Green Fluorescent Protein Marker Research Articles

Unveiling the defensive role of Snakin-3, a member of the subfamily III of Snakin/GASA peptides in potatoes.

The first in-depth characterization of a subfamily III Snakin/GASA member was performed providing experimental evidence on promoter activity and subcellular localization and unveiling a role of potato Snakin-3 in defense Snakin/GASA proteins share 12 cysteines in conserved positions in the C-terminal region. Most of them were involved in different aspects of plant growth and development, while a small number of these peptides were reported to have antimicrobial activity or participate in abiotic stress tolerance. In potato, 18 Snakin/GASA genes were identified and classified into three groups based on phylogenetic analysis. Snakin-1 and Snakin-2 are members of subfamilies I and II, respectively, and were reported to be implicated not only in defense against pathogens but also in plant development. In this work, we present the first in-depth characterization of Snakin-3, a member of the subfamily III within the Snakin/GASA gene family of potato. Transient co-expression of Snakin-3 fused to the green fluorescent protein and organelle markers revealed that it is located in the endoplasmic reticulum. Furthermore, expression analyses via pSnakin-3::GUS transgenic plants showed GUS staining mainly in roots and vascular tissues of the stem. Moreover, GUS expression levels were increased after inoculation with Pseudomonas syringae pv. tabaci or Pectobacterium carotovorum subsp. carotovorum and also after auxin treatment mainly in roots and stems. To gain further insights into the function of Snakin-3 in planta, potato overexpressing lines were challenged against P. carotovorum subsp. carotovorum showing enhanced tolerance to this bacterial pathogen. In sum, here we report the first functional characterization of a Snakin/GASA gene from subfamily III in Solanaceae. Our findings provide experimental evidence on promoter activity and subcellular localization and reveal a role of potato Snakin-3 in plant defense.

In silico design of sgRNA for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis to decrease the infection process to Colletotrichum acutatum in strawberry

Rapid alkalinization factors (RALFs) are ubiquitous cysteine-rich peptides present in plants. They exert diverse functions as hormonal signals in various processes, including cell growth, root elongation, and fertilization. RALF peptides can also act as negative regulators of the plant immune response, inhibiting the formation of the signal receptor complex for immune activation. In Fragaria × ananassa, silencing of FaRALF33 gene plays a key role in the defense against the fungal pathogen Colletotrichum acutatum. In this study, single-guide RNA (sgRNAs) were designed in silico for clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) 9-mediated FaRALF33 gene mutagenesis in F. × ananassa for the reduction of C. acutatum infection. FaRALF33 was compared with homologous RALF33 sequences from other plant species, showing that the amino acid sequence of FaRALF33 presents typical sequences of known RALF peptides in RRILA proteolytic site, in addition to tight clustering presented by FaRALF33 with FvRALF33. The online tool CHOPCHOP provided 73 hits for FaRALF33 gene, selecting two sgRNA sequences for mutagenesis, sgRNA 1 (5’-CGACTCTCCCATCTCTTGGACT-3’) and sgRNA 2 (5’-GCAAGCAACGGCAGCGATCA-3’). The predicted secondary structures of the selected sgRNAs presented efficient structures in targeted mutagenesis. The pCas9-TPC-GFP-2XsgRNA vector for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis was designed in silico with two sgRNA sequences (with Arabidopsis thaliana U6-26 promoters) and a green fluorescent protein marker.

Monocytic involvement in the neuroinflammatory response after retinal injury

Aims/Purpose: Inflammation and disease lead to the recruitment of blood monocyte‐derived macrophages (MDM) to the central nervous system. The role of MDMs following a neuroinflammatory process after a lesion remains elusive because MDMs that enter the neural parenchyma are difficult to distinguish from microglia. The aim of this study is to elucidate the response of recruited MDMs after selective retinal ganglion cell (RGC) injury.Methods: Unilateral complete intra‐orbital optic nerve (ON) crush was performed in adult C57BL/6J LysM‐GFP mice carrying the selective marker green fluorescent protein (GFP) in myeloid MDM but not microglia, 1 week after retrograde labelling of RGCs with the neuronal tracer hydroxystilbamidine (OHSt) from the superior colliculi. This injury model allows to investigate how the retinal mononuclear phagocytic system responds to a neurodegenerative process via the phagocytic activity of its main cellular components, resident microglia and recruited MDM, both in the injured retina and its contralateral uninjured fellow retina, at 5‐, 7‐ or 10‐days post‐lesion.Results: At all‐time points after injury, microglial phagocytic activity was statistically significant in the injured retina compared to the uninjured fellow retina. In comparison to naïve retinas, the number of MDMs increases significantly after injury in both injured and uninjured retinas. In injured retinas the increase is statistically higher. However, only a subset of the recruited MDMs shows phagocytic activity in the injured retinas 10 days after the lesion but not before. In contrast, the population of MDMs in the contralateral uninjured retina shows no signs of phagocytic activity after the lesion.Conclusions: In the injured retina, recruited MDMs show an initial quiescent stage, with no phagocytic activity compatible with a neuroprotective state, followed by a delayed phase with detrimental behaviour for RGC survival. In the uninjured fellow retinas however, MDMs retain a quiescent stage. It is tempting to suggest that following ON injury, recruited MDMs adopt an initial neuroprotective stage followed by a detrimental phagocytic activity in the injured retina, whereas in the uninjured retina MDMs remain in the initial stage.

Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry

BackgroundThe diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry.ResultsHere, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD600) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries.ConclusionWe established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

Characterization of wheat Wrab18 gene promoter and expression analysis under abiotic stress.

Promoters play key roles in plant gene expression in complex and varied natural environments. The type and amount of cis-acting elements in the promoter sequence tend to indicate the response of genes to induction factors. WRAB18 is a group III member of the late embryogenesis abundant (LEA) protein family that performs multiple functions in plant stress physiology. To elucidate the particularly biological effects of WRAB18 on stress, exploration of its promoter sequence is necessary. In this study, the full-length and promoter sequences of Wrab18 were isolated from the Zhengyin 1 cultivar of Triticum aestivum. The gene sequences and cis-acting elements in the promoter were analyzed using the Plant Promoter Database and bioinformatics methods. The results showed that Wrab18 possessed one intron with 100bp, the promoter sequence contained various stress-related cis-acting elements, and the functionality of the promoter was checked using green fluorescent protein (GFP) marker protein expression by transient assay in Nicotiana benthamiana. Furthermore, based on promoter prediction analysis, quantitative real-time fluorescent PCR results confirmed the response of gene expression levels to stress factors. In summary, the promoter sequence of Wrab18 plays a role in plant stress responses, contains multiple cis-acting elements, and provides insights into the role of WRAB18 in plant resilience to stress. This study has guiding significance for further studies of gene function and mechanism of action, and lays a theoretical foundation for improving wheat quality.

Biased pollen transfer by bumblebees favors the paternity of virus-infected plants in cross-pollination.

We used a green fluorescent protein marker gene for paternity analysis to determine if virus infection affected male reproductive success of tomato in bumblebee-mediated cross-pollination under glasshouse conditions. We found that bumblebees that visited flowers of infected plants showed a strong preference to subsequently visit flowers of non-infected plants. The behavior of the bumblebees to move toward non-infected plants after pollinating virus-infected plants appears to explain the paternity data, which demonstrate a statistically significant ∼10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male reproductive success.

Molecular identification and physiological functional analysis of NtNRT1.1B that mediated nitrate long-distance transport and improved plant growth when overexpressed in tobacco.

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for , little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating transport associated with tobacco growth on nutrition. Heterologous expression of NtNRT1.1B in the uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5mM , suggesting a possible molecular function of NtNRT1.1B in the import of into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including , after 3 days' N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, , and even in plants supplied with ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- root influx assays, which showed 15%-20% higher deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for at a K m of around 30-45µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot-stem vascular tissues, and higher in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.

QSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

A Multi-plex Protein Expression System for Production of Complex Enzyme Formulations in Trichoderma reesei.

Historically, heterologous protein production has been challenging using the hyper-cellulolytic fungus, Trichoderma reesei. T. reesei is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for identification of successful transformants. In this work, we have applied the 2A-peptide multi-gene expression system to co-express four enzymes, which include three cellulases, a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1); as well as the enhanced Green Fluorescent Protein, (eGFP), used here as a marker for monitoring expression levels. We designed a new chassis vector, pTrEno-4X-2A for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each of the cellulases was assessed using pNP glycosides and the chromogenic substrate, AZCL-HE-cellulose, which confirmed the functionality of the enzymes. Expression and activity of these enzymes was proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. Although all three cellulase proteins were successfully expressed, their expression levels varied significantly. Specifically, up to an 18-fold difference was observed between the first and the third gene within the 2A-peptide construct, based on protein quantitation. The availability of this new screening tool is expected to greatly impact multi-enzyme applications, such as production of complex commercial enzyme formulations and the study of metabolic pathway enzymes, especially those destined for cell free applications.

Isolation and identification of antagonistic bacteria of Angelica root rot and their mechanism as biological control

Fusarium avenaceum is the predominant pathogen associated with Angelica sinensis root rot, which results in mycotoxin contamination of Angelica, most prominently Enniatin B (ENN B). This study aimed to isolate bacteria capable of combating various phytopathogenic fungi and degrading ENN B to reduce Angelica root rot. Through co-culture with F. avenaceum, the bacterial strains YF and SY89 were isolated from Angelica rhizosphere soil for their antifungal activities. They were identified as Paenibacillus polymyxa and Bacillus tequilensis based on their morphological characteristics and phylogenetic trees constructed using the 16 S rDNA genes sequence. The strains YF and SY89 could produce antimicrobial substance such as surfactin, fengycin, iturin, polyketide synthases and non ribosomal polypeptide synthase detected by Polymerase Chain Reaction (PCR). In addition, strain P. polymyxa YF and B. tequilensis SY89 showed a prominent ability to inhibit synthesis and degrade ENN B in F. avenaceum suspensions and standard samples. The inhibition rate reached 61.93% and 77.64%, respectively, and the degradation rate reached 60.32% and 76.03%, respectively. Angelica pot experiments were conducted to further evaluate the strains YF and SY89 culture ability to promote plant growth and control Angelica root rot to assess its potential agricultural use. The results indicated that strains YF and SY89 could produce IAA and siderophores, which significantly promoted Angelica roots growth. In addition, strains YF and SY89 have the potential to increase the activity of resistant enzymes, thereby inhibiting F. avenaceum infection. The disease index of Angelica roots treated by strain YF and SY89 decreased by 65.38% and 61.54%, respectively. To further elucidate the antagonistic mechanism of YF and SY89, we examined their colonization pattern in the Angelica root using a green fluorescent protein (GFP) marker. The results indicated that YF and SY89 mainly colonized the root surface before migrating into the roots interior part. The present study demonstrated that P. polymyxa YF and B. tequilensis SY89 showed promising prospects for use as a biological control agent against Angelica root rot and ENN B inhibition under field conditions.

Profiling of Single-cell-type-specific MicroRNAs in Arabidopsis Roots by Immunoprecipitation of Root Cell-layer-specific GFP-AGO1.

MicroRNAs (miRNA) are small (21-24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR ) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves. This protocol was validated in: Dev Cell (2022), DOI: 10.1016/j.devcel.2022.03.015 Graphical abstract.

Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense ) and Camelina (Camelina sativa ).

Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6×106 cells from pennycress and 3×106 cells from camelina per gram of fresh leaf tissues. Using a 7.3kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.

Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components

Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.

The effect of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase gene overexpression in the kynurenine pathway on the expression levels of indoleamine 2,3-dioxygenase 1 and interferon-γ in inflammatory conditions: an in vitro study.

The kynurenine pathway (KP) can be involved in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. This study aimed to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line to present more information about the effect of this gene on inflammatory conditions and the KP cycle. The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. To simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24h before transfection, and transfected by Polyethyleneimine(PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and murine Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed 72h after transfection, and Acmsd expression level increased 29-fold (P < 0.001) in transfected LPS-stimulated cells compared to the control group (LPS-stimulated cells that were not transfected). Additionally, increased Acmsd expression level significantly down-regulated Ifn-γ (P < 0.001) and Ido1 (P < 0.01) expression level in transfected LPS-stimulated cells compared to LPS-stimulated cells. Acmsd gene overexpression in inflammatory conditions can reduce the expression levels of the Ido1 gene, and its regulator, Ifn-γ. Consequently, it may be considered as a novel regulatory factor in the KP balance.

Revisiting the regeneration of Stenostomum leucops (Catenulida, Platyhelminthes)

ABSTRACT Stenostomum are small flatworms that live in freshwater and normally reproduce by paratomy. They are basal in Platyhelminthes phylogeny. For more than a century, species of this genus, especially S. leucops, have been used in regeneration studies. However, some contradictory results remain, as there are concerns about which body parts regenerate, and their routes of regeneration. Problems in species identification are a possible explanation for these contradictory results. Regeneration studies were repeated, transforming the worms with vectors carrying an eye-specific GFP (green fluorescence protein) marker in a characterized strain. All regeneration patterns described previously were observed in this strain, showing that these complex regeneration routes are probably associated with the different parts of the body that were cut. Stenostomum species are promising experimental organisms for using a transcriptome approach in regeneration studies.

A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example.

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

Nano-TiO2 aggravates bioaccumulation and developmental neurotoxicity of triphenyl phosphate in zebrafish larvae

This study explored the combined effects of titanium dioxide nanoparticles (nano-TiO2) and triphenyl phosphate (TPhP) on the neurodevelopment of zebrafish larvae as well as the underlying mechanisms. With this regard, zebrafish embryos were exposed to nano-TiO2 of 100 μg·L−1, TPhP of 0, 8, 24, 72, and 144 μg·L−1, or their combinations until 120 h post-fertilization (hpf). Results indicated 100 μg·L−1 nano-TiO2 alone to be nontoxic to zebrafish larvae. However, obvious developmental toxicity manifested as inhibition of surviving rate, heart rate and body length as well as increased malformation was observed in the higher concentrations of TPhP (72 and 144 μg·L−1) alone and the co-exposure groups. Additionally, results suggested that nano-TiO2 significantly enhanced the bioaccumulation of TPhP in zebtafish larvae, and thus aggravated the abnormities of spontaneous movement and swimming behavior in zebrafish larvae induced by TPhP. Nano-TiO2 also exacerbated the TPhP-induced inhibition of the axonal growth on the secondary motor neuron, and aggravated the TPhP-induced decrease on expressions of neuron-specific green fluorescent protein (GFP) and neuronal marker genes (ngn1 and elavl3). Further, the content of neurotransmitter serotonin was not altered by TPhP alone exposure, but was decreased significantly in the co-exposure group of 144 μg·L−1 TPhP and nano-TiO2. Our data indicated that nano-TiO2 might aggravate the neuron abnormities and serotonin system dysfunction by enhancing the TPhP accumulation, leading to exacerbated abnormal locomotors in zebrafish larvae.

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System.

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-β Precursor Protein.

Alzheimer’s disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-β precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.

Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF.The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%.We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.