Transgenic Mutant Research Articles

Three Camellia sinensis glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanidins.

Biochemical, transgenic, and genetic complementation data demonstrate that three glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanins in plant cells. Flavonoids are compounds in tea (Camellia sinensis) that confer the characteristic astringent taste of tea beverages; these compounds have numerous benefits for human health. In plant cells, flavonoids are synthesized in different locations within the cytoplasm and are then transported and finally stored in vacuoles. To date, the mechanism involved in the intracellular transport of flavonoids in tea has not been well elucidated. In this study, we report the functional characterization of three cDNAs encoding glutathione S-transferases (CsGSTs) of C. sinensis, namely, CsGSTa, CsGSTb, and CsGSTc. The expression profiles of CsGSTa and CsGSTb were positively correlated with the accumulation of flavonols, anthocyanins and proanthocyanins in tea tissues and cultivars. These three recombinant CsGSTs showed a high affinity for flavonols (kaempferol-3-O-glucoside and quercetin-3-O-glucoside) and anthocyanin (cyanidin-3-O-glucoside) in vitro but had no or weak affinity for epicatechin. In vivo, CsGSTa, CsGSTb and CsGSTc fully or partially restored the storage of anthocyanins and proanthocyanidins in transgenic tt19 mutants. Metabolic profiling revealed that the contents of anthocyanins, flavonols, and proanthocyanidins were increased in the transgenic petals of Nicotiana tabacum. Taken together, all data showed that CsGSTa, CsGSTb, and CsGSTc are associated with the storage of anthocyanins, flavonols, and proanthocyanins in C. sinensis cells.

Depletion of extracellular ATP affects the photosystem II photochemistry and the role of salicylic acid in this process

In the present work, treatment with AMP-PCP (β,γ-methyleneadenosine-5'-triphosphate - a competitive inhibitor of the extracellular ATP pool) or apyrase (an ATP-degrading enzyme) decreased the PSII operating efficiency (ΦPSII), photochemical quenching (qp), and maximum quantum efficiency of PSII photochemistry (Fv/Fm) of Arabidopsis leaves illuminated with high light intensity. Mutation of extracellular ATP receptor, DORN1, also caused decreases of ΦPSII, qp, and Fv/Fm when the leaves were exposed to high light. Either the treatment with AMP-PCP and apyrase or the mutation in DORN1 increased the salicylic acid content in the illuminated leaves. Salicylic acid deficiency in NahG transgenic mutant made the PSII photochemistry more sensitive to AMP-PCP or apyrase. These results indicate that extracellular ATP depletion can affect the PSII photochemistry, whereas salicylic acid can relieve this effect.

A single residue change in the product of the chrysanthemum gene TPL1-2 leads to a failure in its repression of flowering

The TPL/TPR co-repressor is involved in many plant signaling pathways, including those regulating the switch from vegetative to reproductive growth. Here, a TPL homolog (TPL 1-2) was isolated from chrysanthemum. Its product was found to be deposited in the nucleus. The abundance of TPL1-2 transcript varied across the plant, with its highest level being recorded in the stem apex, and its lowest in the root and stem. In the leaf, the abundance of TPL1-2 transcript was highest at dusk in plants exposed to long days, and at dawn in those exposed to short days. Site-directed mutagenesis was used to induce an N176H mutation in TPL1-2. The constitutive expression in Arabidopsis thaliana of the wild type and the mutated alleles of TPL1-2 had a contrasting effect on flowering time, with the mutant transgene expressors flowering later than the wild type transgene expressors. The flowering-related genes FT, TSF, FUL and AP1 were all more strongly transcribed in the mutant transgene expressors than in the wild type transgene expressors.

Cloning of Cia7 Gene and Generation of Transgenic Mutants of Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii is a green haploid microalga that stands as an exemplary model organism in the study of heavy metal tolerance and homeostasis of photosynthetic organisms. Within C. reinhardtii's genome resides Cia7, a novel gene that encodes a protein (CIA7) of 104 amino acids with a conserved sequence of four cysteine residues. Given that thiol moieties are essential to metal‐binding proteins, it has been hypothesized that CIA7 might play a role in heavy metal homeostasis of C. reinhardtii. In the path of elucidating the function of the gene, previous studies have focused on exposing C. reinhardtii cells to heavy metal stress and metal bioaccumulation studies. This research study, on the other hand, aimed to isolate and overexpress the CIA7 protein in Escherichia coliand in C. reinhardtii. Polymerase chain reaction (PCR) was used to amplify Cia7 from C. reinhardtii's cDNA library. The PCR product was purified and ligated to the maltose‐binding protein expression vector pMAL‐c5x. The recombinant DNA with the Cia7 insert was used to transform E. coli. The presence of the insert was verified by restriction enzyme digests. In this study, we report the protein overexpression of CIA7. The overexpressed protein will be used for the in vitro studies of the novel protein, CIA7. In addition, a recombinant DNA generated by using the psL72 vector and the Cia7 insert was transformed into C. reinhardtii cells (wild type, cc4425 and mutant of Cia7, cc5013). The obtained transgenic algae will be used in the future for metal bioaccumulation analysis. Overall, this study will provide the clones and transgenic algae necessary to characterize the function of the gene and to determine whether Cia7 confers heavy metal resistance to C. reinhardtii.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel murine model for endothelial dysfunction and heart failure with preserved ejection fraction

The prevalence of Heart Failure with Preserved Ejection Fraction (HFpEF) is dramatically increasing and associated with high mortality. HFpEF predominantly affects elderly (> 65 years) hypertensive women with cardiovascular and non-cardiovascular comorbidities such as obesity, diabetes, atrial fibrillation or renal dysfunction. Mechanisms underlying HFpEF are poorly understood today. A new, but still untested paradigm, focusing on the role of endothelial cells (EC), has recently emerged. The ubiquitine ligase PDZRN3 is a component of the non-canonical Wnt pathway and its endothelial expression has been linked to vascular bed stability. Here, we investigated long-term consequences of endothelial PDZRN3 signaling activation, on heart failure. Investigate the etiology and functional consequence of EC dysfunction in HFpEF. We have generated mice which had post-natal restrictive and inducible endothelial expression of Pdzrn3 (iEC-Pdzrn3). EC specific Pdzrn3 overexpression was induced under tamoxifen administration to 5-week-old mice. To potentiate EC dysfunction, mice were fed with high fat diet for 3 months and studied at 6 months by echocardiography, histology and hemodynamic parameters. At 6 months, both iEC-Pdzrn3 female and male cohorts displayed hemodynamic parameters of HFpEF with a significant strong elevation of the end-diastolic pressure compared to their respective littermates (2.7-fold increase for females, P = 0.02, N = 10; and 1.5-fold increase for males, P = 0.04, N = 10) but did not show any other sign of echocardiography dysfunction. In both female and male cohorts, mutants presented low-grade of inflammation characterized by macrophages infiltration. Precocious times and histological parameters are currently under investigation. iEC-Pdzrn3 mice fed with high fat diet develop markers of HFpEF within 6 months of life.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus – rhizobium interaction.

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

APP depletion alters selective pre- and post-synaptic proteins

The normal role of Alzheimer's disease (AD)-linked amyloid precursor protein (APP) in the brain remains incompletely understood. Previous studies have reported that lack of APP has detrimental effects on spines and electrophysiological parameters. APP has been described to be important in synaptic pruning during development. The effect of APP knockout on mature synapses is complicated by this role in development. We previously reported on differential changes in synaptic proteins and receptors in APP mutant AD transgenic compared to wild-type neurons, which revealed selective decreases in levels of pre- and post-synaptic proteins, including of surface glutamate receptors. In the present study, we undertook a similar analysis of synaptic composition but now in APP knockout compared to wild-type mouse neurons. Here we demonstrate alterations in levels of selective pre- and post-synaptic proteins and receptors in APP knockout compared to wild-type mouse primary neurons in culture and brains of mice in youth and adulthood. Remarkably, we demonstrate selective increases in levels of synaptic proteins, such as GluA1, in neurons with APP knockout and with RNAi knockdown, which tended to be opposite to the reductions seen in AD transgenic APP mutant compared to wild-type neurons. These data reinforce that APP is important for the normal composition of synapses.

CRISPR-Cas9 mediated targeted disruption of FAD2\u20132 microsomal omega-6 desaturase in soybean (Glycine max.L)

BackgroundRecent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis.Results Here we demonstrated an efficient sequence specific integration/mutation of FAD2–2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2–2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2–2 loci. The integration of FAD2–2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis.ConclusionHigh frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.

A Zebrafish JAK1-A634D Gain-of-Function Model Provides New Insights into the Pathogenesis of Familial Hypereosinophilia

Introduction: JAK/STAT signaling is of major importance for hematopoiesis and immune function. A heterozygous JAK1 c.1901C>A de novo point mutation was identified in three members of a family (a mother and two male sons). These patients presented with a unique clinical phenotype of severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, and failure to thrive. Treatment with ruxolitinib, a JAK1/2 inhibitor, resulted in reduced eosinophilia and improved growth (Del Bel et al. JACI 2017). However, the role of this JAK1 mutation in hematopoiesis remains unclear.Methods: ubi-JAK1-WT or A634D-P2A-sfGFP-pA zebrafish transgenic lines were generated incorporating the human genes. Both of these constructs were injected into embryos, but the survival rate for the JAK1-A634D embryos was very low. Six hour post-fertilization (hpf) embryos expressing the mutant transgene were treated with 1 µM ruxolitinib for 24h, which dramatically increased survival. Whole-mount in situ hybridization (WISH) was used to assess changes in hematopoietic stem cells/blood cell populations due to wild-type or JAK1-A634D expression at 24, 28, 36 and 48 hpf using a battery of blood lineage specific probes. O-dianisidine staining was employed to evaluate hemoglobin levels. ImageJ cell counter pluggin was used for cell quantification. Gata1 and o-dianisidine staining intensity was analyzed using Ilastik and CellProfiler. Six groups of 30-50 embryos were analyzed by RNA sequencing (RNAseq): control, JAK1-WT and JAK1-A634D at 28 and 36 hpf. RNA was extracted using Trizol. RNAseq data was analyzed with edgeR and enrichment analysis was performed using DAVID 6.8.Results: JAK1-A634D was toxic to zebrafish embryos but they survived to sexual maturity post-ruxolitinib therapy. F1 embryos from JAK1-A634D founders with high expression of the transgene exhibited abnormal development (70% of embryos) which could be reduced to 12% of embryos following 24h exposure to 1 µM ruxolitinib. This rescue suggests that JAK1-A634D is highly active in zebrafish and its activity needs to be under tight regulation for normal development. Both JAK1-WT and A634D mutant fish demonstrated increased HSPCs (c-myb/runx1 expression) at 36 hpf (p<0.0001) through 48 hpf (A634D>WT>controls; p=0.0308 and p=0.0045, respectively). pu.1/spi1 early myeloid expression showed a similar expression pattern at 28 hpf, A634D>WT>controls; p<0.0001 for both). At 24 and 28 hpf, granulocytes expressing myeloperoxidase (mpx) were increased in both JAK1-WT and JAK1-A634D mutants, but particularly high in JAK1-A634D embryos at 48 hpf (p<0.0001). Macrophages marked by l-plastin (lcp) expression, showed similar cell numbers in JAK1-WT and controls at 24 and 28 hpf, but a significant decrease in JAK1-A634D embryos (p<0.0001). An increase of carboxypeptidase A5 positive mast cells was observed in both JAK1-WT and JAK1-A634D embryos at 48 hpf compared with controls (p<0.0001). We observed an increase of gata1a expression at 24 hpf in A634D embryos (p=0.0058, p=0.0065), but o-dianisidine staining at 48 hpf revealed a decrease in hemoglobin in both JAK1 lines (p<0.0001). The latter data is supported by RNAseq results revealing decreased expression of hemoglobin-associated genes at 36 hpf in both transgenic lines. RNAseq revealed a total of 365 genes differentially expressed, with enrichment for genes coding for proteosomal and ribosomal proteins. In the JAK-1WT we found 79 genes upregulated and 115 downregulated; in the JAK1-A634D, 94 genes were upregulated and 77 were downregulated. JAK-STAT pathway genes were upregulated in the A634D mutant (p<0.05), as well as a trend for genes associated with hematopoiesis (p=0.0633) and negative regulation of cell proliferation (p=0.0658).Conclusions: JAK1-A634D plays an important role in zebrafish development and specifically impacts the myeloid lineage, particularly the granulocyte branch, from which eosinophils arise. Ruxolitinib can revert the arrested development phenotype, demonstrating the specificity of this targeted therapy. Our model provides the first in vivo evidence of the effects of the JAK1-A634D mutation on hematopoiesis and will provide further insight in understanding the cause of familial hypereosinophilia. DisclosuresNo relevant conflicts of interest to declare.

Engineering Non-cellulosic Polysaccharides of Wood for the Biorefinery.

Non-cellulosic polysaccharides constitute approximately one third of usable woody biomass for human exploitation. In contrast to cellulose, these substances are composed of several different types of unit monosaccharides and their backbones are substituted by various groups. Their structural diversity and recent examples of their modification in transgenic plants and mutants suggest they can be targeted for improving wood-processing properties, thereby facilitating conversion of wood in a biorefinery setting. Critical knowledge on their structure-function relationship is slowly emerging, although our understanding of molecular interactions responsible for observed phenomena is still incomplete. This review: (1) provides an overview of structural features of major non-cellulosic polysaccharides of wood, (2) describes the fate of non-cellulosic polysaccharides during biorefinery processing, (3) shows how the non-cellulosic polysaccharides impact lignocellulose processing focused on yields of either sugars or polymers, and (4) discusses outlooks for the improvement of tree species for biorefinery by modifying the structure of non-cellulosic polysaccharides.

Antiageing properties of Damaurone D in Caenorhabditis elegans.

This study was conducted to evaluate the longevity potential of damaurone D (DaD), a component of the damask rose, in the animal model Caenorhabditis elegans. To investigate the effect of DaD on the longevity, lifespan assay was carried out. Fluorescence intensity of transgenic mutants was quantified to test the expression levels of stress proteins. A genetic study using single gene knockout mutants was designed to determine the target genes of DaD. DaD prolonged the mean lifespan of wild-type nematodes by 16.7% under normal conditions and also improved their stress endurance under thermal, osmotic, and oxidative stress conditions. This longevity-promoting effect could be attributed to in vivo antioxidant capacity and its up-regulating effects on the expressions of stress-response proteins such as SOD-3 and HSP-16.2. In addition, DaD treatment attenuated food intake, body length, lipofuscin accumulation and age-dependent decline of motor ability. Gene-specific mutant studies showed the involvement of genes such as daf-2, age-1, and daf-16. These results suggest that DaD has beneficial effects on the longevity, and thus it can be a valuable plant origin lead compound for the development of nutraceutical preparations targeting ageing and ageing-related diseases.

Neuroinflammatory mechanisms in amyotrophic lateral sclerosis pathogenesis

Neuroinflammation is increasingly recognized as an important mediator of disease progression in patients with amyotrophic lateral sclerosis (ALS), and is characterized by reactive central nervous system (CNS) microglia and astroglia as well as infiltrating peripheral monocytes and lymphocytes. Anti-inflammatory and neuroprotective factors sustain the early phase of the disease whereas inflammation becomes proinflammatory and neurotoxic as disease progression accelerates. Initially, motor neurons sustain injuries through multiple mechanisms resulting from harmful mutations causing disruptions of critical intracellular pathways. Injured motor neurons release distress signal(s), which induce inflammatory processes produced by surrounding glial cells in the CNS as well as peripheral innate and adaptive immune cells. This review will focus on mechanisms of neuroinflammation and their essential contributions in ALS pathogenesis. Regulatory T lymphocytes (Tregs) are a subpopulation of immunosuppressive T lymphocytes that become reduced and dysfunctional as the disease progresses in ALS patients. Their degree of dysfunction correlates with the extent and rapidity of the disease. Treg numbers are boosted in transgenic mutant SOD1 (mSOD1) mice through the passive transfer of Tregs or through treatment with an interleukin-2/ interleukin-2 monoclonal antibody complex and rapamycin. Treating the transgenic mice with either of these modalities delays disease progression and prolongs survival. In addition, Treg function is restored when dysfunctional Tregs are isolated from ALS patients and expanded ex vivo in the presence of interleukin-2 and rapamycin. Based on these findings, a first-in-human phase 1 trial has been completed in which expanded autologous Tregs were infused back into ALS patients as a potential treatment. The infusions were safe and shown to 'hit target' by enhancing both Treg numbers and suppressive functions. A delicate balance between anti-inflammatory and proinflammatory factors modulates the rates of disease progression and survival times in ALS. Tipping the balance toward the anti-inflammatory mediators shows promise in slowing the progression of this devastating disease.

DIFERENCIAS ENTRE EL MEJORAMIENTO GENÉTICO CLÁSICO DEL MAÍZ Y EL MEJORAMIENTO POR INGENIERÍA GENÉTICA

&#x0D; &#x0D; &#x0D; En este ensayo se analizan dos aseveraciones: a) no hay diferencias fundamentales entre el mejoramiento genético clásico (MGC) o fitomejoramiento, y el mejoramiento por ingeniería genética (MIG); este último es una extensión del primero pero es más preciso, por lo que da mayor seguridad al consumidor; y b) la transformación por ingeniería genética es equivalente a una mutación natural. Se concurre con la afirmación de que el MGC y el MIG persiguen objetivos que pueden ser complementarios; sin embargo, sus fundamentos, métodos e implicaciones biológicas son fundamentalmente distintos: el MGC funciona dentro de los límites de la compatibilidad sexual, usando a la diversidad genética de la especie como fuente de ADN favorable, lo que le confiere la precisión y mecanismos de recombinación de la reproducción sexual. El MIG recurre al ADN foráneo integrado en una quimera transgénica, que se inserta con imprecisión conocida en el espacio cromosómico del transformando, y genera un diferente locus en cada evento transgénico independiente. Tal dispersión puede conducir a la acumulación de quimeras transgénicas en sus progenies, y posibles efectos deletéreos. Al excluir a los caracteres fenotípicos de tipo cuantitativo como el rendimiento, el MIG depende de los avances logrados por el MGC para desarrollar fenotipos superiores. También se concurre con la aseveración de que la inserción transgénica es una mutación con diferentes implicaciones para el genoma del maíz. La expresión de cada alelo es regulada por el genoma y epigenoma para activarse o desactivarse de acuerdo con un plan de crecimiento de desarrollo del genotipo. En cambio, el mutante transgénico se expresa somáticamente (en todas las células de la planta, sin pausa).&#x0D; &#x0D; &#x0D;

Ectopic Expression of Two FOREVER YOUNG FLOWER Orthologues from Cattleya Orchid Suppresses Ethylene Signaling and DELLA Results in Delayed Flower Senescence/Abscission and Reduced Flower Organ Elongation in Arabidopsis

Two orthologues of Arabidopsis FOREVER YOUNG FLOWER (FYF), CaFYF1 and CaFYF2, were identified from Cattleya intermedia. To investigate the function of these two genes, we performed ectopic expression of CaFYF1/2 in Arabidopsis. Delayed flower senescence and abscission were observed in 35S::CaFYF1/2 transgenic Arabidopsis plants. Furthermore, once CaFYF1/2 was fused with the strong repressor domain SRDX, severe delayed flower senescence and abscission were observed in 35S::CaFYF1/2+SRDX transgenic Arabidopsis plants. In contrast, when 35S::CaFYF1/2 was converted to a potent activator by fusion with the VP16-AD motif, flower senescence and abscission were promoted in these 35S::CaFYF1/2+VP16 transgenic dominant-negative mutant Arabidopsis plants. These results indicated that similar to Arabidopsis FYF, CaFYF1/2 also act as repressors in controlling floral organ senescence and abscission in transgenic Arabidopsis plants. The delayed senescence and abscission of the flower organs in 35S::CaFYF1/2 and 35S::CaFYF1/2+SRDX transgenic Arabidopsis plants were unaffected by ethylene treatment. Genes of the ethylene signaling and abscission-associated pathways, such as EDF1/2/3/4, BOP1/2, and IDA, were repressed in 35S::CaFYF1/2 and 35S::CaFYF1/2+SRDX transgenic Arabidopsis plants. Furthermore, 35S::CaFYF1/2 and 35S::CaFYF1/2+SRDX transgenic Arabidopsis plants showed additional morphological defects, such as short sepals and petals, which were correlated with the upregulation of the DELLA genes RGA, GAI, RGL1, and RGL2. These results suggested a possible role for Cattleya orchid CaFYF1/2 in controlling floral senescence/abscission by suppressing ethylene signaling and abscission-associated genes as well as controlling flower organ elongation through negative regulation of GA response by activating the expression of the DELLA genes during flower development.

Assessment of the mutagenic potential of para-chloroaniline and aniline in the liver, spleen, and bone marrow of Big Blue® rats with micronuclei analysis in peripheral blood.

Splenic tumors have been reported in rat cancer bioassays with para-chloroaniline (PCA) and aniline. Development of these tumors is hypothesized to be due to hematotoxicity via the formation of methemoglobin (MetHb) and not direct DNA reactivity. To evaluate the mode of action (MOA) for tumor formation a transgenic rodent (TGR) in vivo gene mutation assay in Big Blue® TgF344 rats was performed with parallel micronuclei analysis in peripheral blood. Male rats were gavaged daily for 28 d to 0.5, 15, and 60 mg/kg PCA and 100 mg/kg aniline, the base molecular structure of PCA. On test day 10, the 60 mg/kg PCA dose was reduced to 30 mg/kg due to toxicity. On test day 4 and 29 peripheral blood micronucleus analysis was performed and on test day 29 clinical chemistry, hematology, and MetHb measurements were taken. At study termination, on test day 31, spleen, bone marrow, and liver (control tissue) were analyzed for cII transgene mutant frequency (MF). Repeat gavage exposure to PCA and aniline for 28 d did not produce an increase in cII transgene MF in analyzed tissues. An increase in micronuclei was seen at both time points at ≥15 mg/kg PCA and 100 mg/kg aniline. At the same dose levels, significant reductions in red blood cells, increases in absolute reticulocytes (ABRET), and increased levels of MetHb were observed. Together these results support that generation of micronuclei and tumorigenicity following exposure to PCA and aniline is due to compensatory mechanisms (e.g. increased cellular turnover) and not direct DNA reactivity. Environ. Mol. Mutagen. 59:785-797, 2018. © 2018 Wiley Periodicals, Inc.

Mutagenic properties of dimethylaniline isomers in mice as evaluated by comet, micronucleus and transgenic mutation assays

BackgroundThe carcinogenic potential of dimethylaniline (DMA) isomers in rodents and humans has been previously reported, and there is sufficient evidence for the carcinogenicity of 2,6-DMA in experimental animals. The target organ of carcinogenesis of 2,6-DMA is the nasal cavity. In the current study, six DMA isomers, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-DMA, were evaluated for mutagenic properties.ResultsMale ddY mice (3/group) were treated intragastrically (i.g.) with 200 mg/kg of one of the six DMAs, and a comet assay was performed on samples of bone marrow, kidney, liver and lung at 3 and 24 h after the treatment. Positive responses were observed in the kidney, liver and lungs of mice from all of the DMA treatment groups after 3 h and in the bone marrow of mice treated with either 3,4- or 3,5-DMA after 3 h; however, these effects were diminished at the 24 h time point. The micronucleus induction in the bone marrow was analysed in the same mouse at 24 h after the treatment. No induction of micronucleated polychromatic erythrocytes was observed after treatment with any of the DMAs.Male transgenic Muta™ mice (five/group) were treated i.g. with 2,5-, 2,6- or 3,5-DMA at 100 mg/kg bw weekly for 4 weeks, and the lacZ and the cII mutation frequencies were examined in the nasal cavity, liver and bone marrow at 7 days after the last treatment. Statistically significant increases in the mutation frequencies of the lacZ and/or cII genes were observed in the nasal cavity of 2,5-DMA or 2,6-DMA treated mice. Sequence analysis showed increased incidences of AT to GC and GC to TA mutations in the nasal tissues.ConclusionsThese findings suggest that the carcinogenic activities of DMAs are associated with mutagenic events.

DAF-21/Hsp90 is required for C. elegans longevity by ensuring DAF-16/FOXO isoform A function

The FOXO transcription factor family is a conserved regulator of longevity and the downstream target of insulin/insulin-like signaling. In Caenorhabditis elegans, the FOXO ortholog DAF-16A and D/F isoforms extend lifespan in daf-2 insulin-like receptor mutants. Here we identify the DAF-21/Hsp90 chaperone as a longevity regulator. We find that reducing DAF-21 capacity by daf-21(RNAi) initiated either at the beginning or at the end of larval development shortens wild-type lifespan. daf-21 knockdown employed from the beginning of larval development also decreases longevity of daf-2 mutant and daf-2 silenced nematodes. daf-16 loss-of-function mitigates the lifespan shortening effect of daf-21 silencing. We demonstrate that DAF-21 specifically promotes daf-2 and heat-shock induced nuclear translocation of DAF-16A as well as the induction of DAF-16A-specific mRNAs, without affecting DAF-16D/F localization and transcriptional function. DAF-21 is dispensable for the stability and nuclear import of DAF-16A, excluding a chaperone-client interaction and suggesting that DAF-21 regulates DAF-16A activation upstream of its cellular traffic. Finally, we show a selective requirement for DAF-21 to extend lifespan of DAF-16A, but not DAF-16D/F, transgenic daf-2 mutant strains. Our findings indicate a spatiotemporal determination of multiple DAF-21 roles in fertility, development and longevity and reveal an isoform-specific regulation of DAF-16 activity.

B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy

BackgroundIn the pig-to-baboon artery patch model with no immunosuppressive therapy, a graft from an α1,3-galactosyltransferase gene-knockout (GTKO) pig elicits a significant anti-nonGal IgG response, indicating sensitization to the graft. A costimulation blockade-based regimen, e.g., anti-CD154mAb or anti-CD40mAb, prevents sensitization. However, neither of these agents is currently FDA-approved. The aim of the present study was to determine the efficacy of FDA-approved agents on the T and B cell responses. MethodsArtery patch xenotransplantation in baboons was carried out using GTKO/CD46 pigs with (n = 2) or without (n = 1) the mutant transgene for CIITA-knockdown. Immunosuppressive therapy consisted of induction with ATG and anti-CD20mAb, and maintenance with different combinations of CTLA4-Ig, tacrolimus, and rapamycin. In addition, all 3 baboons received daily corticosteroids, the IL-6R blocker, tocilizumab, at regular intervals, and the TNF-α blocker, etanercept, for the first 2 weeks. Recipient blood was monitored for anti-nonGal antibody levels by flow cytometry (using GTKO/CD46 pig aortic endothelial cells), and mixed lymphocyte reaction (MLR). CD22+B cell profiles (naïve [IgD+/CD27−], non-switched memory [IgD+/CD27+], and switched memory [IgD−/CD27+] B cell subsets) were measured by flow cytometry. At 6 months, the baboons were euthanized and the grafts were examined histologically. ResultsNo elicited anti-pig antibodies developed in any baboon. The frequency of naïve memory B cells increased significantly (from 34% to 90%, p = 0.0015), but there was a significant decrease in switched memory B cells (from 17% to 0.5%, p = 0.015). MLR showed no increase in the proliferative T cell response in those baboons that had received CTLA4-Ig (n = 2). Histological examination showed few or no features of rejection in any graft. ConclusionsThe data suggest that immunosuppressive therapy with only FDA-approved agents may be adequate to prevent an adaptive immune response to a genetically-engineered pig graft, particularly if CTLA4-Ig is included in the regimen, in part because the development of donor-specific memory B cells is inhibited.

Jasmonic Acid-Dependent Defenses Play a Key Role in Defending Tomato Against Bemisia tabaci Nymphs, but Not Adults.

The silverleaf whitefly Bemisia tabaci is an important and invasive crop pest in many countries. Previous laboratory studies with Arabidopsis demonstrated that B. tabaci can suppress jasmonic acid (JA) defenses and thereby enhance B. tabaci performance. Whether B. tabaci can suppress JA-regulated host plant defenses in field is unknown. In the present study, we found that, relative to wild-type (WT) tomato plants, transgenic tomato mutants that activated JA defenses (35s::prosys) or impaired JA defenses (spr-2 and def-1) did not affect the survival or reproduction of B. tabaci adults in growth chamber experiments. In contrast, tomato mutants that activated JA defenses slowed B. tabaci nymphal development, while mutants that impaired JA defenses accelerated nymphal development. These effects of JA defenses on nymphal development were also documented under semi-field conditions. Changes in the expression of defense genes and in the production of phytohormones indicated that B. tabaci adults can suppress JA-dependent defenses after infestation for >72 h. The suppression of JA was correlated with the induction of salicylic acid (SA) in B. tabaci-infested leaves under laboratory and under semi-field conditions. If SA signaling was blocked, JA accumulation increased in infested leaves and B. tabaci nymphal development was delayed. These results indicate that, although JA signaling helps in mediating tomato responses against B. tabaci nymphs, B. tabaci can inhibit JA biosynthesis and its action in an SA-dependent manner.