Toxicity mechanism of Cu2+ ion individually and in combination with Zn2+ ion in characterizing the molecular changes of Staphylococcus aureus studied using FTIR coupled with chemometric analysis

Copper and zinc have a high binding affinity with a Staphylococcus aureus bacterial community. This causes a change in the biomolecular composition of S. aureus. Our study aims at understanding the resistance mechanism of Cu and Zn either or in various combinations using FTIR and chemometric techniques. Zn toxicity resulted in a significant change in lipid content (3100-2800 cm⁻¹) compared to Cu. A significant decrease in protein content is observed for Cu treatment in the amide region. The bio-concentration factor shows a higher value for Cu compared to Zn. The increase in band area of carbohydrates moieties 1059 cm⁻¹ shows the secretion of EPS due to Cu toxicity. A significant change in nucleic acid compositions was noted in the region1200-900 cm⁻¹ due to Zn treatment. Secondary structural change in protein shows β sheet formation. The result of the finding shows Cu has greater toxicity than Zn. Further toxicity effects were greatly enhanced for metal mixtures ratio (Cu:2Zn). This shows Zn exhibits synergism effect with Cu. The obtained ROC (receiver operating characteristic) curve area gives good reliability of the experiments. The study attempts to understand the mechanism of toxicity removal of Cu and Zn metal mixtures by bacterial population using FTIR coupled with chemometric techniques. Open in a separate windowGraphical abstract     

Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment

AbstractThe development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.Graphic abstract      

Comparison Study of Synthesized Red (or Blood) Orange Peels and Juice Extract-Nanoflowers and Their Antimicrobial Properties on Fish Pathogen (Yersinia ruckeri)

AbstractIn this work, we synthesized blood orange peel extract-copper (II) (Cu²⁺) ions nanoflower (NFs) and blood orange juice extract-copper (II) (Cu²⁺) ions nanoflower examine their antimicrobial properties on the fish pathogen (Yersinia ruckeri). The main compounds of the blood orange peel extract and the blood orange juice extract were organic components, and the copper (II) (Cu^{2 +}) ions were inorganic components. BOPE-Cu^{2 +} nanoflowers are quite compact, porous, and uniform as compared to BOJE-Cu²⁺ nanoflowers. Scanning Electron Microscopy, Fourier Transform Infrared spectrometry, and Energy-Dispersive X-ray spectroscopy were used to observe the structures of the NFs. The findings of FT-IR show Cu–O and Cu–N bonds in NF, which may be an indicator of the development of NFs. Although the antimicrobial actions of BOPE-hNFs and BOJE-hNFs against Yersinia ruckeri (NCTC 12,268) have been confirmed.Graphic Abstract      

A Novel Gene Controlling the Timing of Courtship Initiation in Drosophila melanogaster

Over the past 35 years, developmental geneticists have made impressive progress toward an understanding of how genes specify morphology and function, particularly as they relate to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless protein function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal courtship and mating behavior. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. We describe the results of feminization of the Tre1-expressing neurons, as well as the effects on courtship behavior of mutation of Tre1. In addition, we show that Tre1 is expressed in a sexually dimorphic pattern in the central and peripheral nervous systems and investigate the role of the Tre1 cells in mate identification.     

Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family’s function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.     

The Drosophila wings apart Gene Anchors a Novel,Evolutionarily Conserved Pathway of Neuromuscular Development

wings apart (wap) is a recessive, semilethal gene located on the X chromosome in Drosophila melanogaster, which is required for normal wing-vein patterning. We show that the wap mutation also results in loss of the adult jump muscle. We use complementation mapping and gene-specific RNA interference to localize the wap locus to the proximal X chromosome. We identify the annotated gene CG14614 as the gene affected by the wap mutation, since one wap allele contains a non-sense mutation in CG14614, and a genomic fragment containing only CG14614 rescues the jump-muscle phenotypes of two wap mutant alleles. The wap gene lies centromere-proximal to touch-insensitive larva B and centromere-distal to CG14619, which is tentatively assigned as the gene affected in introverted mutants. In mutant wap animals, founder cell precursors for the jump muscle are specified early in development, but are later lost. Through tissue-specific knockdowns, we demonstrate that wap function is required in both the musculature and the nervous system for normal jump-muscle formation. wap/CG14614 is homologous to vertebrate wdr68, DDB1 and CUL4 associated factor 7, which also are expressed in neuromuscular tissues. Thus, our findings provide insight into mechanisms of neuromuscular development in higher animals and facilitate the understanding of neuromuscular diseases that may result from mis-expression of muscle-specific or neuron-specific genes.     

The Arabidopsis ZED1-Related Kinase Genomic Cluster Is Specifically Required for Effector-Triggered Immunity

CRISPR/Cas9-mediated deletion of an Arabidopsis gene cluster encoding eight kinases supports their immunity-specific roles in sensing pathogenic effectors.

Dear Editor,ZED1-related kinases (ZRKs) associate with the nucleotide binding, Leu-rich repeat (NLR) protein HOPZ-ACTIVATED RESISTANCE1 (ZAR1) to mediate effector-triggered immunity (ETI) against at least three distinct families of pathogenic effector proteins. However, it is unknown whether ZRKs specifically function in ETI or whether they also have additional roles in immunity and/or development. Eight ZRKs are clustered in the Arabidopsis (Arabidopsis thaliana) genome, including the three members with known roles in ETI. Here, we show that an ∼14-kb CRISPR-mediated deletion of the Arabidopsis ZRK genomic cluster specifically affects ETI, with no apparent defects in pattern-recognition-receptor–triggered immunity (PTI) or development.Phytopathogens deliver effector proteins into plant cells that suppress PTI and promote the infection process (Jones and Dangl, 2006). In turn, plants have evolved NLRs that recognize effectors, leading to an ETI response. This recognition often occurs indirectly, whereby NLRs monitor host “sensor” proteins for effector-induced perturbations (Khan et al., 2016). In the absence of their respective NLRs, some of these sensors are effector virulence targets that modulate immunity and development, while others appear to be decoys that mimic virulence targets, with ETI-specific roles (van der Hoorn and Kamoun, 2008; Khan et al., 2018).The ZAR1 NLR recognizes at least six type-III secreted effector (T3SE) families from bacterial phytopathogens. This remarkable immunodiversity appears to be conveyed through associations with members of the receptor-like cytoplasmic kinase XII-2 (RLCK XII-2) family, which all display characteristics of atypical kinases (Lewis et al., 2013; Roux et al., 2014). The ZAR1-mediated ETI responses against the Pseudomonas syringae T3SEs HopZ1a and HopF1r (formerly HopF2a) require ZED1 and ZRK3, whereas recognition of the Xanthomonas campestris T3SE AvrAC requires ZRK1/RKS1 (Lewis et al., 2013; Wang et al., 2015; Seto et al., 2017). ZRKs currently have no ascribed functions outside of ZAR1-associated ETI responses and are therefore considered decoy sensors or adaptors (Lewis et al., 2013; Wang et al., 2015; Khan et al., 2018). However, functional redundancy may exist among members of the ZRK family, masking phenotypes of individual mutants beyond gene-for-gene–type ETI responses (Lewis et al., 2013). We therefore utilized the CRISPR/Cas9 system to knock out the Arabidopsis genomic region containing eight of the 13 members of the RLCK XII-2, including all ZRK genes known to contribute to ETI, to investigate any non-ETI roles of ZRKs. The 14-kb ZRK gene cluster in Arabidopsis Col-0 plants includes ZRK1, ZRK2, ZRK3, ZRK4, ZED1, ZRK6, ZRK7, and ZRK10. A CRISPR/Cas9-mediated deletion of 13.3 kb was accomplished by designing guide RNAs flanking the ends of the ZRK gene cluster, which would result in a double-stranded break on both sides of the ZRK cluster, leaving only the 5′ end 63 nucleotides (21 amino acids) of ZRK10 and the 3′ end 118 nucleotides (39 amino acids) of ZRK7 (Fig. 1A). We obtained a T1 individual (zrk_1.11) homozygous for the deletion, as well as a T1 individual heterozygous for the mutation (zrk_1.10; Fig. 1B), from which we obtained homozygous T2 (zrk_2.11) and T3 (zrk_3.10) plants, respectively. Sequencing results from zrk_2.11 confirmed that the expected region had been deleted (Supplemental Fig. S1). Plants homozygous for the ZRK gene cluster deletion were morphologically indistinguishable from wild-type Col-0 plants, as well as zar1-1 plants (Fig. 1C). In addition, zrk plant fresh weight did not significantly differ from Col-0 plants (Supplemental Fig. S2), indicating that the ZRK cluster does not play a major role in vegetative plant development.Open in a separate windowFigure 1.Deletion of the ZRK gene cluster results in loss of ZRK-mediated ETI and does not significantly alter vegetative growth. A, Representation of ZRK gene cluster before (top) and after (bottom) CRISPR/Cas9-mediated deletion depicting guide RNAs and primers used for genotyping (see Supplemental Methods S1). ZRK KO primers (magenta) were used to confirm the deletion of the ZRK cluster, while ZRK3 primers (green) were used to check if the ZRK cluster was still present in T1 individuals. B, PCR genotyping for deletion of ZRK gene cluster. Amplification of product by ZRK3 F + R primers indicates lack of deletion; amplification by ZRK KO F + R indicates deletion has occurred. Examples for wild type (WT), heterozygous (HT; zrk_1.10), and homozygous for the deletion (HM KO; zrk_1.11) are shown. T1 lines (zrk_1.10 and zrk_1.11) are compared to wild-type Col-0. C, Uninfected morphology of homozygous zrk KO plants (zrk_3.10 or zrk_2.11) compared to Col-0 and zar1-1 plants. Bar = 1 cm. D, Phenotypes of zrk_2.11 plants 7 d after being sprayed with PtoDC3000(hopZ1a; left) or PtoDC3000(hopF1r; right) relative to wild-type Col-0 and zar1-1 plants. Plant immunity and disease image-based quantification of disease symptoms is presented in Supplemental Figure S3A (Laflamme et al., 2016).Next, we wanted to confirm that the deletion of the ZRK gene cluster compromised ZRK-mediated ETI responses. We sprayed the zrk_2.11 line with PtoDC3000(hopZ1a) or PtoDC3000(hopF1r), as both T3SEs require a ZRK as well as the NLR ZAR1 for their recognition in Arabidopsis (Lewis et al., 2013; Seto et al., 2017). We observed that the zrk_2.11 line was susceptible to both PtoDC3000(hopZ1a) and PtoDC3000(hopF1r), and this susceptibility was to the same level as zar1-1 plants as quantified by plant immunity and disease image-based quantification (Fig. 1D, Supplemental Fig. S3, A and C; Laflamme et al., 2016). We observed a similar phenotype for the zrk_3.10 line, confirming that the ZRK cluster deletion compromised ZRK-mediated ETI responses (Supplemental Fig. S3, B and C). Furthermore, the ZAR1-mediated ETI responses against the P. syringae T3SEs HopBA1a, HopX1i, and HopO1c were also lost in zrk_2.11, demonstrating the ZRK-dependence of these ETI responses (Supplemental Fig. S4; Laflamme et al., 2020). To ensure that the ZRK gene cluster deletion specifically impacted ZRK-related ETI responses, the zrk_3.10 and zrk_2.11 lines were also sprayed with PtoDC3000(avrRpt2), an ETI elicitor that does not require a ZRK or ZAR1 for its recognition (Mackey et al., 2003). zrk_3.10 and zrk_2.11 plants remained resistant to PtoDC3000(avrRpt2), indicating that the ZRK gene cluster deletion specifically impacts ZRK-mediated ETI responses (Supplemental Fig. S3C). In addition, growth of virulent PtoDC3000 on the zrk_3.10 and zrk_2.11 lines was unchanged compared to wild-type Col-0 plants, indicating that the ZRKs within this cluster likely do not represent virulence targets (Supplemental Fig. S5).We then examined whether knocking out the ZRK gene cluster impacted PTI. We first measured induction of peroxidase (POX) enzyme activity, as POX enzymes are produced in response to PTI (Mott et al., 2018). After treatment with the PTI elicitor flg22, addition of the POX substrate 5-aminosalicylic acid produces a brown end-product in the presence of active POX enzymes, which is quantified by reading at an optical density of 550 nm (OD₅₅₀; Mott et al., 2018). Twenty h after leaf discs were treated with flg22, zrk_3.10 and zrk_2.11 plants showed the same level of PTI-associated POX activity as wild-type Col-0 plants (Fig. 2A). To further examine the role of the ZRK gene cluster in PTI, we quantified the growth of PtoDC3000ΔhrcC, which is defective in T3SE secretion and is sensitive to altered host PTI responses under high humidity conditions such as those used in our growth assays (Guo et al., 2009; Xin et al., 2016). Growth of PtoDC3000ΔhrcC on zrk_3.10 and zrk_2.11 plants was not significantly different compared to wild-type Col-0 plants (Fig. 2B). In addition, we monitored reactive oxygen species (ROS) production and found that zrk_3.10 and zrk_2.11 plants did not show a significant difference in the ROS burst observed in wild-type Col-0 plants (Fig. 2, C and D). Finally, we treated seedlings with flg22, and found that growth of zrk_3.10 and zrk_2.11 seedlings was inhibited by the same amount as in wild-type Col-0 seedlings, indicative of a similar induction of PTI responses (Fig. 2, E and F; Gómez-Gómez et al., 1999). Together, these results indicate that the ZRK gene cluster does not play a significant role in Arabidopsis PTI responses.Open in a separate windowFigure 2.Deletion of the ZRK gene cluster does not alter pattern-recognition-receptor–triggered immune responses. A, Response to the PTI elicitor flg22 measured by POX activity. Activity from leaf discs was quantified 20 h after treatment with 1 μm of flg22 at a measurement of OD₅₅₀ (n = 6; Mott et al., 2018). B, Bacterial growth of the T3SS-compromised PtoDC3000ΔhrcC on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants 3-d postinoculation. Plants were domed for the duration of the experiment (n = 8). C, Response of Col-0, zrk KO plants (zrk_3.10 and zrk_2.11), and fls2 to the PTI elicitor flg22 measured using luminol-based detection of ROS over a time course of 60 min, with relative light units measured every 2 min (n = 12). D, Boxplots of total relative light units over a period of 30 min from treatments in C (n = 12). E, Growth inhibition of seedlings 7 d after treatment with 1 μm of flg22. F, Seedling growth inhibition was quantified by measuring fresh weight of flg22-treated seedlings as a percentage of water-treated controls (n = 4). Error bars in A, B, C, D, and F, represent se. Lowercase letters represent significantly different statistical groups by Tukey’s honest significant difference test (P < 0.05). Experiments were replicated three times with similar results.Overall, our results support an ETI-specific role for ZRKs in Arabidopsis, acting as sensors of the ZAR1 NLR. Structural insights have revealed important residues required for ZAR1-ZRK1 complex formation, and these are conserved across the RLCK XII-2 family, which includes ZRKs outside the genomic cluster (Supplemental Fig. S6; Lewis et al., 2013; Wang et al., 2019). This suggests that the ZRKs outside this genomic cluster may also play a similar role as ZAR1 sensors. As such, the ZRK family would have evolved to mimic and/or interact with the numerous kinase virulence targets of pathogenic effectors, thereby expanding the surveillance potential of ZAR1.Supplemental DataThe following supplemental materials are available.

• Supplemental Figure S1. Sequencing confirmation of the ZRK gene cluster deletion.
• Supplemental Figure S2. Fresh weight of zrk knockout (KO) plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants.
• Supplemental Figure S3. ZRK gene cluster deletion specifically compromises ZRK-dependent ETI responses.
• Supplemental Figure S4. ZRK gene cluster compromises the ZAR1-dependent ETI responses against HopBA1a, HopO1c, and HopX1i.
• Supplemental Figure S5. Bacterial growth of the virulent PtoDC3000 strain on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Col-0 (wild-type Col-0) plants 0- and 3-d post-inoculation via syringe infiltration.
• Supplemental Figure S6. Multiple sequence alignment of RLCK XII-2 family shows high conservation of putative ZAR1-interacting residues.
• Supplemental Methods S1. Generation and characterization of ZRK cluster deletion lines.

     

Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.     

Interactive role of zinc and iron lysine on Spinacia oleracea L. growth,photosynthesis and antioxidant capacity irrigated with tannery wastewater

AbstractUntreated wastewater contains toxic amounts of heavy metals such as chromium (Cr), which poses a serious threat to the growth and physiology of plants when used in irrigation. Though, Cr is among the most widespread toxic trace elements found in agricultural soils due to various anthropogenic activities. To explore the interactive effects of micronutrients with amino acid chelators [iron-lysine (Fe-lys) and zinc-lysine (Zn-lys)], pot experiments were conducted in a controlled environment, using spinach (Spinacia oleracea L.) plant irrigated with tannery wastewater. S. oleracea was treated without Fe and Zn-lys (0 mg/L Zn-lys and 0 mg/L Fe-lys) and also treated with various combinations of (interactive application) Fe and Zn-lys (10 mg/L Zn-lys and 5 mg/L Fe-lys), when cultivated at different levels [0 (control) 33, 66 and 100%) of tannery wastewater in the soil having a toxic level of Cr in it. According to the results, we have found that, high concentration of Cr in the soil significantly (P < 0.05) reduced plant height, fresh biomass of roots and leaves, dry biomass of roots and leaves, root length, number of leaves, leaf area, total chlorophyll contents, carotenoid contents, transpiration rate (E), stomatal conductance (gs), net photosynthesis (PN), and water use efficiency (WUE) and the contents of Zn and Fe in the plant organs without foliar application of Zn and Fe-lys. Moreover, phytotoxicity of Cr increased malondialdehyde (MDA) contents in the plant organs (roots and leaves), which induced oxidative damage in S. oleracea manifested by the contents of hydrogen peroxide (H₂O₂) and membrane leakage. The negative effects of Cr toxicity could be overturned by Zn and Fe-lys application, which significantly (P < 0.05) increase plant growth, biomass, chlorophyll content, and gaseous exchange attributes by reducing oxidative stress (H₂O₂, MDA, EL) and increasing the activities of various antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX). Furthermore, the supplementation of Zn and Fe-lys increased the contents of essential nutrients (Fe and Zn) and decreased the content of Cr in all plant parts compared to the plants cultivated in tannery wastewater without application of Fe-lys. Taken together, foliar supplementation of Zn and Fe-lys alleviates Cr toxicity in S. oleracea by increased morpho-physiological attributes of the plants, decreased Cr contents and increased micronutrients uptake by the soil, and can be an effective in heavy metal toxicity remedial approach for other crops.Graphic abstract     

Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution of Drosophila centrosomin

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins.     

Genome sequence of the lupin-nodulating Bradyrhizobium sp. strain WSM1417

Bradyrhizobium sp. strain WSM1417 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen (N₂) fixing root nodule of Lupinus sp. collected in Papudo, Chile, in 1995. However, this microsymbiont is a poorly effective N₂ fixer with the legume host Lupinus angustifolius L.; a lupin species of considerable economic importance in both Chile and Australia. The symbiosis formed with L. angustifolius produces less than half of the dry matter achieved by the symbioses with commercial inoculant strains such as Bradyrhizobium sp. strain WSM471. Therefore, WSM1417 is an important candidate strain with which to investigate the genetics of effective N₂ fixation in the lupin-bradyrhizobia symbioses. Here we describe the features of Bradyrhizobium sp. strain WSM1417, together with genome sequence information and annotation. The 8,048,963 bp high-quality-draft genome is arranged in a single scaffold of 2 contigs, contains 7,695 protein-coding genes and 77 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Copper(II) Complexes with N,O-Donor Ligands and Ofloxacin Drug as Antibacterial,DNA Interacting,Cytotoxic and SOD Mimic Agent

Abstract

The N,O-donor bidentate ligands (L¹–L⁷) derived from the reaction between chalcones and pyridinium salt of 2-acetyl furan were synthesized and characterized by IR and NMR spectroscopic techniques. Their complexes [1–7] of Cu(II) were synthesized and characterized by elemental analysis, magnetic measurements, TG analyses, IR and mass spectroscopy. Synthesized complexes were carried out for their biological elucidation using different biological experiments like minimum inhibitory concentration, DNA binding and cleavage study, cytotoxicity, and antiradical activity. Efficient cleavage of pUC19 DNA was observed for all the test complexes than the reference drug.

Graphical Abstract

Increase in DNA chain length and hence the relative viscosity as the complexes binds to DNA via intercalative mode and involves a strong stacking interaction between an aromatic chromophore and the DNA base pair.Electronic supplementary materialThe online version of this article (doi:10.1007/s12088-015-0525-9) contains supplementary material, which is available to authorized users.     

Genetic Complexity in a Drosophila Model of Diabetes-Associated Misfolded Human Proinsulin

Drosophila melanogaster has been widely used as a model of human Mendelian disease, but its value in modeling complex disease has received little attention. Fly models of complex disease would enable high-resolution mapping of disease-modifying loci and the identification of novel targets for therapeutic intervention. Here, we describe a fly model of permanent neonatal diabetes mellitus and explore the complexity of this model. The approach involves the transgenic expression of a misfolded mutant of human preproinsulin, hINS^C96Y, which is a cause of permanent neonatal diabetes. When expressed in fly imaginal discs, hINS^C96Y causes a reduction of adult structures, including the eye, wing, and notum. Eye imaginal discs exhibit defects in both the structure and the arrangement of ommatidia. In the wing, expression of hINS^C96Y leads to ectopic expression of veins and mechano-sensory organs, indicating disruption of wild-type signaling processes regulating cell fates. These readily measurable “disease” phenotypes are sensitive to temperature, gene dose, and sex. Mutant (but not wild-type) proinsulin expression in the eye imaginal disc induces IRE1-mediated XBP1 alternative splicing, a signal for endoplasmic reticulum stress response activation, and produces global change in gene expression. Mutant hINS transgene tester strains, when crossed to stocks from the Drosophila Genetic Reference Panel, produce F₁ adults with a continuous range of disease phenotypes and large broad-sense heritability. Surprisingly, the severity of mutant hINS-induced disease in the eye is not correlated with that in the notum in these crosses, nor with eye reduction phenotypes caused by the expression of two dominant eye mutants acting in two different eye development pathways, Drop (Dr) or Lobe (L), when crossed into the same genetic backgrounds. The tissue specificity of genetic variability for mutant hINS-induced disease has, therefore, its own distinct signature. The genetic dominance of disease-specific phenotypic variability in our model of misfolded human proinsulin makes this approach amenable to genome-wide association study in a simple F₁ screen of natural variation.     

Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.