Non-transgenic Siblings Research Articles

CsMYB60 Confers Enhanced Resistance to Fusarium solani by Increasing Proanthocyanidin Biosynthesis in Cucumber.

Root rot caused by Fusarium solani is one of the most common fungal diseases in cucumber (Cucumis sativus). Proanthocyanidins (PAs) are known to play important roles in inhibiting the growth of phytopathogens. In addition, CsMYB60 is a known positive regulator of flavonol and PA biosynthesis in cucumber. However, it remains unclear that whether PAs can inhibit the growth of F. solani and whether CsMYB60 serves as a target gene for increasing resistance to phytopathogens in cucumber. In this study, we demonstrated that PAs (or their building block, catechin) could increase the resistance of cucumber seedlings to F. solani both invitro and invivo. The addition of catechins, or crude leaf extracts treated with different concentrations of catechins in culture medium, could significantly inhibit the hyphal growth of F. solani. On the other hand, cucumber seedlings treated with catechins showed higher resistance to F. solani than the seedlings of control group. Moreover, transgenic cucumber seedlings overexpressing CsMYB60, with the observed accumulation of PAs, were more resistant to F. solani than the nontransgenic siblings. Therefore, our results suggest that PAs (or catechin) can serve as a biological control agent to protect cucumber plants from the infection of F. solani. More importantly, CsMYB60 holds great promise as a target gene to confer disease resistance during the molecular breeding in cucumber.

Mark-release-recapture experiment in Burkina Faso demonstrates reduced fitness and dispersal of genetically-modified sterile malaria mosquitoes

Every year, malaria kills approximately 405,000 people in Sub-Saharan Africa, most of them children under the age of five years. In many countries, progress in malaria control has been threatened by the rapid spread of resistance to antimalarial drugs and insecticides. Novel genetic mosquito control approaches could play an important role in future integrated malaria control strategies. In July 2019, the Target Malaria consortium proceeded with the first release of hemizygous genetically-modified (GM) sterile and non-transgenic sibling males of the malaria mosquito Anopheles coluzzii in Burkina Faso. This study aimed to determine the potential fitness cost associated to the transgene and gather important information related to the dynamic of transgene-carrying mosquitoes, crucial for next development steps. Bayesian estimations confirmed that GM males had lower survival and were less mobile than their wild type (WT) siblings. The estimated male population size in Bana village, at the time of the release was 28,000 - 37,000. These results provide unique information about the fitness and behaviour of released GM males that will inform future releases of more effective strains of the A. gambiae complex.

Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes. Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose, triglycerides, glucose, and protein in young (3–5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides (AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

Growth Differences of Growth Hormone Transgenic Female and Male Channel Catfish, Ictalurus punctatus, Grown in Earthen Ponds to Sexual Maturation.

This study compared growth performance between female and male transgenic channel catfish, Ictalurus punctatus, containing channel catfish growth hormone full-length cDNA driven by the ocean pout antifreeze protein promoter, opAFP-ccGH, the rainbow trout metallothionein promoter, rtMT-ccGH, or both constructs, and their non-transgenic siblings in earthen ponds at 16 and 48months of age. Body weight between the transgenic and their non-transgenicsiblings differed (P < 0.001) at all ages. Transgenic F2 opAFP-ccGH grew 1.51- to 2.58-, F2 rtMT-ccGH grew 1.44- to 2.99- and F1fish transgenic for both constructs grew 1.36- to 2.92- fold larger than their non-transgenicsibling controls, depending upon age and sex. Body weight of the transgenic GH males was significantly higher than those of the transgenic GH females at 16months of age (P < 0.001). However, body weight of the transgenic GH females was significantly higher (P < 0.001) compared with those of the transgenic GH males at 48months of age, but not for the double transgenics (P > 0.05). In the case of non-transgenic GH siblings, males were larger than females at both 16 and 48months of age (P < 0.001). Sexually dimorphic responses to GH transgenes were the opposite after sexual maturation. When critically low dissolved oxygen levels were encountered, survival of transgenic male and female opAFP-ccGH channel catfish was lower than that of controls (P = 0.004), as well as rtMT-ccGH females (P = 0.11), which is not surprising since the largest fish are most likely to succumb during an oxygen depletion.

Cloning of embryos

Recent research clearly shows that fusion genes can be microinjected into a pronucleus of an ovum and integrate into the pig genome. Animals with such fusion genes are called 'transgenic'. The percentage of injected ova that developed into transgenic pigs varied among experiments from 0.31% to 1.73%. The percentage of transgenic pigs that expressed the fusion gene ranged from 17% to 100%. Eleven different regulatory sequences have been used for fusion genes transferred into pigs. Some of these regulatory sequences directed strong gene expression, but control over level of expression was inadequate. Other regulatory sequences directed weak expression, but imparted only brief spikes of induced expression. The predominant gene coding sequences transferred were for growth-related hormones. Elevation of growth hormone (GH) in expressing transgenic pigs enhanced plasma concentrations of insulin-like growth factor-I (IGF-I), insulin, and glucose, improved feed efficiency about 15%, and markedly reduced subcutaneous fat compared to nontransgenic siblings. Growth rate was enhanced in some transgenic GH pigs but not in others, possibly due to dietary limits. The 'over-expression' of GH was detrimental to the general health of most transgenic pigs. The most prevalent problems were lethargy, lameness, and gastric ulcers. Gilts that expressed foreign GH genes were anoestrous. Boars that expressed foreign GH genes lacked libido, but their semen was fertile when used by artificial insemination. Six different fusion genes have been transmitted from transgenic founders to progeny. Most of the transgenic pigs that produced progeny transmitted the fusion gene as an autosomal dominant trait to about half of their progeny. Regulatory sequences that will permit full control of gene expression must be developed before the full potential of gene transfer in pigs can be realized.

Increased Akt signaling in the fat body of Anopheles stephensi extends lifespan and increases lifetime fecundity through modulation of insulin-like peptides

Insulin-like peptides (ILPs) and the insulin/insulin-like growth factor 1 signaling (IIS) cascade regulate numerous physiological functions, including lifespan, reproduction, immunity, and metabolism, in diverse eukaryotes. We previously demonstrated that in female Anopheles stephensi and Aedes aegypti mosquitoes, activation of the IIS cascade in the fat body led to a significant increase in lifespan. In this work, we elucidated two putative mechanisms in A. stephensi behind the observed lifespan extension and assessed whether this lifespan extension confers an overall fitness advantage to the mosquito. Specifically, we demonstrated that increased Akt signaling in the mosquito fat body following a blood meal significantly suppressed the expression of ILP2 in the head. Moreover, overexpression of active Akt in the fat body altered the expression of a putative insulin binding protein ortholog, Imaginal morphogenesis protein-Late 2 (Imp-L2), in response to transgene expression. Combined, these two factors may act to reduce overall levels of circulating ILP2 or other ILPs in the mosquito, in turn conferring increased survival. We also examined the impact increased fat body IIS had on lifetime fecundity and demonstrated that transgenic female mosquito populations had higher lifetime fecundity relative to non-transgenic sibling controls. These studies provide new insights into the complex hormonal and molecular mechanisms regulating the interplay between IIS, aging, and reproduction in this important vector of human malaria parasites.

NAC-type transcription factors regulate accumulation of starch and protein in maize seeds.

Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids.

Plasmodium falciparum (Haemosporodia: Plasmodiidae) and O'nyong-nyong Virus Development in a Transgenic Anopheles gambiae (Diptera: Culicidae) Strain.

Transgenic Anopheles gambiae Giles (Diptera: Culicidae) mosquitoes have been developed that confer sexual sterility on males that carry a transgene encoding a protein which cuts ribosomal DNA. A relevant risk concern with transgenic mosquitoes is that their capacity to transmit known pathogens could be greater than the unmodified form. In this study, the ability to develop two human pathogens in these transgenic mosquitoes carrying a homing endonuclease which is expressed in the testes was compared with its nontransgenic siblings. Infections were performed with Plasmodium falciparum (Welch) and o’nyong-nyong virus (ONNV) and the results between the transgenic and nontransgenic sibling females were compared. There was no difference observed with ONNV isolate SG650 in intrathoracic infections or the 50% oral infectious dose measured at 14 d postinfection or in mean body titers. Some significant differences were observed for leg titers at the medium and highest doses for those individuals in which virus titer could be detected. No consistent difference was observed between the transgenic and nontransgenic comparator females in their ability to develop P. falciparum NF54 strain parasites. This particular transgene caused no significant effect in the ability of mosquitoes to become infected by these two pathogens in this genetic background. These results are discussed in the context of risk to human health if these transgenic individuals were present in the environment.

Is Saglin a mosquito salivary gland receptor for Plasmodium falciparum?

BackgroundSaglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the salivary glands of Anopheles gambiae and has been considered an essential receptor for sporozoites (SPZ) of Plasmodium berghei and Plasmodium falciparum (Pf), allowing SPZ to recognize, bind to, and infect mosquito salivary glands. Spatial and temporal patterns of Saglin expression reported here, however, suggest that this model does not fully describe the Saglin–SPZ interaction.ResultsSaglin protein was detected by indirect immunofluorescence microscopy only in the medial and proximal-lateral lobes, but not in the distal-lateral lobes, of the salivary glands of An. gambiae; the pattern of expression was independent of mosquito age or physiological state. These results were confirmed by steady-state Saglin transcript and protein expression using qRT-PCR and Western-blot analysis, respectively. Saglin was localized to the basal surface of the cells of the medial lobes and was undetectable elsewhere (intracellularly, on the lateral or apical membranes, the cells’ secretory vacuoles, or in the salivary duct). In the cells of the proximal lateral lobes of the salivary glands, Saglin was distinctly intracellular and was not localized to any of the cell surfaces. Transgenic Anopheles stephensi were produced that expressed An. gambiae Saglin in the distal lateral lobes of the salivary gland. Additional Saglin expression did not enhance infection by PfSPZ compared to non-transgenic siblings fed on the same gametocyte-containing blood meal.ConclusionsThe absence of Saglin in the distal lateral lobes of the salivary glands, a primary destination for SPZ, suggests Saglin is not an essential receptor for Plasmodium SPZ. The lack of any correlation between increased Saglin expression in the distal lateral lobes of the salivary glands of transgenic An. stephensi and PfSPZ infection is also consistent with Saglin not being an essential salivary gland receptor for Plasmodium SPZ.

A Novel Dietary Source of EPA and DHA: Metabolic Engineering of an Important Freshwater Species—Common Carp by fat1-Transgenesis

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are essential for neural development and human health. The n-3 PUFAs are mainly obtained from marine fish by dietary intake. Freshwater fish species usually contain low level of n-3 PUFAs due to the lack of n-3 PUFAs in their food chain. In this study, we report on the substantial production of EPA and DHA in a globally important freshwater fish species, common carp (Cyprinus carpio). This was achieved by introducing an "all-fish" transgene CA:fat1 containing the fish codon-optimized omega-3 desaturase gene (fat1) driven by the common carp β-actin promoter (CA). Through a sperm sample screening method, we successfully generated fat1-positive F1 transgenic population with high efficiency. In F1 population, the muscle contents of ALA (18:3n-3), EPA and DHA were significantly increased when compared with non-transgenic siblings. Thereafter, four independent F2 heterozygous lines were obtained from 4 F1 transgenic males and a detailed comparison of fatty acids profile and growth performance was carried out for these 4 lines. All fat1-transgenic common carps from 4 lines showed an evident decrease in n-6 PUFA contents and a substantial increase in n-3 PUFA contents, among which line 4 stands out, showing a statistically significant increase in all 4 types of n-3 PUFAs including ALA (4.4-fold increase, p < 0.001), EPA (4.8-fold increase, p < 0.01), C22:5n-3 (DPA, 2.4-fold increase, p < 0.05), and DHA (1.9-fold increase, p < 0.05). Therefore, the line 4 was selected as the optimized breeding stock for further study, and the proximate nutrition composition and PUFA synthesis pathway were analyzed. Our study demonstrates that in the transgenic group, the muscular lipid content did not change, while fat accumulations in the internal organs and especially in the liver were significantly decreased as a result of hyperactivation of fatty acid oxidation process. Finally, we conclude that the "all-fish" CA:fat1-transgenic freshwater fish-common carp-can serve as a novel healthy dietary source of n3-PUFAs, especially EPA and DHA.

GUCY2D Cone-Rod Dystrophy-6 Is a "Phototransduction Disease" Triggered by Abnormal Calcium Feedback on Retinal Membrane Guanylyl Cyclase 1.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Dorsal spine evolution in threespine sticklebacks via a splicing change in MSX2A

BackgroundDorsal spine reduction in threespine sticklebacks (Gasterosteus aculeatus) is a classic example of recurrent skeletal evolution in nature. Sticklebacks in marine environments typically have long spines that form part of their skeletal armor. Many derived freshwater populations have evolved shorter spines. Changes in spine length are controlled in part by a quantitative trait locus (QTL) previously mapped to chromosome 4, but the causative gene and mutations underlying the repeated evolution of this interesting skeletal trait have not been identified.ResultsRefined mapping of the spine length QTL shows that it lies near the MSX2A transcription factor gene. MSX2A is expressed in developing spines. In F1 marine × freshwater fish, the marine allele is preferentially expressed.Differences in expression can be attributed to splicing regulation. Due to the use of an alternative 5 ′ splice site within the first exon, the freshwater allele produces greater amounts of a shortened, non-functional transcript and makes less of the full-length transcript. Sequence changes in the MSX2A region are shared by many freshwater fish, suggesting that repeated evolution occurs by reuse of a spine-reduction variant.To demonstrate the effect of full-length MSX2A on spine length, we produced transgenic freshwater fish expressing a copy of marine MSX2A. The spines of the transgenic fish were significantly longer on average than those of their non-transgenic siblings, partially reversing the reduced spine lengths that have evolved in freshwater populations.ConclusionsMSX2A is a major gene underlying dorsal spine reduction in freshwater sticklebacks. The gene is linked to a separate gene controlling bony plate loss, helping explain the concerted effects of chromosome 4 on multiple armor-reduction traits. The nature of the molecular changes provides an interesting example of morphological evolution occurring not through a simple amino acid change, nor through a change only in gene expression levels, but through a change in the ratio of splice products encoding both normal and truncated proteins.

Predation, metabolic priming and early life-history rearing environment affect the swimming capabilities of growth hormone transgenic rainbow trout.

The period of first feeding, when young salmonid fishes emerge from natal stream beds, is one fraught with predation risk. Experiments conducted in semi-natural stream mesocosms have shown that growth hormone transgenic salmonids are at greater risk of predation than their non-transgenic siblings, due partly to the higher metabolic demands associated with transgenesis, which force risky foraging behaviours. This raises questions as to whether there are differences in the swim-performance of transgenic and non-transgenic fishes surviving predation experiments. We tested this hypothesis in wild-origin rainbow trout (Oncorhynchus mykiss) that were reared from first feeding in semi-natural stream mesocosms characterized by complex hydrodynamics, the presence of predators and oligotrophic conditions. Using an open-flume raceway, we swam fish and measured their capacity for burst-swimming against a sustained flow. We found a significant genotype effect on burst-performance, with transgenic fish sustaining performance longer than their wild-type siblings, both in predator and predator-free stream segments. Importantly, this effect occurred before differences in growth were discernable. We also found that mesocosm-reared fish had greater burst-performance than fish reared in the controlled hatchery environment, despite the latter being unexposed to predators and having abundant food. Our results suggest a potential interaction between predation and metabolic priming, which leads to greater burst capacity in transgenic trout.

GUY1 confers complete female lethality and is a strong candidate for a male-determining factor in Anopheles stephensi

Despite their importance in sexual differentiation and reproduction, Y chromosome genes are rarely described because they reside in repeat-rich regions that are difficult to study. Here, we show that Guy1, a unique Y chromosome gene of a major urban malaria mosquito Anopheles stephensi, confers 100% female lethality when placed on the autosomes. We show that the small GUY1 protein (56 amino acids in length) causes female lethality and that males carrying the transgene are reproductively more competitive than their non-transgenic siblings under laboratory conditions. The GUY1 protein is a primary signal from the Y chromosome that affects embryonic development in a sex-specific manner. Our results have demonstrated, for the first time in mosquitoes, the feasibility of stable transgenic manipulation of sex ratios using an endogenous gene from the male-determining chromosome. These results provide insights into the elusive M factor and suggest exciting opportunities to reduce mosquito populations and disease transmission.

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.

Food intake and appetite control in a GH-transgenic zebrafish.

The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. The regulatory network for GH is complex and includes many central and peripheral endocrine factors as well as that from the environment. It is known that GH transgenesis results in increased growth, food intake, and consequent metabolic rates in fishes. However, the manner in which GH transgenesis alters the energetic metabolism in fishes has not been well explored. In order to elucidate these consequences, we examined the effect of GH overexpression on appetite control mechanisms in a transgenic zebrafish (Danio rerio) model. To this, we analyzed feeding behavior and the expression of the main appetite-related genes in two different feeding periods (fed and fasting) in non-transgenic (NT) and transgenic (T) zebrafish as well as glycaemic parameters of them. Our initial results have shown that NT males and females present the same feeding behavior and expression of main appetite-controlling genes; therefore, the data of both sexes were properly grouped. Following grouped data analyses, we compared the same parameters in NT and T animals. Feeding behavior results have shown that T animals eat significantly more and faster than NT siblings. Gene expression results pointed out that gastrointestinal (GT) cholecystokinin has a substantial contribution to the communication between peripheral and central control of food intake. Brain genes expression analyses revealed that T animals have a down-regulation of two strong and opposite peptides related to food intake: the anorexigenic proopiomelanocortin (pomc) and the orexigenic neuropeptide Y (npy). The down-regulation of pomc in T when compared with NT is an expected result, since the decrease in an anorexigenic factor might keep the transgenic fish hungry. The down-regulation of npy seemed to be contradictory at first, but if we consider the GH's capacity to elevate blood glucose, and that NPY is able to respond to humoral factors like glucose, this down-regulation makes sense. In fact, our last experiment showed that transgenics presented elevated blood glucose levels, confirming that npy might responded to this humoral factor. In conclusion, we have shown that GT responds to feeding status without interference of transgenesis, whereas brain responds to GH transgenesis without any effect of treatment. It is clear that transgenic zebrafish eat more and faster, and it seems that it occurs due to pomc down-regulation, since npy might be under regulation of the humoral factor glucose.

The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato.

Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.

Increased Akt signaling in the mosquito fat body increases adult survivorship

Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A. stephensi and Ae. aegypti Akt in the fat body of transgenic mosquitoes led to activation of the downstream signaling molecules forkhead box O (FOXO) and p70 S6 kinase in a tissue and blood meal-specific manner. In both species, increased Akt signaling in the fat body after blood feeding significantly increased adult survivorship relative to nontransgenic sibling controls. In A. stephensi, survivorship was increased by 15% to 45%, while in Ae. aegypti, it increased 14% to 47%. Transgenic mosquitoes fed only sugar, and thus not expressing active Akt, had no significant difference in survivorship relative to nontransgenic siblings. Expression of active Akt also increased expression of fat body vitellogenin, but the number of viable eggs did not differ significantly between transgenic and nontransgenic controls. This work demonstrates a novel mechanism of enhanced survivorship through increased Akt signaling in the fat bodies of multiple mosquito genera and provides new tools to unlock the molecular underpinnings of aging in eukaryotic organisms.

The effect of Bt-transgene introgression on plant growth and reproduction in wild Brassica juncea.

This study aims to investigate the relative plant growth and reproduction of insect-resistant and susceptible plants following the introgression of an insect-resistance Bt-transgene from Brassica napus, oilseed rape, to wild Brassica juncea. The second backcrossed generation (BC2) from a single backcross family was grown in pure and mixed stands of Bt-transgenic and non-transgenic siblings under two insect treatments. Various proportions of Bt-transgenic plants were employed in mixed stands to study the interaction between resistant and susceptible plants. In the pure stands, Bt-transgenic BC2 plants performed better than non-transgenic plants with or without insect treatments. In mixed stands, Bt-transgenic BC2 plants produced fewer seeds than their non-Bt counterparts at low proportions of Bt-transgenic BC2 plants in the absence of insects. Reproductive allocation of non-transgenic plants marginally increased with increasing proportions of Bt-transgenic plants under herbivore pressure, which resulted in increased total biomass and seed production per stand. The results showed that the growth of non-transgenic plants was protected by Bt-transgenic plants under herbivore pressure. The Bt-transgene might not be advantageous in mixed stands of backcrossed hybrids; thus transgene introgression would not be facilitated when herbivorous insects are not present. However, a relatively large initial population of Bt-transgenic plants might result in transgene persistence when target herbivores are present.