Molecular analysis of eight splicing variants in the hydroxymethylbilane synthase gene.

Background: Molecular genetic testing is the most sensitive and specific method to confirm acute intermittent porphyria (AIP), a rare autosomal dominant disease, caused by Hydroxymethylbilane synthase (HMBS) gene mutation. According to the Human Gene Mutation Database (HGMD), approximately 20% of the reported HMBS gene variants affect pre-RNA splicing. Thus, the ensuing challenge is how to decipher the pathogenicity of these splicing variants. Methods: Using next-generation sequencing, we identified a novel heterozygous variant in the HMBS gene (c.160 + 5G>C) from a Chinese family with AIP. And, previously, seven HMBS variants (c.33 + 5G>A, c.88-16_88-4del, c.88-2A>G, c.161-1G>C, c.652-1G>A, c.772-2A>G and c.772-1G>C) have been reported to be linked with AIP. Herein, we performed a valid and novel in vitro minigene assay to analyze the pathogenicity of these eight splicing variants. Results: By minigene assay in 293 T cell experiments, we demonstrated that all eight variants caused splicing defects in the pre-mRNA of the HMBS gene: c.160 + 5G>C (intron3p_141bp retention), c.33 + 5G>C(intron1p_91bp retention), c.88-16_88-4del and c.88-2A>G (Exon3p_15bp deletion), c.161-1G>C (Exon4p_18bp deletion), c.652-1G>A (Exon11p_1bp deletion), c.772-2A>G and c.772-1G>C (intron11q_104bp retention or Exon12p_4bp deletion).Encouragingly, the c.160 + 5G>C RNA sequencing from peripheral blood lymphocytes was consistent with the minigene assay result. Conclusion: We have made a pioneering attempt to apply minigene in vitro validation to the HMBS gene to evaluate the splicing effect of eight variants, including a novel splice variant (c.160 + 5G>C). This study provides a molecular basis for future research on the pathogenesis and gene therapy of AIP.

Clinical feature and genetic analysis of HMBS gene in Chinese patients with acute intermittent porphyria: a systematic review.

Background: Early detection and diagnosis are important crucial to prevent life-threatening acute attacks in patients with acute intermittent porphyria (AIP). We aim to provide comprehensive data on the clinical and hydroxymethylbilane synthase (HMBS) gene variant characteristics and genotype-phenotype association of Chinese patients with AIP in order to improve clinicians' knowledge of AIP and reduce misdiagnosis and mistaken treatment. Methods: We searched the literature on Chinese patients with AIP in PubMed, Web of Science, Wiley Online Library, ScienceDirect and Chinese literature databases up to August 2023 in our analysis to explore the clinical and HMBS gene variant characteristics of Chinese patients with AIP. Results: A total of 41 original articles associated with Chinese AIP patients were included for analysis: 97 variants were detected in 160 unrelated families, including 35 missense, 29 frameshift, 24 splicing and 9 nonsense variants, with c.517C>T being the most common variant. Clinical data were reported in 77 of 160 patients: Most of them were female (67/77) and the age was 28.8 ± 9.9 years. The most common symptom was abdominal pain (73/77, 94.8%), followed by central nervous system symptoms (45/77, 58.4%). 13.0% (10/77) of patients experienced psychiatric symptoms. Hyponatremia was the most common electrolyte abnormality (42/77). 31 patients received carbohydrate loading therapy, and 30 of them were improved. 6 patients were treated with carbohydrate loading combined with hemin therapy and 5 eventually improved. All variants causing premature stop codons, frameshifts or enzyme activity center may experience more severe clinical phenotypes such as seizures, respiratory paralysis, intracranial hemorrhage disorder or respiratory failure. Conclusion: The most common presenting symptom in Chinese AIP patients was abdominal pain, followed by central nervous system symptoms. The HMBS gene analysis in Chinese AIP patients revealed that the heterogeneity is strong and the most common variant was missense mutation, with c.517C>T being the most common variant. The genotype-phenotype association helps guide clinical diagnosis and treatment. However, the treatment for AIP in China is limited and monolithic, and more attention needs to be paid to the treatment.

A papain-like cysteine protease-released small signal peptide confers wheat resistance to wheat yellow mosaic virus.

Wheat yellow mosaic virus (WYMV), a soil-borne pathogen, poses a serious threat to global wheat production. Here, we identify a WYMV resistance gene, TaRD21A, that belongs to the papain-like cysteine protease family. Through genetic manipulation of TaRD21A expression, we establish its positive role in the regulation of wheat to WYMV resistance. Furthermore, our investigation shows that the TaRD21A-mediated plant antiviral response relies on the release of a small peptide catalyzed by TaRD21A protease activity. To counteract wheat resistance, WYMV-encoded nuclear inclusion protease-a (NIa) suppress TaRD21A activity to promote virus infection. In resistant cultivars, a natural variant of TaRD21A features a glycine-to-threonine substitution and this substitution enables the phosphorylation of threonine, thereby weakening the interaction between NIa and TaRD21A, reinforcing wheat resistance against WYMV. Our study not only unveils a WYMV resistance gene but also offers insights into the intricate mechanisms underpinning resistance against WYMV.

HMBS gene mutations and hydroxymethylbilane synthase activity in acute intermittent porphyria: A systematic review.

BACKGROUND: Acute intermittent porphyria (AIP) is caused by a partial deficiency of hydroxymethylbilane synthase and affects heme biosynthesis. Mutations in the HMBS gene result in HMBS deficiency. AIP is a rare disease, and there been insufficient studies on it. This report describes the molecular epidemiology of HMBS gene defects and hydroxymethylbilane synthase activity levels in classical AIP. METHODS: Databases of PubMed, CNKI, and Wang Fang Database were searched for eligible studies to investigate HMBS gene mutations in peripheral blood samples and HMBS activity in erythrocytes of patients with classical AIP. Relevant studies published up to July 15, 2023, from several databases were independently searched and selected by 2 reviewers. Accuracy data and relevant information were extracted from each eligible study by 2 independent researchers and analyzed using statistical software. RESULTS: After pooling the accuracy data from 232 patients of the 15 eligible studies, 90.5% (210/232) of AIP patients had decreased erythrocyte hydroxymethylbilane synthase activity (<70%), and 96 different mutations were identified in 232 patients, including 33 missense (34.4%), 27 splice (28.1%), 19 deletion (19.8%), 8 nonsense (8.3%), 9 insertion (9.4%) mutations. Residual enzyme activities (%) for different groups of type were expressed using mean and 95% confidence interval (95% CI): missense (51.2, 48.5-53.9), splice (57.5, 52.0-59.1), deletion (54.9, 50.7-59.1), nonsense (52.2, 44.4-60.0), insertion (53.2, 47.4-59.0), group analysis P = .17. Subgroups of missense mutations, domain 1 (50.2, 46.0-54.4), domain 2 (52.8, 49.1-56.4), and domain 3 (49.2, 38.3-60.0), Subgroup analysis, P = .62. CONCLUSION: Different mutation types and mutation positions are not associated with the level of hydroxymethylbilane synthase activity. Erythrocyte hydroxymethylbilane synthase activity is often reduced to half of normal in patients with AIP, and the enzyme activity assay has a high diagnostic value in AIP. AIP is highly molecularly heterogeneous, with missense mutations being the most common, followed by splice mutations. R173W and G111R are high-frequency mutations and have been found in multiple families from different countries.

Measurement of Transgenes Copy Number in Wheat Plants Using Droplet Digital PCR.

Genetic transformation is a powerful method for the investigation of gene function and improvement of crop plants. The transgenes copy number in the transgenic line is involved in gene expression level and phenotypes. Additionally, identification of transgene zygosity is important for quantitative assessment of phenotype and for tracking the inheritance of transgenes in progeny generations. Several methods have been developed for estimating the transgene copy number, including southern blot assay and quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization, although convincing and reliable, is a time-consuming method through which the examination of the copy number is challenging in species with large genomes like wheat plants. Although qPCR is potentially simpler to perform, its results lack accuracy and precision, especially to distinguish between one and two copy events in transgenic plants with large genomes. The droplet digital PCR (ddPCR)-based method for investigation of transgenes copy number has been widely used in an array of crops. In this method, the specific primers to amplify target transgenes and reference genes are used as a single duplexed reaction, which is divided into tens of thousands of nanodroplets. The copy number in independent transgenic lines is determined by detection and quantification of droplets using sequence-specific fluorescently labeled probes. This method offers superior accuracy and reliability with a low cost and scalability as other PCR techniques in the investigation of transgenes copy number. This protocol was validated in: Mol Plant (2021), DOI: 10.1016/j.molp.2021.03.022 Graphical abstract Flow chart for the ddPCR protocol.

Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA).

In China, wheat yellow mosaic disease is mostly caused by wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV). If wheat is co-infected with these two viruses, it can cause severe yellow mosaic symptoms and yield losses. Early detection of viruses is crucial for preventing disease in the field. In this study, we optimized a sensitive, specific reverse transcription recombinase polymerase amplification (RT-RPA) detection method for two viruses, WYMV and CWMV. Two sets of primers were designed based on the capsid protein (CP)-encoding genes of the two viruses, and the reaction conditions were determined. The RT-RPA method, which amplified the target amplicon by a handheld reaction mixture for 20 min, was more sensitive than PCR-CP in the detection of WYMV. Finally, the RT-RPA method was performed on 110 randomly selected field samples, demonstrating its applicability to samples from different regions and specificity for co-infected samples. This study not only describes an improved method for detecting WYMV and CWMV using RT-RPA but also demonstrates the potential of this method, which could be applied under field conditions.

Changes in soil fungal communities after onset of wheat yellow mosaic virus disease.

Rhizosphere-associated microbes have important implications for plant health, but knowledge of the association between the pathological conditions of soil-borne virus-infected wheat and soil microbial communities, especially changes in fungal communities, remains limited. We investigated the succession of fungal communities from bulk soil to wheat rhizosphere soil in both infected and healthy plants using amplicon sequencing methods, and assessed their potential role in plant health. The results showed that the diversity of fungi in wheat rhizosphere and bulk soils significantly differed post wheat yellow mosaic virus disease onset. The structure differences in fungal community at the two wheat health states or two compartment niches were evident, soil physicochemical properties (i.e., NH4 +) contribute to differences in fungal community structure and alpha diversity. Comparison analysis showed Mortierellomycetes and Dothideomycetes as dominant communities in healthy wheat soils at class level. The genus Pyronemataceae and Solicoccozyma were significantly are significantly enriched in rhizosphere soil of diseased plant, the genus Cystofilobasidium, Cladosporium, Mortierella, and Stephanonectria are significantly enriched in bulk soil of healthy plant. Co-occurrence network analysis showed that the fungi in healthy wheat soil has higher mutual benefit and connectivity compared with diseased wheat. The results of this study demonstrated that the occurrence of wheat yellow mosaic virus diseases altered both fungal community diversity and composition, and that NH4 + is the most important soil physicochemical factor influencing fungal diversity and community composition.

Ni-Catalyzed Reductive Fluoroalkylacylation of Alkynes for the Steroselective Synthesis of Fluoroalkylated Enones.

A Ni-catalyzed three-component reductive fluoroalkylacylation of alkynes with fluoroalkyl halides and acyl chlorides is presented. This dicarbofunctionalization provides an efficient method for the synthesis of fluoroalkyl-incorporated enones under mild conditions with high yields and excellent regioselectivity and stereoselectivity.

Effect of sediment burial depth on the control of sedimentary phosphorus release by iron/aluminum co-modified calcite and strategy for overcoming the negative effect of sediment burial.

After placing an active capping material on surface sediments, the capping layer will be buried by the newly formed sediment. In this research, the influence of sediment burial depth on the performance of iron/aluminum co-modified calcite (FeAlCAL) to suppress sedimentary phosphorus (P) release into overlaying water (OL-water) was studied. Furthermore, in order to find out the strategy for overcoming the negative effect of sediment burial, the efficiencies and mechanisms of three different FeAlCAL treatments (one-time FeAlCAL capping with 3 cm sediment burial, multiple FeAlCAL capping with 1 cm sediment burial, and amendment of top 3 cm sediment with FeAlCAL) in the inhibition of sediment P release were contrastively studied. The results showed that with the increase of sediment burial depth, the efficiency of FeAlCAL to block the release of sediment P into OL-water gradually decreased until the FeAlCAL lost the ability to hinder sediment-P release. In contrast to the one-time FeAlCAL capping in the presence of 3 cm sediment burial, the multiple FeAlCAL capping in the presence of 1 cm sediment burial and amendment of top 3 cm sediment with FeAlCAL both effectively prevented the release of P from sediment into OL-water. All results of this work suggest that although sediment burial can negatively affect the ability of FeAlCAL in the inhibition of sediment P release into OL-water and the negative effect becomes stronger as the sediment burial depth increases, the transformation of the application mode of FeAlCAL from one-time capping to multiple capping or from capping to amendment can overcome the negative influence of sediment burial.

Genome-wide identification and analysis of wheat LRR-RLK family genes following Chinese wheat mosaic virus infection.

Background: As the largest plant receptor-like protein kinase (RLK) superfamily, the 21 leucine-rich repeat receptor-like kinases (LRR-RLKs) family are involved in plant 22 growth, development, and stress responses. However, the functions of LRR-RLKs in 23 wheat immunity remain unknown. Results: In the current study, 929 LRR-RLKs were identified in Triticum aestivum 25 genome database using the BLAST and hidden Markov models (HMM) approach and 26 divided into 14 clades. Chromosomal localization and synteny analysis revealed that 27 TaLRR-RLKs were randomly distributed on all chromosomes with 921 collinear 28 events. Through the cis-acting elements analysis, we observed that TaLRR-RLKs 29 participated in hormone response, light response, development, metabolism, and 30 response to environmental stress. The transcript level of 14 random selected 31 TaLRR-RLKs from each subfamily was regulated by plant hormone treatment and 32 Chinese wheat mosaic virus (CWMV) infection. The function of TaLRR-RLKs in 33 wheat resistance to CWMV infection was further investigated by virus-induced gene 34 silencing assay. Additionally, the accumulation of MeJA response genes, as well as 35 CWMV RNA were not changed in the TaLRR-RLK silencing plants under MeJA 36 treatment. Conclusions: Our results demonstrated that TaLRR-RLKs play an important role in 38 wheat resistance to viral infection via hormone signals and lay the groundwork for the 39 functional study of TaLRR-RLKs in wheat.

Effectiveness and mechanism of aluminum/iron co-modified calcite capping and amendment for controlling phosphorus release from sediments.

The effectiveness and mechanism of aluminum/iron co-modified calcite (Al/Fe-CA) for the control of phosphorus (P) liberation from sediments was investigated. The results showed that Al/Fe-CA possessed good sorption performance for phosphate, and the maximum phosphate sorption capacity for Al/Fe-CA could reach 27.0 mg/g. The major mechanisms involved the surface adsorption of phosphate on calcite, the precipitation between phosphate and Ca2+ leached from calcite, and the ligand exchange between Al/Fe-bound hydroxyl groups and phosphate to form the Al-O-P and Fe-O-P inner-sphere complexes. The re-releasing risk of Al/Fe-CA-bound P under the circumstances of normal pH (5-9) and reducing environment was very low. Al/Fe-CA addition could significantly reduce the risk of P releasing from sediment to overlying water (OL-water), and the inactivation of mobile P, reactive soluble P (SRP) and diffusive gradient in thin-films (DGT)-labile P in sediment by Al/Fe-CA had a great part in the suppression of sediment-P liberation to OL-water by the Al/Fe-CA amendment. Al/Fe-CA capping and fabric-wrapped Al/Fe-CA capping both could greatly reduce the risk of P releasing from sediment into OL-water, and the formation of a static layer with low concentrations of SRP and DGT-labile P in the upper sediment was the key to sustaining a high P controlling efficiency. When the applied mode of Al/Fe-CA varied from capping to amendment, although the inactivation efficiency of DGT-labile P in the overlying water and upper sediment by Al/Fe-CA would decrease to a certain degree, the inactivation efficiency of DGT-labile P in the lower sediment by Al/Fe-CA would increase. Results of this study suggest that Al/Fe-CA has the high potential to be used as an active capping or amendment material for the management of internal P loading in surface water bodies.

Nutritional metabolites in Brassica rapa subsp. chinensis var. parachinensis (choy sum) at three different growth stages: Microgreen, seedling and adult plant.

Choy sum is a commonly consumed Asian green leafy brassica vegetable. A comprehensive spectrum of nutritional important metabolites, including amino acids, plant sugars, essential minerals, vitamins (A, B9, E, and K1) and glucosinolates were systematically quantified using LC-QQQ-MS, GC-QQQ-MS and ICP-MS. Significant metabolic profile shifts were observed during the three major developmental stages (microgreen, seedling and adult) studied. Primary metabolites, especially essential amino acids decreased while most plant sugars increased from microgreens to seedlings. Carotenoids, such as violaxanthin, neoxanthin, together with vitamin K1 were higher in the seedlings whereas CHO-folate vitamers and ß-cryptoxanthin were much lower in adult plants. Most essential minerals were concentrated in the microgreens, while sodium increased in adult plants. Aliphatic glucosinolates in microgreens were converted to indolic glucosinolates in the seedlings and further to aromatic glucosinolates in the adults. Overall findings reveal that most of the nutritional metabolites were concentrated either in the microgreens or seedlings.

A comparative investigation on absorption performances of three expanded graphite-based complex materials for toluene.

Three kinds of expanded graphite-based complex materials were prepared to absorb toluene by dispersing plant oil, animal oil and mineral oil on the surface of expanded graphite, respectively. These complex materials were characterized by scanning electronic micrograph, contact angle meter and Brunauer-Emmett-Teller surface area. And their absorption capacities for toluene were comparatively investigated. The results showed that the surfaces of the three types of sorbents were very hydrophobic and nonporous, but they all had excellent absorption capacities for toluene. And their absorption capacities were proportional to the toluene concentration in streams and decreased differently with increasing the absorption temperature. It was noteworthy that the absorption capacities varied with the unsaturated degree of the complex materials and kept unchanged under different relative humidities of streams. Moreover, the regeneration experiments showed that after 15-run regeneration the absorption capacities of expanded graphite modified by mineral oil almost kept unchanged, while that of expanded graphite loaded plant oil and animal oil dropped by 157 and 93.6 mg g(-1), respectively. The losses of their absorption capacities were ascribed to the destruction of their unsaturated carbon bounds.