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Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

The malfunctioning of the brain synucleins is associated with pathogenesis of Parkinson’s disease. Synucleins’ ability to modulate various pre-synaptic processes suggests their modifying effects on the electroencephalogram (EEG) recorded from different brain structures. Disturbances in interrelations between them are critical for the onset and evolution of neurodegenerative diseases. Recently, we have shown that, in mice lacking several synucleins, differences between the frequency spectra of EEG from different brain structures are correlated with specificity of synucleins’ combinations. Given that EEG spectra are indirect characteristics of inter-structural relations, in this study, we analyzed a coherence of instantaneous values for EEGs recorded from different structures as a direct measure of “functional connectivity” between them. Methods: EEG data from seven groups of knock-out (KO) mice with combined deletions of alpha, beta, and gamma synucleins versus a group of wild-type (WT) mice were compared. EEG coherence was estimated between the cortex (MC), putamen (Pt), ventral tegmental area (VTA), and substantia nigra (SN) in all combinations. Results: EEG coherence suppression, predominantly in the beta frequency band, was observed in KO mice versus WT littermates. The suppression was minimal in MC-Pt and VTA-SN interrelations in all KO groups and in all inter-structural relations in mice lacking either all synucleins or only beta synuclein. In other combinations of deleted synucleins, significant EEG coherence suppression in KO mice was dominant in relations with VTA and SN. Conclusion: Deletions of the synucleins produced significant attenuation of intra-cerebral EEG coherence depending on the imbalance of different types of synucleins.

Abstract Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Three decades following the introduction of the first Rb knockout (KO) mouse model, the role of this critical protein in regulating brain development during embryogenesis and beyond remains a major scientific interest. Rb is a tumor suppressor gene known as the master regulator of the G1/S checkpoint and control of cell cycle progression in stem and progenitor cells, but also their differentiated progeny. Here, we review the recent literature about the various Rb conditional Knockout (cKO) and inducible Knockout (iKO) models studied thus far, highlighting how findings should always be interpreted in light of the model and context under inquiry especially when studying the role of Rb in neuronal survival. There is indeed evidence of age-specific, cell type-specific and region-specific effects following Rb KO in the embryonic and the adult mouse brain. In terms of modeling neurodegenerative processes in human diseases, we discuss cell cycle re-entry (CCE) as a candidate mechanism underlying the increased vulnerability of Rb-deficient neurons to cell death. Notably, mouse models may limit the extent to which CCE due to Rb inactivation can mimic the pathological course of these disorders, such as Alzheimer's disease. These remarks ought to be considered in future research when studying the consequences of Rb inactivation on neuronal generation and survival in rodents and their corresponding clinical significance in humans.

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.

ABSTRACTBCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. Results The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100–200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon–intron structure, with a 70–80% success rate.

Rabbits have been domesticated for meat, wool, and fur production, and have also been cherished as a companion, artistic inspiration, and an experimental model to study many human diseases. In the present study, the muscle mass negative regulator gene myostatin (MSTN) was knocked out in rabbits at two novel sites in exon3, and the function of these mutations was determined in subsequent generations. The prominent double muscle phenotype with hyperplasia or hypertrophy of muscle fiber was observed in the MSTN-KO rabbits, and a similar phenotype was confirmed in the F1 generation. Moreover, the average weight of 80-day-old MSTN-KO rabbits (2,452 ± 63 g) was higher than that of wild-type rabbits (2,393.2 ± 106.88 g), and also the bodyweight of MSTN-KO rabbits (3,708 ± 43.06g) was significantly higher (P < 0.001) at the age of 180 days than wild-type (WT) rabbits (3,224 ± 48.64g). In MSTN-KO rabbits, fourteen rabbit pups from the F1 generation and thirteen from the F2 generation stably inherited the induced MSTN gene mutations. Totally, 194 pups were produced in the F1 generation of which 49 were MSTN-KO rabbits, while 47 pups were produced in the F2 generation of which 20 were edited rabbits, and the ratio of edited to wild-type rabbits in the F2 generation was approximately 1:1. Thus, we successfully generated a heritable double muscle buttocks rabbits via myostatin mutation with CRISPR/Cas9 system, which could be valuable in rabbit's meat production and also a useful animal model to study the development of muscles among livestock species and improve their important economic traits as well as the human muscle development-related diseases.

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.

mRNAs have been found to undergo substantial selective degradation during the late stages of spermiogenesis. However, the mechanisms regulating this biological process are unknown. In this report, we have identified Tex13a, a spermatid-specific gene that interacts with the CCR4–NOT complex and is implicated in the targeted degradation of mRNAs encoding particular structural components of sperm. Deletion of Tex13a led to a delayed decay of these mRNAs, lowered the levels of house-keeping genes, and ultimately lowered several key parameters associated with the control of sperm motility, such as the path velocity (VAP, average path velocity), track speed (VCL, velocity curvilinear), and rapid progression.

We previously reported Israa (immune-system-released activating agent), a novel gene nested in intron 6 of the mouse Zmiz1 gene. Zmiz1 is involved in several functions such as fertility and T cell development and its knockout leads to non-viable embryos. We also reported ISRAA's expression in lymphoid organs, particularly in the thymus CD3+ T cells during all developmental stages. In addition, we showed that ISRAA is a binding partner of Fyn and Elf-1 and regulates the expression of T cell activation-related genes in vitro. In this paper, we report the generation and characterization of an Israa−/− constitutive knockout mouse. The histological study shows that Israa−/− mice exhibit thymus and spleen hyperplasia. Israa−/− derived T cells showed increased proliferation compared to the wild-type mice T cells. Moreover, gene expression analysis revealed a set of differentially expressed genes in the knockout and wild-type animals during thymus development (mostly genes of T cell activation pathways). Immunological phenotyping of the thymocytes and splenocytes of Israa−/- showed no difference with those of the wild-type. Moreover, we observed that knocking out the Zmiz1 intron embedded Israa gene does not affect mice fertility, thus does not disturb this Zmiz1 function. The characterization of the Israa−/- mouse confirms the role ISRAA plays in the expression regulation of genes involved in T cell activation established in vitro. Taken together, our findings point toward a potential functional interrelation between the intron nested Israa gene and the Zmiz1 host gene in regulating T cell activation. This constitutively Israa−/− mice can be a good model to study T cell activation and to investigate the relationship between host and intron-nested genes.

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human.

While both risk-taking and avoidant behaviors are necessary for survival, their imbalanced expression can lead to impulse-control and anxiety disorders, respectively. In laboratory rodents, the conflict between risk proneness and anxiety can be studied by using their innate fear of heights. To explore this aspect in detail and investigate venturesome behavior, here we used a “Himalayan Bridge,” a rat-adapted version of the suspended wire bridge protocol originally developed for mice. The apparatus is composed of two elevated scaffolds connected by bridges of different lengths and stability at 1 m above a foam rubber-covered floor. Rats were allowed to cross the bridge to reach food, and crossings, pawslips, turnabouts, and latencies to cross were measured. Given the link between risky behavior and adolescence, we used this apparatus to investigate the different responses elicited by a homecage mate on the adolescent development of risk-taking behavior. Thus, 24 wild-type (WT) subjects were divided into three different housing groups: WT rats grown up with WT adult rats; control WT adolescent rats (grown up with WT adolescents), which showed a proclivity to risk; and WT rats grown up with an adult rat harboring a truncated mutation for their dopamine transporter (DAT). This latter group exhibited risk-averse responses reminiscent of lower venturesomeness. Our results suggest that the Himalayan Bridge may be useful to investigate risk perception and seeking; thus, it should be included in the behavioral phenotyping of rat models of psychiatric disorders and cognitive dysfunctions.

Genetic manipulation is one of the indispensable techniques to examine gene functions both in vitro and in vivo. In particular, cardiovascular phenotypes such as blood pressure cannot be evaluated in vitro system, necessitating the creation of transgenic or gene-targeted knock-out and knock-in experimental animals to understand the pathophysiological roles of specific genes on the disease conditions. Although genome-wide association studies (GWAS) in various human populations have identified multiple genetic variations associated with increased risk for hypertension and/or its complications, the causal links remain unresolved. Genome-editing technologies can be applied to many different types of cells and organisms for creation of knock-out/knock-in models. In the post-GWAS era, it may be more worthwhile to validate pathophysiological implications of the risk variants and/or candidate genes by creating genome-edited organisms.

Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before. To this purpose, it is necessary to introduce the system into early stage zygotes or to edit cells followed by somatic cell nuclear transfer. In this review, several strategies for pig knock-out gene editing, using the CRISPR-Cas9 system, will be summarized, as well as genotyping methods and different delivery techniques to introduce these tools into the embryos. Finally, the best approaches to produce homogeneous, biallelic edited animals will be discussed.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Abstract Background Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD” (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. Results Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. Conclusions We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals.

Age-associated loss of retinal ganglion cells (RGCs) causes visual deficits, but there is not yet any therapeutic agent to prevent the loss of these cells. Herein, we report that apelin, an endogenous peptide ligand of APJ receptor, is protective against the age-related loss of RGCs in mice. The mRNA expression of apelin was reduced in the retina of old mice compared with that in young mice, whereas retinal APJ expression increased with age. Immunofluorescence staining showed that APJ was present in RGCs and their surrounding cells expressed apelin. In addition, both functional and histological analyses demonstrated that apelin deficiency accelerated the loss of RGCs associated with age in mice. These results suggest that endogenous apelin plays a protective role against the degeneration of RGCs and that the apelinergic axis may be a new target for preventing age-related visual impairment.

Problems associated with the abuse of amphetamine-type stimulants, including methamphetamine (MA), pose serious health and socioeconomic issues world-wide. While it is well-established that MA’s psychopharmacological effects involve interactions with monoamine neurotransmission, accumulating evidence from animal models implicates dysregulated glutamate in MA addiction vulnerability and use disorder. Recently, we discovered an association between genetic vulnerability to MA-taking and increased expression of the glutamate receptor scaffolding protein Homer2 within both the shell and core subregions of the nucleus accumbens (NAC) and demonstrated a necessary role for Homer2 within the shell subregion in MA reward and reinforcement in mice. This report extends our earlier work by interrogating the functional relevance of Homer2 within the NAC core for the conditioned rewarding and reinforcing properties of MA. C57BL/6J mice with a virus-mediated knockdown of Homer2b expression within the NAC core were first tested for the development and expression of a MA-induced conditioned place-preference/CPP (four pairings of 2 mg/kg MA) and then were trained to self-administer oral MA under operant-conditioning procedures (5–80 mg/L). Homer2b knockdown in the NAC core augmented a MA-CPP and shifted the dose-response function for MA-reinforced responding, above control levels. To determine whether Homer2b within NAC subregions played an active role in regulating MA reward and reinforcement, we characterized the MA phenotype of constitutive Homer2 knockout (KO) mice and then assayed the effects of virus-mediated overexpression of Homer2b within the NAC shell and core of wild-type and KO mice. In line with the results of NAC core knockdown, Homer2 deletion potentiated MA-induced CPP, MA-reinforced responding and intake, as well as both cue- and MA-primed reinstatement of MA-seeking following extinction. However, there was no effect of Homer2b overexpression within the NAC core or the shell on the KO phenotype. These data provide new evidence indicating a globally suppressive role for Homer2 in MA-seeking and MA-taking but argue against specific NAC subregions as the neural loci through which Homer2 actively regulates MA addiction-related behaviors.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1–3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12–14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

In many complex systems one observes the formation of medium-level structures, whose detection could allow a high-level description of the dynamical organization of the system itself, and thus to its better understanding. We have developed in the past a powerful method to achieve this goal, which however requires a heavy computational cost in several real-world cases. In this work we introduce a modified version of our approach, which reduces the computational burden. The design of the new algorithm allowed the realization of an original suite of methods able to work simultaneously at the micro level (that of the binary relationships of the single variables) and at meso level (the identification of dynamically relevant groups). We apply this suite to a particularly relevant case, in which we look for the dynamic organization of a gene regulatory network when it is subject to knock-outs. The approach combines information theory, graph analysis, and an iterated sieving algorithm in order to describe rather complex situations. Its application allowed to derive some general observations on the dynamical organization of gene regulatory networks, and to observe interesting characteristics in an experimental case.

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.

This paper analyses the impact of different volatility structures on a range of traditional option pricing models for the valuation of call down and out style barrier options. The construction of a Risk-Neutral Probability Term Structure (RNPTS) is one of the main contributions of this research, which changes in parallel with regard to the Volatility Term Structure (VTS) in the main and traditional methods of option pricing. As a complementary study, we propose the valuation of options by assuming a constant or historical volatility. The study implements the GARCH (1,1) model with regard to the continuously compound returns of the DAX XETRA Index traded at daily frequency. Current methodology allows for obtaining accuracy forecasts of the realized market barrier option premiums. The paper highlights not only the importance of selecting the right model for option pricing, but also fitting the most accurate volatility structure.

Background/Aims: Pannexin-1 (Panx1) is an ATP release channel that is ubiquitously expressed and coupled to several ligand-gated receptors. In isolated cardiac myocytes, Panx1 forms large conductance channels that can be activated by Ca2+ release from the sarcoplasmic reticulum. Here we characterized the electrophysiological function of these channels in the heart in vivo, taking recourse to mice with Panx1 ablation. Methods: Cardiac phenotyping of Panx1 knock-out mice (Panx1-/-) was performed by employing a molecular, cellular and functional approach, including echocardiography, surface and telemetric ECG recordings with QT analysis, physical stress testing and quantification of heart rate variability. In addition, an in vivo electrophysiological study entailed programmed electrical stimulation using an intracardiac octapolar catheter. Results: Panx1 deficiency results in a higher incidence of AV-block, delayed ventricular depolarisation, significant prolongation of QT- and rate corrected QT-interval and a higher incidence of atrial fibrillation after intraatrial burst stimulation. Conclusion: Panx1 seems to play an important role in murine cardiac electrophysiology and warrants further consideration in the context of hereditary forms of atrial fibrillation.

Growth-regulating factor (GRF) interacting factors (GIFs) are involved in several developmental processes in Arabidopsis. We previously showed that upregulation of OsGIF1 expression improves rice grain size. However, whether OsGIF1 is involved in other developmental processes remains unclear. Here, we report pleiotropic effects of OsGIF1 on rice organ size regulation. Overexpression and functional knock-out via a CRISPR/Cas9 strategy revealed that OsGIF1 not only positively regulates the sizes of rice leaf, stem, and grain but also influences rice reproduction. Expression profiles based on both qRT-PCR and GUS (β-glucuronidase) histochemical staining suggested that OsGIF1 is differentially expressed across various rice tissues, consistent with its roles in regulating the development of multiple rice organs. Additionally, we found that OsGIF1-GFP localized preferentially in the nucleus, which supports its proposed role as a transcriptional cofactor. Further histological analysis suggested that OsGIF1 affected rice organ size possibly by regulating cell size. Our results suggest that OsGIF1 plays important roles in vegetative and reproductive developmental processes, with important implications for rice breeding.

Rate of ovulation (i.e. fecundity) is largely influenced by both genetic and environmental factors. The ovarian growth factorsincluding members of bone morphogenetic proteins (BMPs) play a central role in determining ovulation quota and litter size.Naturally occurring mutation in sheep and knock-out and knock–down studies in murine indicated the importance of bonemorphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9) and bone morphogenetic protein receptor 1B(BMPR1B) genes in mammals. These factors have major regulatory roles during the gonadotrophin-independent and -dependent stages of follicle development. Understanding of BMPs in reproduction assists in the treatment of infertility/sterility in animals.

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

The role of sex and androgen receptors (ARs) for social preference and social memory is rather unknown. In this study of mice we compared males, females and males lacking ARs specifically in the nervous system, ARNesDel, with respect to social preference, assessed with the three-chambered apparatus test, and social recognition, assessed with the social discrimination procedure. In the social discrimination test we also evaluated the tentative importance of the sex of the stimulus animal. Novel object recognition and olfaction were investigated to complement the results from the social tests. Gene expression analysis was performed to reveal molecules involved in the effects of sex and androgens on social behaviors. All three test groups showed social preference in the three-chambered apparatus test. In both social tests an AR-independent sexual dimorphism was seen in the persistence of social investigation of female conspecifics, whereas the social interest towards male stimuli mice was similar in all groups. Male and female controls recognized conspecifics independent of their sex, whereas ARNesDel males recognized female but not male stimuli mice. Moreover, the non-social behaviors were not affected by AR deficiency. The gene expression analyses of hypothalamus and amygdala indicated that Oxtr, Cd38, Esr1, Cyp19a1, Ucn3, Crh and Gtf2i were differentially expressed between the three groups. In conclusion, our results suggest that ARs are required for recognition of male but not female conspecifics, while being dispensable for social investigation towards both sexes. In addition, the AR seems to regulate genes related to oxytocin, estrogen and William’s syndrome.

TAU is a microtubule-associated protein that under pathological conditions such as Alzheimer’s disease forms insoluble, filamentous aggregates. When twenty years after TAU’s discovery the first TAU transgenic mouse models were established, one declared goal that was achieved was the modelling of authentic TAU aggregate formation in the form of neurofibrillary tangles. However, as we review here, it has become increasingly clear that TAU causes damage much before these filamentous aggregates develop. In fact, because TAU is a scaffolding protein, increased levels and an altered subcellular localisation (due to an increased insolubility and impaired clearance) result in the interaction of TAU with cellular proteins with which it would otherwise either not interact or do so to a lesser degree, thereby impairing their physiological functions. We specifically discuss the non-axonal localization of TAU, the role phosphorylation has in TAU toxicity and how TAU impairs mitochondrial functions. A major emphasis is on what we have learned from the four available TAU knock-out models in mice, and the knock-out of the TAU/MAP2 homologue PTL-1 in worms. It has been proposed that in human pathological conditions such as Alzheimer’s disease, a rare toxic TAU species exists which needs to be specifically removed to abrogate TAU’s toxicity and restore neuronal functions. However, what is toxic in one context may not be in another, and simply reducing, but not fully abolishing TAU levels may be sufficient to abrogate TAU toxicity.

Abstract Background Polyunsaturated fatty acids (PUFA) have diverse biological effects, from promoting inflammation to preventing cancer and heart disease. Growing evidence suggests that individual PUFA may have independent effects in health and disease. The individual roles of the two essential PUFA, linoleic acid (LA) and α-linolenic acid (ALA), have been difficult to discern from the actions of their highly unsaturated fatty acid (HUFA) downstream metabolites. This issue has recently been addressed through the development of the Δ-6 desaturase knock out (D6KO) mouse, which lacks the rate limiting Δ-6 desaturase enzyme and therefore cannot metabolize LA or ALA. However, a potential confounder in this model is the production of novel Δ-5 desaturase (D5D) derived fatty acids when D6KO mice are fed diets containing LA and ALA, but void of arachidonic acid. Objective The aim of the present study was to characterize how the D6KO model differentially responds to diets containing the essential n-6 and n-3 PUFA, and whether the direct provision of downstream HUFA can rescue the phenotype and prevent the production of D5D fatty acids. Methodology Liver and serum phospholipid (PL) fatty acid composition was examined in D6KO and wild type mice fed i) 10% safflower oil diet (SF, LA rich) ii) 10% soy diet (SO, LA+ALA) or iii) 3% menhaden oil +7% SF diet (MD, HUFA rich) for 28 days (n = 3-7/group). Results Novel D5D fatty acids were found in liver PL of D6KO fed SF or SO-fed mice, but differed in the type of D5D fatty acid depending on diet. Conversely, MD-fed D6KO mice had a liver PL fatty acid profile similar to wild-type mice. Conclusions Through careful consideration of the dietary fatty acid composition, and especially the HUFA content in order to prevent the synthesis of D5D fatty acids, the D6KO model has the potential to elucidate the independent biological and health effects of the parent n-6 and n-3 fatty acids, LA and ALA.

Abstract Background Urea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice. Methods UT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining. Results The UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis. Conclusions Long-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.