KED gene expression in early response to wounding stress in tomato plants.

The wounding-responsive KED gene, named for its coding for a lysine (K), glutamic acid (E), and aspartic acid (D)-rich protein, is widely present among land plants. However, little is known about its regulation or function. In this study, we found that transcription of the tomato (Solanum lycopersicum) KED gene, SlKED, was rapidly and transiently elevated by wounding or ethephon treatment. Compared to the wild-type plants, the CRISPR/Cas9-mediated SlKED knockout plants did not exhibit altered expression patterns for genes involved in hormone biosynthesis or stress signaling, suggesting a lack of pleiotropic effect on other stress-responsive genes. Conversely, jasmonic acid did not appear to directly regulate SlKED expression. Wounded leaves of the KED-lacking plants exhibited higher binding of Evans blue dye than the wild-type, indicating a possible role for KED in healing damaged tissues. The SlKED knockout plants showed a similar dietary effect as the wild-type on the larval growth of tobacco hornworm. But a higher frequency of larval mandible (mouth) movement was recorded during the first 2 minutes of feeding on the wounded KED-lacking SlKED knockout plants than on the wounded KED-producing wild-type plants, probably reflecting an initial differential response by the feeding larvae to the SlKED knockout plants. Our findings suggest that SlKED may be an ethylene-mediated early responder to mechanical stress in tomato, acting downstream of the wound stress response pathways. Although its possible involvement in response to other biotic and abiotic stresses is still unclear, we propose that SlKED may play a role in plant's rapid, short-term, early wounding responses, such as in cellular damage healing.

Evolutionary analysis of KED-rich proteins in plants.

During the course of evolution, organisms have developed genetic mechanisms in response to various environmental stresses including wounding from mechanical damage or herbivory-caused injury. A previous study of wounding response in the plant tobacco identified a unique wound-induced gene, aptly named KED due to its coding for a protein that has an unusually high content of amino acids lysine (K), glutamic acid (E) and aspartic acid (D). However, by far little is known about this intriguing gene. In this study, we investigated the evolutionary aspects of the KED-rich coding genes. We found that a consistent pattern of wound-induced KED gene expression is maintained across representative species of angiosperm and gymnosperm. KED genes can be identified in species from all groups of land plants (Embryophyta). All the KED proteins from vascular plants (Tracheophyta) including angiosperm, gymnosperm, fern and lycophyte share a conserved 19-amino acid domain near the C-terminus, whereas bryophytes (moss, liverwort and hornwort) possess KED-rich, multi-direct-repeat sequences that are distinct from the vascular plant KEDs. We detected KED-rich sequences in Charophyta species but not in Chlorophyta wherever genome sequences are available. Our studies suggest diverse and complex evolution pathways for land plant KED genes. Vascular plant KEDs exhibit high evolutionary conservation, implicating their shared function in response to wounding stress. The extraordinary enrichment of amino acids K, E and D in these groups of distinct and widely distributed proteins may reflect the structural and functional requirement for these three residues during some 600 million years of land plant evolution.

Elevated somatic mutation and evidence of genomic instability in veterans with Gulf War illness.

AIMS: Gulf War illness (GWI) is thought to be associated with exposures experienced by soldiers deployed in the 1991 Gulf War. A major question is how these exposures continue to influence the health of these individuals three decades later. One potentially permanent effect of such exposures is the induction of genetic mutations. We investigated whether veterans with GWI exhibited persistently elevated levels of somatic mutation. MATERIALS AND METHODS: We applied the blood-based glycophorin A (GPA) somatic mutation assay to a cohort of veterans diagnosed with GWI and a set of both concurrent and historic age-matched controls. This assay quantifies red blood cells with a phenotype consistent with loss of one allele at the genetic determinant for the MN blood group, the GPA gene. KEY FINDINGS: As a population, those affected with GWI exhibited an uninduced mutation frequency at the GPA locus that was effectively twice that observed in controls, a result that was statistically significant. This result was influenced by an increase in the incidence of individuals with aberrantly high mutation frequencies, seemingly higher than would be expected by dose extrapolation and consistent with the induction of localized genomic instability in the hematopoietic bone marrow stem cells. When these "outliers" were removed from consideration, the remaining affected population retained a significantly higher mean allele loss mutation frequency, suggesting that both dose-dependent bone marrow genotoxicity and induction of genomic instability are contributing to the elevation in mutation frequency in these affected veterans. SIGNIFICANCE: This study provides evidence that manifestation of GWI is associated with increased cumulative exposure to agents capable of inducing persistent mutations in bone marrow stem cells. Whether these mutations are involved in the clinical aspects of the condition or are simply biomarkers of overall exposure has yet to be determined. The increased incidence of genomic instability suggests that this persistent mutation can have important delayed effects on cellular integrity.

Expression of a Tardigrade Dsup Gene Enhances Genome Protection in Plants.

DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.

Tissue-Preferential Activity and Induction of the Pepper Capsaicin Synthase PUN1 Promoter by Wounding, Heat and Metabolic Pathway Precursor in Tobacco and Tomato Plants.

A promoter is an essential structural component of a gene that controls its transcription activity in different development stages and in response to various environmental stimuli. Knowledge of promoter functionality in heterologous systems is important in the study of gene regulation and biotechnological application. In order to explore the activity of the pepper capsaicin synthase gene (PUN1) promoter, gene constructs of pPUN1::GUS (for ß-glucuronidase) and pPUN1::NtKED (for a tobacco wound-responsive protein) were introduced into tobacco and tomato, respectively, and their activities were examined. Higher levels of GUS staining intensity and transcription were detected in ovary, anther and pollen than other tissues or organs in tobacco plants. Likewise, transgenic tomato fruits had a higher level of pPUN1::NtKED gene expression than the leaf and flower. The PUN1-driven gene expression can be transiently induced by wounding, heat (40 °C) and the capsaicinoid biosynthetic pathway precursor phenylalanine. When compared to the reported pPUN1::GUS-expressing Arabidopsis, the PUN1 promoter exhibited a more similar pattern of activities among pepper, tobacco and tomato, all Solanaceae plants. Our results suggest the potential utility of this tissue-preferential and inducible promoter in other non-pungent Solanaceae plants for research of gene function and regulation as well as in the biotechnological applications.

Tobacco plants expressing the maize nitrate transporter ZmNrt2.1 exhibit altered responses of growth and gene expression to nitrate and calcium.

BACKGROUND: Nitrate uptake is a highly regulated process. Understanding the intricate interactions between nitrate availability and genetically-controlled nitrate acquisition and metabolism is essential for improving nitrogen use efficiency and increasing nitrate uptake capacity for plants grown in both nitrate-poor and nitrate-enriched environments. In this report, we introduced into tobacco (Nicotiana tabacum) the constitutively expressed maize high-affinity transporter ZmNrt2.1 gene that would bypass the tight control for the endogenous nitrate-responsive genes. By using calcium inhibitors and varying levels of NO3-, Ca2+ and K+, we probed how the host plants were affected in their nitrate response. RESULTS: We found that the ZmNrt2.1-expressing plants had better root growth than the wild type plants when Ca2+ was deficient regardless of the nitrate levels. The growth restriction associated with Ca2+-deficiency can be alleviated with a high level of K+. Furthermore, the transgenic plants exhibited altered expression patterns of several endogenous, nitrate-responsive genes, including the high- and low-affinity nitrate transporters, the Bric-a-Brac/Tramtrack/Broad protein BT2 and the transcription factor TGA-binding protein TGA1, in responding to treatments of NO3-, K+ or inhibitors for the calcium channel and the cytosolic Ca2+-regulating phospholipase C, as compared to the wild type plants under the same treatments. Their expression was not only responsive to nitrate, but also affected by Ca2+. There were also different patterns of gene expression between roots and shoots. CONCLUSION: Our results demonstrate the ectopic effect of the maize nitrate transporter on the host plant's overall gene expression of nitrate sensing system, and further highlight the involvement of calcium in nitrate sensing in tobacco plants.

Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development.

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENT Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in ∼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

Enantioselective cascade biocatalysis for deracemization of 2-hydroxy acids using a three-enzyme system.

BACKGROUND: Enantiopure 2-hydroxy acids are key intermediates for the synthesis of pharmaceuticals and fine chemicals. We present an enantioselective cascade biocatalysis using recombinant microbial cells for deracemization of racemic 2-hydroxy acids that allows for efficient production of enantiopure 2-hydroxy acids. RESULTS: The method was realized by a single recombinant Escherichia coli strain coexpressing three enzymes: (S)-2-hydroxy acid dehydrogenase, (R)-2-keto acid reductase and glucose dehydrogenase. One enantiomer [(S)-2-hydroxy acid] is firstly oxidized to the keto acid with (S)-2-hydroxy acid dehydrogenase, while the other enantiomer [(R)-2-hydroxy acid] remains unchanged. Then, the keto acid obtained reduced to the opposite enantiomer with (R)-2-keto acid reductase plus cofactor regeneration enzyme glucose dehydrogenase subsequently. The recombinant E. coli strain coexpressing the three enzymes was proven to be a promising biocatalyst for the cascade bioconversion of a structurally diverse range of racemic 2-hydroxy acids, giving the corresponding (R)-2-hydroxy acids in up to 98.5 % conversion and >99 % enantiomeric excess. CONCLUSIONS: In summary, a cascade biocatalysis was successfully developed to prepare valuable (R)-2-hydroxy acids with an efficient three-enzyme system. The developed elegant cascade biocatalysis possesses high atom efficiency and represents a promising strategy for production of highly valued (R)-2-hydroxy acids.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

Comparative studies of two methods for miRNA isolation from milk whey.

MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS(®) followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS(®) followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100).

[Progress on the miRNA related with mammary gland development and lactation].

MicroRNAs (miRNAs) are non-coding RNAs that play important roles in post transcriptional regulation. They are involved in the regulation of mammary gland development and lactation. In this paper, we summarized the expression pattern of miRNAs which varied with tissues and lactation stages, and the functions of several miRNAs are also briefly reviewed. The objective of this work is to give reference for further study on miRNAs in mammary gland, and to provide theoretical basis and ideas for the use of miRNAs in improving healthy development of mammary gland and regulating the efficiency of lactation and the quality of milk.

Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth.

Understanding neurite growth regulation remains a seminal problem in neurobiology. During development and regeneration, neurite growth is modulated by neurotrophin-activated signaling endosomes that transmit regulatory signals between soma and growth cones. After injury, delivering neurotrophic therapeutics to injured neurons is limited by our understanding of how signaling endosome localization in the growth cone affects neurite growth. Nanobiotechnology is providing new tools to answer previously inaccessible questions. Here, we show superparamagnetic nanoparticles (MNPs) functionalized with TrkB agonist antibodies are endocytosed into signaling endosomes by primary neurons that activate TrkB-dependent signaling, gene expression and promote neurite growth. These MNP signaling endosomes are trafficked into nascent and existing neurites and transported between somas and growth cones in vitro and in vivo. Manipulating MNP-signaling endosomes by a focal magnetic field alters growth cone motility and halts neurite growth in both peripheral and central nervous system neurons, demonstrating signaling endosome localization in the growth cone regulates motility and neurite growth. These data suggest functionalized MNPs may be used as a platform to study subcellular organelle localization and to deliver nanotherapeutics to treat injury or disease in the central nervous system.

Characterization of wild-type and mutant vaccinia virus M2L proteins' abilities to localize to the endoplasmic reticulum and to inhibit NF-kappaB activation during infection.

Proinflammatory molecules are important for attracting immune effector cells to localized areas of viral infection. One such cellular mechanism facilitating this response is the NF-kappaB transcription factor. While wild-type vaccinia virus expresses multiple products to inhibit NF-kappaB during infection, the attenuated deletion mutant MVA lacks this ability. However, introduction of the wild-type M2L ORF into the MVA genome will re-establish the parental phenotype. As the M2L protein is unique to poxviruses, we characterized it to elucidate its mechanism to quell an inflammatory response. It was discovered that the M2L protein possesses motifs characteristic of ER-localized proteins: an N-terminal signal peptide sequence, C-terminal endoplasmic reticulum (ER) retention and retrieval sequences, and N-linked glycosylation sites. Indeed, the M2L protein was demonstrated to be N-linked glycosylated and expressed early during infection. Furthermore, confocal microscopic analysis revealed that the M2L protein co-localized with cellular ER proteins. Organelle location also affects M2L protein function: the elimination of the N-terminal leader sequence from the M2L protein compromised both its ER location and its ability to inhibit virus-induced NF-kappaB activation. There is only partial ER localization when a second mutant M2L protein lacking potential endoplasmic reticulum retention signal is expressed. However, this C-terminal deleted mutant protein is compromised in its ability to inhibit NF-kappaB activation. Determination of the ER location of the M2L proteins provides important insights for its function in future investigations.

Poxviral regulation of the host NF-kappaB response: the vaccinia virus M2L protein inhibits induction of NF-kappaB activation via an ERK2 pathway in virus-infected human embryonic kidney cells.

Exposure of eukaryotic cells to viruses will activate the host NF-kappaB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-kappaBeta activation in infected 293T cells. In this virus-host system, the extents of NF-kappaBeta-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-kappaB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the "wild-type" NF-kappaB-inhibitory phenotype, as indicated by decreased NF-kappaB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-kappaBeta response.

The vaccinia virus K1L gene product inhibits host NF-kappaB activation by preventing IkappaBalpha degradation.

Vaccinia virus wild-type strains such as Ankara and WR synthesize proteins capable of inhibiting the activation of host NF-kappaB, a family of transcription factors that regulate the expression of inflammatory genes. In contrast, an infection by the attenuated MVA strain, whose genome lacks many immunoregulatory genes present in the DNA of its Ankara parent, induces NF-kappaB activation. Insertion of NF-kappaB inhibitory genes into the MVA DNA, then, would alter the MVA phenotype. By this method, a 5.2-kb region of Ankara DNA containing the K1L gene and two other genes that are absent in the MVA genome that was identified as NF-kappaB was inhibited in cells infected with the MVA/5.2kb virus. To determine if K1L was responsible, the relevant biological properties of both a recombinant MVA containing a copy of the WR strain's K1L (MVA/K1L) and a WR deletion mutant lacking the K1L gene (DeltaK1L) were examined. Indeed, unlike its progenitor, the altered MVA halted degradation of the host regulatory protein IkappaBalpha-a key event in the pathway of transcriptional activation by NF-kappaB factors. Moreover, MVA/K1L gained the ability to repress artificially contrived and natural NF-kappaB-regulated expression of a transfected luciferase and the cellular tumor necrosis factor gene, respectively. In contrast, although these functions could also be performed by WR, the DeltaK1L virus lost these abilities. Thus, one apparent molecular function of K1L is to prevent IkappaBalpha degradation. This impediment to NF-kappaB-induced host proinflammatory gene expression, in turn, might enhance virus survival.