Ligand-Mediated Regulation of the Chemical/Thermal Stability and Catalytic Performance of Isostructural Cobalt(II) Coordination Polymers.

Regulating the chemical/thermal stability and catalytic activity of coordination polymers (CPs) to achieve high catalytic performance is topical and challenging. The CPs are competent in promoting oxidative cross-coupling, yet they have not received substantial attention. Here, the ligand effect of the secondary ligand of CPs for oxidative cross-coupling reactions was investigated. Specifically, four new isostructural CPs [Co(Fbtx)1.5(4-R-1,2-BDC)]n (denoted as Co-CP-R, Fbtx = 1,4-bis(1,2,4-triazole-1-ylmethyl)-2,3,5,6-tetrafluorobenzene, 4-R-1,2-BDC = 4-R-1,2-benzenedicarboxylate, R = F, Cl, Br, CF3) were prepared. It was found that in the reactions of oxidative amination of benzoxazoles with secondary amines and the oxidative coupling of styrenes with benzaldehydes, both the chemical and thermal stabilities of the four Co-CPs with the R group followed the trend of -CF3 > -Br > -Cl > -F. Density functional theory (DFT) calculations suggested that the difference in reactivity may be ascribed to the effect of substituent groups on the electron transition energy of the cobalt(II) center of these Co-CPs. These findings highlight the secondary ligand effect in regulating the stability and catalytic performance of coordination networks.

Engineering of oxygen vacancy defect in CeO2 through Mn doping for toluene catalytic oxidation at low temperature.

Catalytic oxidation is considered a highly effective method for the elimination of volatile organic compounds. Oxygen vacancy defect engineering in a catalyst is considered an effective approach for high-performance catalysts. Herein, a series of doped MnxCe1-xO2 catalysts (x = 0.05-0.2) with oxygen vacancy defects were synthesized by doping low-valent Mn in a CeO2 lattice. Different characterization techniques were utilized to inspect the effect of doping on oxygen vacancy defect generation. The characterization results revealed that the Mn0.15Ce0.85O2 catalyst has the maximum oxygen vacancy concentration, leading to increased active oxygen species and enhanced oxygen mobility. Thus, Mn0.15Ce0.85O2 catalyst showed an excellent toluene oxidation activity with 90% toluene conversion temperature (T90) of 197 °C at a weight hourly space velocity of 40,000 mL g-1 h-1 as compared to undoped CeO2 (T90 = 225 °C) and Ce based oxides in previous reports. In addition, the Mn0.15Ce0.85O2 catalyst displayed strong recyclability, water resistant ability and long-time stability. The in situ DRIFT results showed that the Mn0.15Ce0.85O2 catalyst has a robust oxidation capability as toluene is quickly adsorbed and actuated as compared to CeO2. Thus, the present work lays the foundation for designing a highly active catalyst for toluene elimination from the environment.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. RESULTS: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. CONCLUSIONS: In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Rapid detection of goose astrovirus genotypes 2 using real-time reverse transcription recombinase polymerase amplification.

BACKGROUND: Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. RESULTS: The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/µL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. CONCLUSIONS: The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV).

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

Identification through fine mapping and verification using CRISPR/Cas9-targeted mutagenesis for a minor QTL controlling grain weight in rice.

KEY MESSAGE: A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect. The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9-2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8-9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.

Recent Progress in Rice Broad-Spectrum Disease Resistance.

Rice is one of the most important food crops in the world. However, stable rice production is constrained by various diseases, in particular rice blast, sheath blight, bacterial blight, and virus diseases. Breeding and cultivation of resistant rice varieties is the most effective method to control the infection of pathogens. Exploitation and utilization of the genetic determinants of broad-spectrum resistance represent a desired way to improve the resistance of susceptible rice varieties. Recently, researchers have focused on the identification of rice broad-spectrum disease resistance genes, which include R genes, defense-regulator genes, and quantitative trait loci (QTL) against two or more pathogen species or many isolates of the same pathogen species. The cloning of broad-spectrum disease resistance genes and understanding their underlying mechanisms not only provide new genetic resources for breeding broad-spectrum rice varieties, but also promote the development of new disease resistance breeding strategies, such as editing susceptibility and executor R genes. In this review, the most recent advances in the identification of broad-spectrum disease resistance genes in rice and their application in crop improvement through biotechnology approaches during the past 10 years are summarized.

Control of Thousand-Grain Weight by OsMADS56 in Rice.

Grain weight and size are important traits determining grain yield and influencing grain quality in rice. In a previous study, a quantitative trait locus controlling thousand-grain weight (TGW) in rice, qTGW10-20.8, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for qTGW10-20.8, OsMADS56 encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the OsMADS56 locus, NILs carrying the OsMADS56 allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the OsMADS56 allele of Teqing in 2018 and 2020, respectively. Using OsMADS56 knock-out mutants and overexpression transgenic plants, OsMADS56 was validated as the causal gene for qTGW10-20.8. Compared with the recipients, the TGW of the knock-out mutants was reduced by 6.0-15.0%. In these populations, decreased grain weight and size were associated with a reduction in the expression of OsMADS56. In transgenic populations of OsMADS56 driven by a strong constitutive promoter, grain weight and size of the positive plants were significantly higher than those of the negative plants. Haplotype analysis showed that the Teqing-type allele of OsMADS56 is the major type presented in cultivated rice and used in variety improvement. Cloning of OsMADS56 provides a new gene resource to improve grain weight and size through molecular design breeding.

Development of a recombinase polymerase amplification fluorescence assay to detect feline coronavirus.

Feline coronavirus (FCoV) is classified into two pathotypes: the avirulent feline enteric coronavirus (FECV), and the virulent feline infectious peritonitis virus (FIPV). Rapid pathogen detection, which is efficient and convenient, is the best approach for early confirmatory diagnosis. In this study, we first developed and evaluated a rapid recombinase polymerase amplification (RPA) detection method for FCoV that can detect FCoV within 15 min at 39 °C. The detection limit of that assay was 233 copies/µL DNA molecules per reaction. The specificity was high: it did not cross-react with canine distemper virus (CDV), canine coronavirus (CCoV), canine adenovirus (CAV), feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). This assay was evaluated using 42 clinical samples (30 diarrhea samples and 12 ascites samples). The coincidence rate between FCoV-RPA and RT-qPCR for detection in clinical samples was 95.2%. In summary, FCoV-RPA analysis provides an efficient, rapid, and sensitive detection method for FCoV.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

Genome-Wide Identification of QTLs for Grain Protein Content Based on Genotyping-by-Resequencing and Verification of qGPC1-1 in Rice.

To clarify the genetic mechanism underlying grain protein content (GPC) and to improve rice grain qualities, the mapping and cloning of quantitative trait loci (QTLs) controlling the natural variation of GPC are very important. Based on genotyping-by-resequencing, a total of 14 QTLs were detected with the Huanghuazhan/Jizi1560 (HHZ/JZ1560) recombinant inbred line (RIL) population in 2016 and 2017. Seven of the fourteen QTLs were repeatedly identified across two years. Using three residual heterozygote-derived populations, a stably inherited QTL named as qGPC1-1 was validated and delimited to a ~862 kb marker interval JD1006-JD1075 on the short arm of chromosome 1. Comparing the GPC values of the RIL population determined by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods, high correlation coefficients (0.966 and 0.983) were observed in 2016 and 2017. Furthermore, 12 of the 14 QTLs were identically identified with the GPC measured by the two methods. These results indicated that instead of the traditional KND method, the rapid and easy-to-operate NIRS was suitable for analyzing a massive number of samples in mapping and cloning QTLs for GPC. Using the gel-based low-density map consisted of 208 simple sequence repeat (SSR) and insert/deletion (InDel) markers, the same number of QTLs (fourteen) were identified in the same HHZ/JZ1560 RIL population, and three QTLs were repeatedly detected across two years. More stably expressed QTLs were identified based on the genome resequencing, which might be attributed to the high-density map, increasing the detection power of minor QTLs. Our results are helpful in dissecting the genetic basis of GPC and improving rice grain qualities through molecular assisted selection.

High-throughput Luminex xMAP assay for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus and rabbit rotavirus.

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.

Development of a real-time loop-mediated isothermal amplification assay for detection of porcine circovirus 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.

Importance of the Interaction between Heading Date Genes Hd1 and Ghd7 for Controlling Yield Traits in Rice.

Appropriate flowering time is crucial for successful grain production, which relies on not only the action of individual heading date genes, but also the gene-by-gene interactions. In this study, influences of interaction between Hd1 and Ghd7 on flowering time and yield traits were analyzed using near isogenic lines derived from a cross between indica rice cultivars ZS97 and MY46. In the non-functional ghd7ZS97 background, the functional Hd1ZS97 allele promoted flowering under both the natural short-day (NSD) conditions and natural long-day (NLD) conditions. In the functional Ghd7MY46 background, Hd1ZS97 remained to promote flowering under NSD conditions, but repressed flowering under NLD conditions. For Ghd7, the functional Ghd7MY46 allele repressed flowering under both conditions, which was enhanced in the functional Hd1ZS97 background under NLD conditions. With delayed flowering, spikelet number and grain weight increased under both conditions, but spikelet fertility and panicle number fluctuated. Rice lines carrying non-functional hd1MY46 and functional Ghd7MY46 alleles had the highest grain yield under both conditions. These results indicate that longer growth duration for a larger use of available temperature and light does not always result in higher grain production. An optimum heading date gene combination needs to be carefully selected for maximizing grain yield in rice.

Development of a sensitive and specific xMAP assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses.

BACKGROUND: A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. METHOD: The microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. . RESULTS: This xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek's disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively. CONCLUSION: This study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination.

Development of an xTAG-multiplex PCR array for the detection of four avian respiratory viruses.

Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 102- 3.52 × 103copies/µL. We examined 60 clinical specimens and found 18 positive for respiratory viruses. Our result demonstrated that xTAG-multiplex PCR method is a high-throughput, rapid, specific and sensitive assay for use in epidemiological studies and clinical detection of avian respiratory pathogens.

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) is one of the most common viral pathogens that circulate widely in captive mouse colonies. A molecular biology detection method would be a useful tool to use in an integrated program to monitor and prevent TMEV infection and transmission. Thus, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect TMEV infection. The sensitivity of the RT-RPA assay approached 8 copies per reaction, which is equivalent to the sensitivity of RT-qPCR reactions. This assay did not detect RNA extracts from other murine pathogens included in this study or TMEV negative samples. Brain tissues and contaminated biological materials were used to assess the clinical performance of the RT-RPA. The detection results of RT-RPA and RT-qPCR were very similar, except that a contaminated biological material sample which was positive by RT-qPCR, with a CT value of 38, was negative by RT-RPA. In summary, the developed RT-RPA assay offers a rapid, sensitive and specific alternative method for monitoring of TMEV, especially in resource-limited conditions.

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus.

BACKGROUND: Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV. RESULTS: The detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA. CONCLUSIONS: A broadly reactive RT-RPA assay was successfully established for MNV detection.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses.

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.