant biomass. The treatment also depressed leaf photosynthesis and transport of 13C assimilates; however, the impact of stress on these activities was alleviated under elevated CO2 concentration and such alleviation was promoted when si...

Phosphorous (P) fertilization is the major mineral nutrient yield determinant among legume crops. However, legume crops vary widely in the ability to take up and use P during deficiency. The aim here was to compare P uptake and translocation, biological nitrogen fixing ability and photosynthetic rate among mashbean (Vigna aconitifolia cv. ‘Mash-88’), mungbean (Vigna radiata cv. ‘Moong-6601’) and soybean (Glycine max L. cv. ‘Tamahomare’) during deficiency in hydroponics. Two treatments, the withdrawal of P from the solution (P-deprivation) and continued P at 160 μM (P sufficient) were effected at the pod initiation stage. Plants were grown for 20 days. Short-term labeling with 32P showed the uptake and distribution of P into plant parts. Withdrawal of P from the solution reduced biomass, photosynthetic activity, and nitrogen fixing ability in mungbean, and mashbean more than in soybean. P deprivation decreased P accumulation more than N accumulation. The decrease was more severe in mungbean and mashbean than soybean. More P was translocated and distributed into leaves in soybean than in mungbean and mashbean. Leaf P amount was more correlated to leaf area than to photosynthetic rate per unit leaf area among all three legume species. The results indicate that selection for increased efficiency of P utilization and leaf area may be used to improve leguminous crops.

Tomato (Lycopersicon esculentum) plants were grown in hydroponics. At the fruiting stage, N was withdrawn from the growing medium for a period of 19 days and its effects were studied on plant biomass production, photosynthesis, partitioning of 13C and 15N, and changes in the stem and fruit diameters, etc., in order to monitor the mechanism of resource management on the plant parts at low N and prevent excess use of the fertilizer. N-deficiency treatment decreased leaf photosynthesis immediately and affected biomass accumulation of tomato. Conversely, N-deficiency increased stem diameter for a period of two weeks before reducing it below the control. During this period, these results suggest that N deficiency suppresses the source activity more than the sink activity. N-deficiency reduced the amplitude of the circadian pattern of daytime shrinkage and nocturnal expansion of the stem diameter by decreasing the magnitude of the former. Circadian pattern of contraction and expansion of diameter was less evident in the fruit. Under N-deficiency, distribution of 13C and 15N decreased and increased, respectively in fruits. Restricted partitioning of carbon to fruits could be responsible for accumulation of unused assimilates and consequential osmotic adjustment for maintenance of stem water potential. This effect might have precluded contraction of stem diameter of N-deficient plants until the production of assimilates became limiting on account of depression of leaf photosynthesis., This version have some slight differences in wordings from the published version.

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.

Leukemias are a group of cancer types that originate from blood-forming tissues. In this disease, an abnormally large number of immature white blood cells is produced by the bone marrow. The relationship between treatments with plant-derived drugs and leukemia-associated immunophenotypes (LAIPs) of clinically isolated leukemia cells has yet to be established. The aim of the present study was to develop a preliminary clinical prognostic map for commonly expressed LAIPs in patients clinically diagnosed with leukemia, as well as to assess the potential involvement of LAIPs in the response rate to 10 natural products of plant origin. An increased expression of LAIPs, including CD4, CD14, CD33 and CD34, was considered a surrogate marker of the desired response of leukemia cells to treatment with plant-derived drugs. By contrast, the increased expression of the LAIPs, MPO and DR, was associated with poor prognostic outcomes following treatment with the plant-derived drugs. The results showed that 5 of the 10 plant-derived drugs tested induced the expression of several desirable LAIPs biomarkers. These findings clearly highlight the potential treatment efficacy of certain plant-derived drugs against leukemic cell types.

Anthranilate synthase (AS) is a key metabolic enzyme of the tryptophan (Trp) biosynthetic pathway in plants and microbes, and its activity is regulated by feedback inhibition by Trp. A rice gene ( OASA1D ) for a modified α-subunit of anthranilate synthase that is resistant to such feedback inhibition was introduced into azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] by Agrobacterium -mediated transformation followed by selection with hygromycin B. Five independent azuki bean lines expressing OASA1D in which the transgene was fixed in the T 2 generation were obtained. The free Trp level in seeds of these lines (1.3–3.3 mg/g of dry weight) was increased compared with that in wild-type seeds (0.2 mg/g). In contrast, the amount of protein-bound (fixed) Trp in seeds was not affected by expression of the transgene. The AS activity in the transgenic seeds showed a reduced sensitivity to feedback inhibition by Trp or the Trp analog 5-methyltryptophan, indicating that the rice OASA1D subunit was able to interact functionally with the β-subunit of azuki bean AS. These results reveal that transformation with OASA1D is a feasible approach to increase Trp synthesis and accumulation in mature seeds of grain legumes.