Low Cd-accumulating rice grain production through inoculation of germinating rice seeds with a plant growth-promoting endophytic bacterium.

This study investigated the effects of the plant growth-promoting endophytic bacterium Cupriavidus taiwanensis KKU2500-3 on the growth of KDML105 rice plants and cadmium (Cd) accumulation in grains. The rice plants were cultivated in soils with 20 and 50 ppm Cd under greenhouse conditions for two consecutive years. At both levels, Cd reduced rice growth and development. Under Cd stress, KKU2500-3 colonized the root surface and interior of rice plants at the early growth stage, and this colonization remained until the late stage. The colonized bacteria increased the pigment contents but reduced the root-to-aboveground translocation of Cd. In soil with 20 ppm Cd, the phytochelatin content of the bacteria-inoculated rice was lower (32.3-89.3%) than that of uninoculated rice. In soil with 50 ppm Cd, the bacteria-inoculated rice exhibited higher glutathione reductase (5-63%) and proline (5-115%) levels, a higher reduced glutathione (GSH)/0.5 oxidized glutathione (GSSG) ratio (4-212%) and decreased lipid peroxidation (1-19%) compared with uninoculated rice. The root-to-grain translocation factor of inoculated rice in soil with 50 ppm Cd was significantly lower than that of inoculated rice in soil with 20 ppm Cd, and this finding was consistent with the 38.6% and 75.1% reductions in Cd accumulation observed in grains from soils with 20 and 50 ppm Cd, respectively. The Cd content of KDML105 grains grown in soil with 50 ppm Cd was 0.36 ppm, which is below the Codex standard for polished rice (0.4 ppm). The levels of available P, Zn, and SO42- also affect Cd availability in soil, and colonized KKU2500-3 showed varying responses to different Cd levels. Thus, bacterial inoculation, the Cd level and soil properties play important roles in Cd accumulation in KDML105 rice grains. The role of C. taiwanensis KKU2500-3 on the production of low-Cd-accumulating rice in paddy fields contaminated with a range of Cd levels should be further investigated.

Effects of Roasting Conditions on Antibacterial Properties of Vietnamese Turmeric (Curcuma longa) Rhizomes.

Processing with heat treatment has been reported to alter several therapeutic effects of turmeric. In Vietnamese traditional medicine, turmeric has been long used for bacterial infections, and roasting techniques are sometimes applied with this material. However, there have been no studies investigating the effects of these thermal processes on the plant's antibacterial properties. Our study was therefore performed to examine the changes that roasting produced on this material. Slices of dried turmeric were further subjected to light-roasting (80 °C in 20 min) or dark-roasting (160 °C in 20 min) processes. Broth dilution and agar-well diffusion methods were applied to examine and compare the effects of ethanol extracts obtained from non-roasted, light-roasted and dark-roasted samples, on a set of 6 gram-positive and gram-negative bacteria. In both investigations, dark-roasted turmeric was significantly less antibacterial than non-roasted and light-roasted materials, as evident by the higher values of minimum inhibitory concentrations and the smaller diameters of induced inhibitory zones. In addition, dark-roasting was also found to clearly reduce curcumin contents, total polyphenol values and antioxidant activities of the extracts. These results suggest that non-roasting or light-roasting might be more suitable for the processing of turmeric materials that are aimed to be applied for bacterial infections.

Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia.

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.

Response in digestibility, growth performance, and carcass quality of local Kandol pigs to incremental levels of fermented banana stems.

Fermentation of banana stems is a novel method for increasing the digestibility of fibrous porcine rations in the tropics; however, optimal feeding rates have not been determined. An experiment was carried out to determine the response of digestibility, growth performance, and carcass quality in local Kandol pigs to incremental levels of FBS in rations. A total of 20 castrated male pigs were randomly allocated to five diets, containing 0, 400, 500, 600, and 700 g FBS of the ration. Daily feed on offer was provided as 4% of the body weight on a dry matter basis and offered three times per day. As result, the inclusion level of FBS was positively correlated with NR (p < 0.001, r = 0.68). The linear regression equation was y = 0.002x + 2.4276 (R2 = 0.63). Furthermore, it was also correlated with ADG (p < 0.05, r = 0.46) and with FCR (p < 0.05, r = - 0.45). The regression equation was y = 0.0634x + 174.2 (R2 = 0.43) and y = - 0.0009x + 4.6521 (R2 = 0.62) for ADG and FCR, respectively. The correlation between inclusion level of FBS and large intestine weight was also observed (p < 0.05, r = - 0.61). The R2 value was 63. However, there was no any correlation between the inclusion level of FBS and the percentages of hanging carcass, dressing carcass, pH, color score, marbling score of the meat, and WHC (p > 0.05). The economic benefit of using FBS to improve pig diets will depend on the availability of banana stems and the labor and processing costs.

Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.

Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation.

Validation of verbal autopsy methods using hospital medical records: a case study in Vietnam.

BACKGROUND: Information on causes of death (COD) is crucial for measuring the health outcomes of populations and progress towards the Sustainable Development Goals. In many countries such as Vietnam where the civil registration and vital statistics (CRVS) system is dysfunctional, information on vital events will continue to rely on verbal autopsy (VA) methods. This study assesses the validity of VA methods used in Vietnam, and provides recommendations on methods for implementing VA validation studies in Vietnam. METHODS: This validation study was conducted on a sample of 670 deaths from a recent VA study in Quang Ninh province. The study covered 116 cases from this sample, which met three inclusion criteria: a) the death occurred within 30 days of discharge after last hospitalisation, and b) medical records (MRs) for the deceased were available from respective hospitals, and c) the medical record mentioned that the patient was terminally ill at discharge. For each death, the underlying cause of death (UCOD) identified from MRs was compared to the UCOD from VA. The validity of VA diagnoses for major causes of death was measured using sensitivity, specificity and positive predictive value (PPV). RESULTS: The sensitivity of VA was at least 75% in identifying some leading CODs such as stroke, road traffic accidents and several site-specific cancers. However, sensitivity was less than 50% for other important causes including ischemic heart disease, chronic obstructive pulmonary diseases, and diabetes. Overall, there was 57% agreement between UCOD from VA and MR, which increased to 76% when multiple causes from VA were compared to UCOD from MR. CONCLUSIONS: Our findings suggest that VA is a valid method to ascertain UCOD in contexts such as Vietnam. Furthermore, within cultural contexts in which patients prefer to die at home instead of a healthcare facility, using the available MRs as the gold standard may be meaningful to the extent that recall bias from the interval between last hospital discharge and death can be minimized. Therefore, future studies should evaluate validity of MRs as a gold standard for VA studies in contexts similar to the Vietnamese context.

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation.

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.

Design and use of transgenic reporter strains for detecting activity of signaling pathways in Xenopus.

Embryos and larvae of vertebrate species with external development are ideal subjects for investigating the dynamic spatiotemporal activity of developmental signaling pathways. The availability of efficient transgene technologies in Xenopus and zebrafish and the translucency and/or transparency of their embryos and larvae make these two species attractive for direct in vivo imaging of reporter gene expression. In this article we describe the design of efficient signaling reporters, using the Wnt/ß-catenin pathway as a representative example. We define methods for validating the reporter constructs and describe how they can be used to generate stable transgenic lines in Xenopus. We provide efficient methods used in our laboratory for raising the tadpoles and froglets rapidly to sexual maturity. We further discuss how the reporter lines can be used for delineating the dynamic activity of a signaling pathway and how modulators of the pathway can be scrutinized via chemical intervention and the micro-injection of synthetic RNAs or morpholinos. The strategic outline discussed in this paper provides a template for studying other developmental signaling pathways in Xenopus.

Hes4 controls proliferative properties of neural stem cells during retinal ontogenesis.

The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis.

Wnt/beta-catenin signaling is involved in the induction and maintenance of primitive hematopoiesis in the vertebrate embryo.

The formation of primitive (embryonic) blood in vertebrates is mediated by spatio-temporally restricted signaling between different tissue layers. In Xenopus, in which primitive blood originates in the ventral blood island, this involves the secretion of bone morphogenetic protein (BMP) ligands by the ectoderm that signal to the underlying mesoderm during gastrulation. Using novel transgenic reporter lines, we report that the canonical Wnt/ß-catenin pathway is also activated in the blood islands in Xenopus. Furthermore, Wnt-reporter activity was also detected in the blood islands of the mouse yolk sac. By using morpholino-mediated depletion in Xenopus, we identified Wnt4 as the ligand that is expressed in the mesoderm of the ventral blood island and is essential for the expression of hematopoietic and erythroid marker genes. Injection of an inducible Wnt-interfering construct further showed that, during gastrulation, Wnt/ß-catenin signaling is required both in the mesoderm and in the overlying ectoderm for the formation of the ventral blood island. Using recombination assays with embryonic explants, we document that ectodermal BMP4 expression is dependent on Wnt4 signals from the mesoderm. Our results thus reveal a unique role for Wnt4-mediated canonical signaling in the formation and maintenance of the ventral blood island in Xenopus.

HCV RNA Quantification by a Domestic Commercial Assay: A Case Study among People Who Inject Drugs in Vietnam.

The desired performance of nucleic acid testing (NAT) may vary if used for disease diagnosis or for the evaluation of the therapeutic efficacy of a treatment, although in most cases, the same assay is used. However, these tests may not be affordable in many situations including in low/middle income countries that in response have developed domestic assays. Given the example of HCV NAT among people who inject drugs in Vietnam, we aimed at evaluating a domestic assay versus an FDA- and CE-approved assay. This cross-evaluation revealed that (i) the domestic assay had a poorer sensitivity with a threshold of detection above 104 IU/mL, and (ii) the FDA-approved assay had a percentage of false negative results close to 1%. Together, in the present study, the domestic assay had a performance compatible with diagnosis purposes (given that this population was 70% HCV seropositive) but not compatible with HCV treatment monitoring (given that treatment failures are rare and the observed viremia frequently below the threshold of detection). This study highlights the need for a proper evaluation of HCV RNA domestic assays in order to efficiently contribute to the WHO HCV elimination target by 2030.

Transcription factor Zic2 inhibits Wnt/ß-catenin protein signaling.

The Zic transcription factors play critical roles during embryonic development. Mutations in the ZIC2 gene are associated with human holoprosencephaly, but the etiology is still unclear. Here, we report a novel function for ZIC2 as a regulator of ß-catenin·TCF4-mediated transcription. We show that ZIC2 can bind directly to the DNA-binding high mobility group box of TCF4 via its zinc finger domain and inhibit the transcriptional activity of the ß-catenin·TCF4 complex. However, the binding of TCF4 to DNA was not affected by ZIC2. Zic2 RNA injection completely inhibited ß-catenin-induced axis duplication in Xenopus embryos and strongly blocked the ability of ß-catenin to induce expression of known Wnt targets in animal caps. Moreover, Zic2 knockdown in transgenic Xenopus Wnt reporter embryos led to ectopic Wnt signaling activity mainly at the midbrain-hindbrain boundary. Together, our results demonstrate a previously unknown role for ZIC2 as a transcriptional regulator of the ß-catenin·TCF4 complex.

ARVCF depletion cooperates with Tbx1 deficiency in the development of 22q11.2DS-like phenotypes in Xenopus.

The 22q11.2 deletion syndrome is a common dominant genetic disorder characterized by a heterozygous deletion of a cluster of genes on chromosome 22q11.2. TBX1, a transcription factor belonging to the T-box gene family, is a key player in the syndrome. However, heterozygosity of Tbx1 in mouse models does not fully recapitulate the phenotypes characteristic of the disease, which may point to the involvement of other genes in the deleted chromosomal region. Hence, we investigated the contribution of the catenin ARVCF, another gene that is deleted in 22q11.2DS. During Xenopus development, ARVCF mRNA is expressed in the pharyngeal arches and depleting either ARVCF or Tbx1 results in delayed migration of the cranial neural crest cells and in defects in the craniofacial skeleton and aortic arches. Moreover, double depletion of ARVCF and Tbx1 revealed that they act cooperatively, indicating that decreased ARVCF levels may also contribute to 22q11.2DS-associated phenotypes.

A cadmium-tolerant endophytic bacterium reduces oxidative stress and Cd uptake in KDML105 rice seedlings by inducing glutathione reductase-related activity and increasing the proline content.

The effect of the endophytic Cupriavidus taiwanensis KKU2500-3 on the Cd toxicity of KDML105 rice seedlings was investigated in a 10 µM CdCl2 hydroponic system. As demonstrated after bacterial inoculation of germinating rice seeds, KKU2500-3 colonized all rice plant parts. In RB (Rice + KKU2500-3) and RBC (Rice + KKU2500-3+Cd), KKU2500-3 effectively colonized and was detected at a markedly higher number in the root surface and interior than in shoots and leaves. The activities of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) and the proline content in inoculated rice were higher in roots and aboveground tissues. RBC exhibited a higher reduced-to-oxidized glutathione ratio in roots and leaves (3-55%) but a lower malondialdehyde content (8-78%). Phytochelatins (PCs) were detected in all rice tissues, but their levels in RBC were 13-70% lower than those in RC (Rice + Cd), demonstrating that the induction of PCs in rice was unrelated to KKU2500-3. The Cd levels in roots and shoots were lower in RBC than RC, and the root-to-shoot Cd translocation factor was 0.6-62.2% lower. At 30 DAT, the Cd levels in RBC roots and shoots were 30.2% and 73.7% lower, respectively, than those in RC. Colonized KKU2500-3 activated GR and increased the proline content to overcome rice Cd toxicity. These effects may trap Cd in plant cells and reduce its translocation. Hence, KKU2500-3 synergistically interacts with rice to detoxify Cd at early growth stages, and KDML105 rice grains with low Cd accumulation could be produced if this interaction is maintained until late growth stages.

Long-Term Persistence of Mitochondrial DNA Instability among HCV-Cured People Who Inject Drugs.

People who inject drugs (PWID) are a population exposed to many genotoxicants and with a high prevalence of HCV infection. Direct-acting antiviral (DAA) regimens are now widely used to treat chronic HCV infection. Although side effects to treatment are currently rare, the long-term effects such as suspicions of de novo hepatocellular carcinoma (HCC) occurrence or HCC recurrence and cardiac defects are still up for debate. Given the structure of DAAs, the molecules have a potential mitochondrial DNA (mtDNA) genotoxicity. We have previously reported acute mtDNA toxicity of three DAA regimens among PWID with a strong impact on the rate of mtDNA deletion, less on the quantity of mtDNA copy per cell at sustained viral response at 12 weeks (SVR12). Herein, we report the mtDNA parameters nine months after drug discontinuation. We observed that the percentage of the deleted mtDNA genome increased over time. No exposure to any other genotoxicants during this period was associated with a high deletion percentage, suggesting that the replicative advantage of the deleted molecules outweighed their elimination processes. Such observation calls for longer-term follow-up and may contribute to the molecular basis of subclinical side effects of DAA treatments.

Mitochondrial Genotoxicity of Hepatitis C Treatment among People Who Inject Drugs.

Antiviral nucleoside analogues (ANA) are newly used therapeutics acting against the hepatitis C virus (HCV). This class of drug is well known to exhibit toxicity on mitochondrial DNA (mtDNA). People who inject drugs (PWID) are particularly affected by HCV infection and cumulated mitotoxic drug exposure from HIV treatments (antiretrovirals, ARV) and other illicit drugs. This study aims to explore the impact of direct-acting antiviral (DAA) treatments on mtDNA among PWID. A total of 470 actively injecting heroin users were included. We used quantitative PCR on whole blood to determine the mitochondrial copy number per cell (MCN) and the proportion of mitochondrial DNA deletion (MDD). These parameters were assessed before and after DAA treatment. MDD was significantly increased after HCV treatment, while MCN did not differ. MDD was even greater when subjects were cotreated with ARV. In multivariate analysis, we identified that poly-exposure to DAA and daily heroin injection or regular consumption of methamphetamines were positively associated with high MCN loss while DAA and ARV treatments or methadone use were identified as risk factors for having mtDNA deletion. These observations deserve attention since they were previously associated with premature cell ageing or cell transformation and therefore call for a long-term follow-up.

Chicken beta-globin insulator overcomes variegation of transgenes in Xenopus embryos.

Chromatin structure and gene transcription regulation are intimately linked, and mosaic expression of randomly integrated transgenes into the genome is frequently observed. This variegation of transgene expression is likely due to the genomic integration site, which can affect the behavior of the integrated DNA sequence in a positive or a negative way. Insulators are a class of DNA elements that can protect genes from inappropriate signals emanating from their environment by acting as boundaries that prevent the spreading of nearby condensed chromatin that may otherwise silence expression. Here we show that transgenes escape this silencing in Xenopus laevis and Xenopus tropicalis embryos and that a stable, uniform, and heritable expression pattern is obtained when transgenes are flanked with tandem copies of the chicken beta-globin 5'HS4 insulator. Our data also indicate that the insulator confers copy-number-dependent transgene expression and can increase transgene expression from weak regulatory elements. Hence, it will be an invaluable tool for generating stable lines expressing different levels of a particular coding sequence.

ΔN-Tp63 Mediates Wnt/ß-Catenin-Induced Inhibition of Differentiation in Basal Stem Cells of Mucociliary Epithelia.

Mucociliary epithelia provide a first line of defense against pathogens. Impaired regeneration and remodeling of mucociliary epithelia are associated with dysregulated Wnt/ß-catenin signaling in chronic airway diseases, but underlying mechanisms remain elusive, and studies yield seemingly contradicting results. Employing the Xenopus mucociliary epidermis, the mouse airway, and human airway Basal cells, we characterize the evolutionarily conserved roles of Wnt/ß-catenin signaling in vertebrates. In multiciliated cells, Wnt is required for cilia formation during differentiation. In Basal cells, Wnt prevents specification of epithelial cell types by activating ΔN-TP63, a master transcription factor, which is necessary and sufficient to mediate the Wnt-induced inhibition of specification and is required to retain Basal cells during development. Chronic Wnt activation leads to remodeling and Basal cell hyperplasia, which are reversible in vivo and in vitro, suggesting Wnt inhibition as a treatment option in chronic lung diseases. Our work provides important insights into mucociliary signaling, development, and disease.

Transgenic reporter tools tracing endogenous canonical Wnt signaling in Xenopus.

Activation of the canonical Wnt pathway leads to the transcriptional activation of a particular subset of downstream Wnt target genes. To track this localized cellular output in a living organism, reporter constructs can be designed containing multimerized consensus lymphoid enhancer binding factor (LEF)-1/T cell factor (TCF) transcription factor binding sites, generally referred to as TCF optimal promoter (TOP) sites. In Xenopus, several Wnt-responsive reporter systems have been designed containing a number of these TOP sites that, in combination with a minimal promoter, drive the expression of a reporter gene. Following transgenic integration in Xenopus embryos, a Wnt reporter tool reveals the spatiotemporal delineation of endogenous Wnt pathway activities throughout development. Assumed to be a general readout of the Wnt pathway, such reporters can assist in elucidating unknown functional implications in developing Xenopus embryos.

TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis.

Truncating mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are the initiating step in the vast majority of sporadic colorectal cancers, and they underlie familial adenomatous polyposis (FAP) syndromes. Modeling of APC- driven tumor formation in the mouse has contributed substantially to our mechanistic understanding of the associated disease, but additional models are needed to explore therapeutic opportunities and overcome current limitations of mouse models. We report on a novel and penetrant genetic cancer model in Xenopus tropicalis, an aquatic tetrapod vertebrate with external development, diploid genome and short life cycle. Tadpoles and froglets derived from embryos injected with TAL effector nucleases targeting the apc gene rapidly developed intestinal hyperplasia and other neoplasms observed in FAP patients, including desmoid tumors and medulloblastomas. Bi-allelic apc mutations causing frame shifts were detected in the tumors, which displayed activation of the Wnt/ß-catenin pathway and showed increased cellular proliferation. We further demonstrate that simultaneous double bi-allelic mutation of apc and a non-relevant gene is possible in the neoplasias, opening the door for identification and characterization of effector or modifier genes in tumors expressing truncated apc. Our results demonstrate the power of modeling human cancer in Xenopus tropicalis using mosaic TALEN-mediated bi-allelic gene disruption.