Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Limits on scalar-induced gravitational waves from the stochastic background by pulsar timing array observations.

Recently, the NANOGrav, PPTA, EPTA, and CPTA Collaborations independently reported their evidence of the Stochastic Gravitational Waves Background (SGWB). While the inferred gravitational-wave background amplitude and spectrum are consistent with astrophysical expectations for a signal from the population of supermassive black-hole binaries (SMBHBs), the search for new physics remains plausible in this observational window. In this work, we explore the possibility of explaining such a signal by the scalar-induced gravitational waves (IGWs) in the very early universe. We use a parameterized broken power-law function as a general description of the energy spectrum of the SGWB and fit it to the new results of NANOGrav, PPTA and EPTA. We find that this approach can put constraints on the parameters of IGW energy spectrum and further yield restrictions on various inflation models that may produce primordial black holes (PBHs) in the early universe, which is also expected to be examined by the forthcoming space-based GW experiments.

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR.

Objective: This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. Methods: The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. Results: VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. Conclusion: Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

Dauricine alleviates cognitive impairment in Alzheimer's disease mice induced by D-galactose and AlCl3 via the Ca2+/CaM pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. Dysregulation of intracellular Ca2+ homeostasis plays a critical role in the pathological development of AD. Dauricine (DAU) is a bisbenzylisoquinoline alkaloid isolated from Menispermum dauricum DC., which can prevent the influx of extracellular Ca2+ and inhibit the release of Ca2+ from the endoplasmic reticulum. DAU has a potential for anti-AD. However, it is unclear whether DAU can exert its anti-AD effect in vivo by regulating the Ca2+ related signaling pathways. Here, we investigated the effect and mechanism of DAU on D-galactose and AlCl3 combined-induced AD mice based on the Ca2+/CaM pathway. The results showed that DAU (1 mg/kg and 10 mg/kg for 30 days) treatment attenuated learning and memory deficits and improved the nesting ability of AD mice. The HE staining assay showed that DAU could inhibit the histopathological alterations and attenuate neuronal damage in the hippocampus and cortex of AD mice. Studies on the mechanism indicated that DAU decreased the phosphorylation of CaMKII and Tau and reduced the formation of NFTs in the hippocampus and cortex. DAU treatment also reduced the abnormally high expression of APP, BACE1, and Aß1-42, which inhibited the deposition of Aß plaques. Moreover, DAU could decrease Ca2+ levels and inhibit elevated CaM protein expression in the hippocampus and cortex of AD mice. The molecular docking results showed that DAU may have a high affinity with CaM or BACE1. DAU has a beneficial impact on pathological changes in AD mice induced by D-galactose and AlCl3 and may act by negative regulation of the Ca2+/CaM pathway and its downstream molecules such as CaMKII and BACE1.

Research review on the pollution of antibiotic resistance genes in livestock and poultry farming environments.

Increasingly serious pollution of antibiotic resistance genes (ARGs) caused by the abuse of antibiotics in livestock and poultry industry has raised worldwide concerns. ARGs could spread among various farming environmental media through adsorption, desorption, migration, and also could transfer into human gut microbiome by hori-zontal gene transfer (HGT), posing potential threats to public health. However, the comprehensive review on the pollution patterns, environmental behaviors, and control techniques of ARGs in livestock and poultry environments in view of One Health is still inadequate, resulting in the difficulties in effectively assessing ARGs transmission risk and developing the efficient control strategies. Here, we analyzed the pollution characteristics of typical ARGs in various countries, regions, livestock species, and environmental media, reviewed the critical environmental fate and influencing factors, control strategies, and the shortcomings of current researches about ARGs in the livestock and poultry farming industry combined with One Health philosophy. In particular, we addressed the importance and urgency of identifying the distribution characteristics and environmental process mechanisms of ARGs, and developing green and efficient ARG control means in livestock farming environments. We further proposed gaps and prospects for the future research. It would provide theoretical basis for the research on health risk assessment and technology exploitation of alleviating ARG pollution in livestock farming environment.

Comparison of the effects of probiotics, rifaximin, and lactulose in the treatment of minimal hepatic encephalopathy and gut microbiota.

Background: Minimal hepatic encephalopathy (MHE) is an early stage in the pathogenesis of hepatic encephalopathy. Intestinal microbiota is involved in the pathogenesis of hepatic encephalopathy and has become an important therapeutic target. Since there is no unified treatment principle for MHE, this study was conducted to determine the safety and efficacy of different intestinal microecological modulators in the treatment of MHE, and to explore the potential mechanism through intestinal microbiota analysis. Methods: Patients with liver cirrhosis were screened for MHE using psychometric hepatic encephalopathy score test. Patients diagnosed with MHE were enrolled and received probiotics, rifaximin, or lactulose for 4 weeks. Adverse events were recorded. The psychometric hepatic encephalopathy score test was performed after treatment. Samples of blood and stool were collected at entry and 4 weeks. Blood samples were analyzed to assess blood ammonia, liver, kidney, and hemostatic functions. Stool microbiota were sequenced to confirm changes in microbial composition. Results: Of 323 patients with liver cirrhosis, 74 patients were diagnosed with MHE. In all, 54 patients were enrolled and 52 who agree to follow-up were included in analysis. The recovery rates of MHE patients received probiotics, rifaximin, and lactulose were 58.8% (20/34), 45.5% (5/11), and 57.1% (4/7), respectively. Probiotics and rifaximin improved liver function in MHE patients to a certain extent. Taxonomic compositions of gut microbiota in MHE patients were distinct from healthy people before treatment; the differences were significantly reduced after treatment, and the gut microbiota gradually resembled the structure of healthy individuals. We found that the relative abundance of specific taxa associated with anti-inflammatory and good cognitive functions was increased in MHE patients after treatment. Accordingly, metabolic pathways in MHE patients were altered before and after treatment. Downregulated pathways after probiotics treatment included glycometabolism and degradation of aromatic compounds. After lactulose treatment, degradation pathways of arginine and ornithine showed a downward trend. Conclusion: Probiotics, rifaximin, and lactulose are safe and effective in the treatment of MHE, and improve the composition of gut microbiota to some extent.

Soybean GmSAUL1, a Bona Fide U-Box E3 Ligase, Negatively Regulates Immunity Likely through Repressing the Activation of GmMPK3.

E3 ubiquitin ligases play important roles in plant immunity, but their role in soybean has not been investigated previously. Here, we used Bean pod mottle virus (BPMV)-mediated virus-induced gene silencing (VIGS) to investigate the function of GmSAUL1 (Senescence-Associated E3 Ubiquitin Ligase 1) homologs in soybean. When two closely related SAUL1 homologs were silenced simultaneously, the soybean plants displayed autoimmune phenotypes, which were significantly alleviated by high temperature, suggesting that GmSAUL1a/1b might be guarded by an R protein. Interestingly, silencing GmSAUL1a/1b resulted in the decreased activation of GmMPK6, but increased activation of GmMPK3 in response to flg22, suggesting that the activation of GmMPK3 is most likely responsible for the activated immunity observed in the GmSAUL1a/1b-silenced plants. Furthermore, we provided evidence that GmSAUL1a is a bona fide E3 ligase. Collectively, our results indicated that GmSAUL1 plays a negative role in regulating cell death and immunity in soybean.

Fecal Microbiota Transplantation Ameliorates Active Ulcerative Colitis by Downregulating Pro-inflammatory Cytokines in Mucosa and Serum.

Background: Ulcerative colitis (UC) is a multi-factor disease characterized by alternating remission periods and repeated occurrence. It has been shown that fecal microbiota transplantation (FMT) is an emerging and effective approach for UC treatment. Since most existing studies chose adults as donors for fecal microbiota, we conducted this study to determine the long-term efficacy and safety of the microbiota from young UC patient donors and illustrate its specific physiological effects. Methods: Thirty active UC patients were enrolled and FMT were administered with the first colonoscopy and two subsequent enema/transendoscopic enteral tubing (TET) practical regimens in The First Affiliated Hospital of Anhui Medical University in China. Disease activity and inflammatory biomarkers were assessed 6 weeks/over 1 year after treatment. The occurrence of adverse events was also recorded. The samples from blood and mucosa were collected to detect the changes of inflammatory biomarkers and cytokines. The composition of gut and oral microbiota were also sampled and sequenced to confirm the alteration of microbial composition. Results: Twenty-seven patients completed the treatment, among which 16 (59.3%) achieved efficacious clinical response and 11 (40.7%) clinical remission. Full Mayo score and calprotectin dropped significantly and remained stable over 1 year. FMT also significantly reduced the levels of C-reactive protein (CRP), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). The gut microbiota altered significantly with increased bacterial diversity and decreased metabolic diversity in responsive patients. The pro-inflammatory enterobacteria decreased after FMT and the abundance of Collinsella increased. Accordingly, the altered metabolic functions, including antigen synthesis, amino acids metabolism, short chain fatty acid production, and vitamin K synthesis of microbiota, were also corrected by FMT. Conclusion: Fecal microbiota transplantation seems to be safe and effective for active UC patients who are nonresponsive to mesalazine or prednisone in the long-term. FMT could efficiently downregulate pro-inflammatory cytokines to ameliorate the inflammation.

Combination of ß-Aminobutyric Acid and Ca2+ Alleviates Chilling Stress in Tobacco (Nicotiana tabacum L.).

Chilling is a major abiotic factor limiting the growth, development, and productivity of plants. ß-aminobutyric acid (BABA), a new environmentally friendly agent, is widely used to induce plant resistance to biotic and abiotic stress. Calcium, as a signaling substance, participates in various physiological activities in cells and plays a positive role in plant defense against cold conditions. In this study, we used tobacco as a model plant to determine whether BABA could alleviate chilling stress and further to explore the relationship between BABA and Ca2+. The results showed that 0.2 mM BABA significantly reduced the damage to tobacco seedlings from chilling stress, as evidenced by an increase in photosynthetic pigments, the maintenance of cell structure, and upregulated expression of NtLDC1, NtERD10B, and NtERD10D. Furthermore, 0.2 mM BABA combined with 10 mM Ca2+ increased the fresh and dry weights of both roots and shoots markedly. Compared to that with single BABA treatment, adding Ca2+ reduced cold injury to the plant cell membrane, decreased ROS production, and increased antioxidant enzyme activities and antioxidant contents. The combination of BABA and Ca2+ also improved abscisic acid and auxin contents in tobacco seedlings under chilling stress, whereas ethylene glycol-bis (ß-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) reversed the effects of BABA. These findings suggested that BABA enhances the cold tolerance of tobacco and is closely related to the state of Ca2+ signaling.

Comparative expression profiles of host circulating miRNAs in response to Trichinella spiralis infection.

Trichinellosis is an important food-borne parasitic zoonosis throughout the world. At present, the mechanisms of Trichinella spiralis infection remain unclear. Acquiring detailed information on the host-Trichinella interaction would be beneficial for the development of new strategies for trichinellosis control. Circulating miRNAs are stably detectable in the blood of humans and animals infected with parasites. Circulating miRNAs might regulate the expression of target genes in pathological responses during infection and might be novel potential biomarkers of parasitic diseases. In the present study, a total of ten differentially expressed circulating mouse miRNAs with |log2(fold change)| ≥ 1.0 and FDR < 0.01 were found during T. spiralis infection, of which five were upregulated and five were downregulated. GO and KEGG analyses showed that the target genes of the ten miRNAs were enriched in many signalling pathways, especially focal adhesion, MAPK pathway, and so on. The results of qRT-PCR showed that among the five upregulated miRNAs, mmu-miR-467a-3p and mmu-miR-467d-3p expression in mouse serum reached a peak at 30 days post-infection (dpi). The expression of mmu-miR-376b-3p and mmu-miR-664-3p increased significantly at 18 dpi and then decreased at 30 dpi. The expression of mmu-miR-292a-5p gradually decreased from 12 to 30 dpi. Among the 5 downregulated miRNAs, mmu-miR-199a-5p expression was significantly downregulated at 30 dpi, while the expression levels of the other four miRNAs (mmu-miR-455-5p, mmu-miR-125b-5p, mmu-miR-125a-5p, and mmu-miR-615-3p) were significantly lower compared with the control, showing a steady downregulation at different phases of infection. These findings will help to further understand the host-Trichinella interaction and provide promising serum biomarkers for trichinellosis.

[DNA marker-assisted selection of medicinal plants (â ¢)Evaluation of disease resistance of "Miaoxiang Kangqi 1" --a new cultivar of Panax notoginseng].

DNA marked-assisted selection of medicinal plants accelerated the breeding and promotion of new cultivars, and guaranteed the healthy development of Chinese medicinal materials industry. The first disease-resistant cultivar of notoginseng, namely "Miaoxiang Kangqi 1", served as the object of study. We evaluated the Kangqi's resistance of seeds, seedlings and root against the pathological bacteria (Fusarum oxysporum) of root rot. Compared to the traditional cultivars, the disease index of notoginseng seeds declined by 52.0% after inoculation for seven days; the death rate of seedlings and disease index of root respectively decreased by 72.1% and 62.4% after inoculation for 25 days. Additionally, the growth inhibition ratio of notoginseng seeds and seedlings declined after inoculation. The seeds, seedlings and roots of "Miaoxiang Kangqi 1" showed significantly resistant to root rot. The evaluation of disease-resistance of Kangqi provided the basis for the popularization of new cultivar and guaranteed the favoring conduct of notoginseng pollution-free cultivation.