ER-1 deficiency induces inflammation and lipid deposition in meibomian gland and lacrimal gland.

PURPOSE: To investigated the role of estrogen receptor-1 (ER-1) in maintaining homeostasis in ocular surface. METHODS: ER-1-knockout (ER-1KO) mice were studied at 4 months of age. The ocular surface was examined using a slit lamp. Histological alterations in the meibomian gland (MG) and lacrimal gland (LG) were observed with H&E staining. Protein levels of P-ERK, peroxisome proliferator-activated receptor gamma (PPAR-γ), p-NFκB-P65, IL-1ß, aquaporin 5 (AQP-5), fatty acid-binding protein 5 (Fabp5) and K10 were determined by immunofluorescence and Western blotting. Gene expressions of APO-F, APO-E, K10, ELOVL4, PPAR-γ, SCD-1, and SREBP1 were quantified by qPCR. Conjunctival (CJ) goblet cell alterations were detected by PAS staining. Lipid metabolism in MG and LG was assessed using LipidTox. Apoptosis in MG and LG was analyzed through the TUNEL assay. RESULTS: Both male and female ER-1KO mice demonstrated increased corneal fluorescence staining scores. MG showed abnormal lipid metabolism and ductal dilation. LG displayed lipid deposition and reduced AQP-5 expression. CJ experienced goblet cell loss. MG, LG exhibited signs of inflammation and apoptosis. CONCLUSION: ER1 is pivotal for ocular surface homeostasis in both genders of mice. ER1 deficiency induces inflammation and lipid deposition to MG and LG, culminating in dry eye-like manifestations on the ocular surface.

An epi-allele of SMS causes Sanming dominant genic male sterility in rice.

Male sterility is an important trait in rice for hybrid rice (Oryza sativa) breeding. However, the factors involved in dominant male sterility are largely unknown. Here, we identified a gene from Sanming dominant genic male sterile rice, named Sanming dominant male sterility (SMS), and reported that an epi-allele of this locus contributes to male sterility. Segregation analysis attributed dominant male sterility to a single locus, SMS, which we characterized using a male-sterile near isogenic line (NIL) of rice cultivar 93-11. The SMS locus was heterozygous in the male-sterile 93-11 NIL, containing an epi-allele identical to that in 93-11, and an epi-allele identical to that in rice cultivar Nipponbare, which we refer to as SMS9 and SMSN, respectively. SMS9 is silent and hyper-methylated, whereas SMSN is expressed and hypo-methylated in the 93-11 NIL. Overexpressing SMSN led to male sterility. Mutations in SMS rescued the male sterility of the 93-11 NIL. Interestingly, we observed the duplication of SMSN in Nipponbare, but did not observe the duplication of SMS9 in 93-11. Together, these findings suggest that the reduced methylation and enhanced expression of the SMSN epi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.

Cadmium Absorption in Various Genotypes of Rice under Cadmium Stress.

Cadmium (Cd) is a kind of heavy metal. Cadmium pollution in paddy fields will accumulate a large amount of cadmium in rice, which will affect the growth and development of rice. In addition, long-term consumption of rice contaminated with Cd can harm human health. In this study, four rice varieties with high Cd accumulation (S4699, TLY619, JHY1586, QLY155) and four varieties with low Cd accumulation (YY4949, CYJ-7, G8YXSM, MXZ-2) were screened through field experiments for two consecutive years (2021 and 2022) and differences in antioxidant enzyme systems and expression of genes in their organs were analyzed. The total Cd content showed as follows: indica rice > japonica rice, high-Cd-accumulation variety > low-Cd-accumulation variety, and the total Cd content of each organ of rice showed root > stem > leaf > grain. The results of the antioxidant enzyme system showed that the contents of malondialdehyde (MAD), reduced glutathione (GSH), oxidized glutathione (GSSH), and peroxidase (POD) were positively correlated with the total Cd content in rice, and superoxide dismutase (SOD) showed the opposite performance in the leaves. There was no correlation between catalase (CAT) and Cd content, but CAT content decreased in leaves and grains and increased in roots and stems with increasing fertility. Based on this study, RT-qPCR was used to further validate the expression of Cd-uptake-related genes in different rice varieties. It was found that high expression of OsHMA3, OsCCX2, OsNRAMP5, and OsHMA9 genes promoted Cd uptake and translocation in rice, especially in rice varieties with high Cd accumulation. The high expression of OslRT1, OsPCR1, and OsMTP1 genes hindered Cd uptake by rice plants, which was especially evident in low-accumulating Cd rice varieties. These results provide an important theoretical reference and scientific basis for our in-depth study and understanding of the mechanism of cadmium stress tolerance in rice.

OsHUB2 inhibits function of OsTrx1 in heading date in rice.

Heading date is one of the most pivotal agronomic traits for rice (Oryza sativa) yield and adaptation. Little is known about the crosstalk between histone ubiquitination and histone methylation in rice heading date regulation. Here, we reported HISTONE MONOUBIQUITINATION 1 (OsHUB1) and OsHUB2 are involved in heading date regulation via the Hd1 and Ehd1 pathway. Loss of OsHUB1 and OsHUB2 function resulted in early heading under long-day and short-day photoperiods. The expression of Hd3a, RFT1, and Ehd1 was induced and the transcript levels of Hd1, Ghd7, OsCCA1, OsGI, OsFKF1, and OsTOC1 were reduced under long-day conditions, whereas RFT1 and Ehd1 expression was induced in oshub2 mutants under short-day conditions. OsHUB2 interacted with OsTrx1 and repressed the gene expression of OsTrx1. OsHUB2 directly bound to Ehd1 to ubiquitinate H2B at Ehd1, and H2B ubiquitination levels were reduced in oshub2-2 and oshub2-3 mutants. OsTrx1 were highly enriched at Ehd1, and H3K4me3 levels of Ehd1 were upregulated in oshub2-2. Mutations of OsTrx1 in the oshub2-2 background rescued the early-heading phenotype of oshub2-2. The increases in Ehd1 H3K4me3 levels and transcript levels in oshub2-2 mutants were attenuated in oshub2-2 ostrx1-2 double mutants. Together, our results (i) reveal that OsHUB2 represses the function of OsTrx1 and H3K4me3 levels at Ehd1 and (ii) suggest that OsHUB2-mediated H2B ubiquitination plays critical roles together with H3K4me3 in rice heading date regulation.

Chromatin remodeling factors OsYAF9 and OsSWC4 interact to promote internode elongation in rice.

Deposition of H2A.Z and H4 acetylation by SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) and Nucleosome Acetyltransferase of H4 (NuA4) complexes in specific regulatory regions modulates transcription and development. However, little is known about these complexes in Oryza sativa (rice) development. Here, we reported that OsYAF9 and OsSWC4, two subunits of SWR1 and NuA4 complexes, are involved in rice vegetative and reproductive development. Loss of OsYAF9 resulted in reduced height, fewer tillers, fewer pollen grains, and defects in embryogenesis and seed filling. OsYAF9 directly interacted with OsSWC4 in vitro and in vivo. Loss of OsSWC4 function exhibited defects in pollen germination and failure to generate seeds, whereas knockdown of OsSWC4 resulted in reduced height and fewer tillers. The reduced height caused by OsYAF9 mutation and OsSWC4 knockdown was due to shorter internodes and defects in cell elongation, and this phenotype was rescued with gibberellin (GA) treatment, suggesting that both OsYAF9 and OsSWC4 are involved in the GA biosynthesis pathway. OsSWC4 was directly bound to the AT-rich region of GA biosynthesis genes, which in turn accomplished H2A.Z deposition and H4 acetylation at the GA biosynthesis genes with OsYAF9. Together, our study provides insights into the mechanisms involving OsSWC4 and OsYAF9 forming a protein complex to promote rice internode elongation with H2A.Z deposition and H4 acetylation.

H∞ synchronization of delayed neural networks via event-triggered dynamic output control.

This paper investigates H∞ exponential synchronization (ES) of neural networks (NNs) with delay by designing an event-triggered dynamic output feedback controller (ETDOFC). The ETDOFC is flexible in practice since it is applicable to both full order and reduced order dynamic output techniques. Moreover, the event generator reduces the computational burden for the zero-order-hold (ZOH) operator and does not induce sampling delay as many existing event generators do. To obtain less conservative results, the delay-partitioning method is utilized in the Lyapunov-Krasovskii functional (LKF). Synchronization criteria formulated by linear matrix inequalities (LMIs) are established. A simple algorithm is provided to design the control gains of the ETDOFC, which overcomes the difficulty induced by different dimensions of the system parameters. One numerical example is provided to demonstrate the merits of the theoretical analysis.

Epstein-Barr Virus Limits the Accumulation of IPO7, an Essential Gene Product.

Epstein-Barr virus (EBV) encodes more than 40 miRNAs that target cellular mRNAs to aid its infection, replication, and maintenance in individual cells and in its human host. Importin-7 (IPO7), also termed Imp7 or RanBPM7, is a nucleocytoplasmic transport protein that has been frequently identified as a target for two of these viral miRNAs. How the viral life cycle might benefit from regulating IPO7 has been unclear, though. We demonstrate with CRISPR-Cas9 mutagenesis that IPO7 is essential in at least three cells lines and that increasing its levels of expression inhibits growth of infected cells. EBV thus regulates the level of IPO7 to limit its accumulation consistent with its being required for survival of its host cell.

Effects of Cryopreservation Duration on the Outcome of Single-Unit Cord Blood Transplantation.

Cryopreservation is widely used in umbilical cord blood (UCB) banking, yet its impact on progenitor cell function remains largely unaddressed. It is unknown whether long-term cryopreservation affects UCB transplantation outcomes. Herein, we evaluated the impact of UCB age on clinical outcomes and investigated the effect of cryopreservation duration of UCB on hematopoietic potency in 91 patients receiving single cord blood transplantations. UCB cryopreservation duration was 0.7 to 13.4 y. The most common indication of transplant was thalassemia (48%). There was no significant association between cryopreservation duration and neutrophil engraftment probability ( P = 0.475). Cryopreservation duration did not affect the post-thaw viability and subsequent neutrophil engraftment rate. Therefore, UCB units can undergo cryopreservation for at least 8 y with no impact on clinical outcomes.

Isolation and identification of five cold-inducible promoters from Oryza sativa.

MAIN CONCLUSION: Five promoters of the cold-inducible rice genes were isolated. The quantitative and qualitative expression analyses in the high generation transgenic rice suggest that the genes are stably induced by low temperature. Cold-inducible promoters are highly desirable for stress-inducible gene expression in crop genetic engineering. In this study, five rice genes, including OsABA8ox1, OsMYB1R35, OsERF104, OsCYP19-4, and OsABCB5, were found to be transcriptionally induced by cold stress. The promoters of these five genes were isolated, and their activities were identified in various tissues of transgenic rice plants at different growth stages both before and after cold stress. Histochemical staining, quantitative fluorescence assays, and GUSplus gene expression assays in corresponding promoter-GUSplus transgenic rice plants confirmed that the five promoters were cold-inducible with different expression patterns and strengths. The OsABA8ox1 and OsERF104 promoters had very low background expression; in contrast, the OsMYB1R35 promoter had higher basal activity in the roots, and OsCYP19-4 promoter activity was preferentially high in leaves and flowers of untreated transgenic lines. The OsABCB5 promoter had the highest basal activity among the five promoters. After cold induction, the activities of the OsABA8ox1, OsMYB1R35, and OsABCB5 promoters were high in both roots and leaves, slightly lower than that of the constitutively expressed OsActin1 promoter but comparable to that of the AtRD29A promoter. During the cold treatment time course, the activities of OsABA8ox1 and OsABCB5 promoters were quickly up-regulated in the early period and peaked at 24 h, after which the induction level gradually decreased until 48 h. The activities of the OsMYB1R35 and OsCYP19-4 promoters increased under stress in a time-dependent manner, while OsERF104 promoter activity began to increase at 4 h and then decreased strongly. Furthermore, activities' analysis in T3, T4, and T5 homozygous progeny of single-copy plants revealed that five promoters maintained their activities at comparable levels with no evidence of silencing under cold stress. Overall, the five cold-inducible rice promoters described herein could potentially be used in crop biotechnology.

Implementing a novel model for hospice and palliative care in the emergency department: An experience from a tertiary medical center in Taiwan.

Hospice and palliative care has been recognized as an essential part of emergency medicine; however, there is no consensus on the optimal model for the delivery of hospice and palliative care in the emergency department (ED). Therefore, we conducted a novel implementation in a tertiary medical center in Taiwan. In the preintervention period, we recruited a specialist for hospice and palliative medicine in the ED to lead our intervention. In the early stage of the intervention, starting on July 1, 2014, we encouraged and funded ED physicians and nurses to receive training for hospice and palliative medicine and residents of emergency medicine to rotate to the hospice ward. In the late stage of the intervention, we initiated educational programs in the ED, an interdisciplinary meeting with the hospice team every month, sharing information and experience via a cell phone communication app, and setting aside an emergency hospice room for end-of-life patients. We compared the outcomes among pre-, during, and postintervention periods. Compared with 4 in the preintervention period, the cases of do not resuscitate (DNR) per month increased significantly to 30.1 in the early stage of intervention, 23.9 in late stage of intervention, and 34.6 in the postintervention period (all P < .001 compared with the preintervention period). Compared with 10.8% in the preintervention period, the ratio of DNR orders signed in the ED/total DNR orders signed in the study hospital was increased to 17.1% in early stage of intervention, 12.5% in late stage of intervention, and 22.8% in postintervention. Compared with zero in preintervention and early intervention, the cases of consultation with the hospice team increased significantly to 19 cases per month in the late stage of intervention and postintervention. The ability of nurses in hospice and palliative care, including knowledge and the timing and method of consultation with the hospice team, was also significantly improved. We successfully implemented a novel model of hospice and palliative care in the ED via a champion, education, and close collaboration with the hospice team, which could be an important reference for other EDs and intensive care unit in the future.

Dissecting the regulation of EBV's BART miRNAs in carcinomas.

Epstein-Barr virus (EBV) encodes multiple miRNAs known to contribute to its pathogenicity. Previous studies have found that the levels of some EBV miRNAs are 10-100 fold higher in biopsies and in tumor xenografts than in cells grown in culture. We have asked if these increased levels reflect transcriptional enhancement resulting from the tumor microenvironment, selection for increased levels of the EBV genome, or both. We measured the levels of BART miRNAs and their DNA templates in tumor xenografts induced from EBV-positive gastric carcinoma cells and EBV-negative gastric carcinoma cells expressing plasmid replicons encoding these miRNAs. We focused on BART miRNAs which are expressed in all tumors and found that they provide tumors selective growth advantages as xenografts. Stem-loop PCR and real-time PCR revealed that the xenografts expressed both higher levels of some miRNAs and viral DNA templates than did the corresponding cells in culture.

Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe2+/Fe3+ sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection.

Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)-glucose system to design a colorimetric probe for glucose. Moreover, a H2O2·BDBA·AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by the naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0-10 ng/mL PSA at an interval of 2 ng/mL in plasma samples.

Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.

Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis.

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Low-Temperature-Induced Expression of Rice Ureidoglycolate Amidohydrolase is Mediated by a C-Repeat/Dehydration-Responsive Element that Specifically Interacts with Rice C-Repeat-Binding Factor 3.

Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non-nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH), which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT) but not abscisic acid- (ABA) regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (P OsUAH ). Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of P OsUAH . Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE) element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s) subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

Isolation and characterization of three cadmium-inducible promoters from Oryza sativa.

Cadmium (Cd) is an important soil pollutant. Developing genetically engineered crops might be a feasible strategy for Cd decontamination and damage prevention. Both genes and promoters are critical for the effective construction of genetically modified plants. Although many functional genes for Cd tolerance and accumulation have been identified, few reports have focused on plant Cd-inducible promoters. Here, we identified three Cd-inducible genes in the rice genome: two tau class glutathione S-transferase (GSTU) genes, OsGSTU5 and OsGSTU37, and an HSP20/alpha crystallin family protein gene, OsHSP18.6. The promoter sequences were isolated and tested in transgenic rice lines using a GUSplus reporter gene. All of the promoters exhibited low background expression under normal conditions and could be strongly induced by Cd stress. Although their strength was comparable to that of the constitutive OsACTIN promoter under Cd stress, their time-dependent expression patterns under both short- and long-term Cd exposure were markedly different. The responses of the three promoters to other heavy metals were also examined. Furthermore, heavy metal-responsive cis elements in the promoters were computationally analyzed, and regions determining the Cd stress response were analyzed using a series of truncations. Our results indicate that the three Cd-inducible rice promoters described herein could potentially be used in applications aimed at improving heavy metal tolerance in crops or for the bio-monitoring of environmental contamination.

Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system.

The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and "transgene clean" targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.

Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies.

The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a marker for measuring vaccine efficacy and antibody titer. The glycosylation profile of E2 may affect the immunogenicity of the vaccine and the timing of re-vaccination. In this study, a human embryonic kidney cell line was used to secrete fully-glycosylated CSFV E2, which was then coated onto ELISA plates without purification or adjustment. The resulting E2-secreting medium-direct-coating (E2-mDc) ELISA was successfully used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. Compared with a virus neutralization test (as standard), the E2-mDc ELISA was found to be more accurate (90%) than a commercial CSFV antibody diagnostic kit (62%). In conclusion, the mammalian cell-secreted antigen can provide cheap, accurate and effective assays for vaccine efficacy and disease diagnoses.