The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly.

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

Molecular characterization of HMW-GS 1Dx3(t) and 1Dx4(t) genes from Aegilops tauschii and their potential value for wheat quality improvement.

Two x-type high molecular weight glutenin subunits (HMW-GS) in Aegilops tauschii, 1Dx3(t) and 1Dx4(t) were identified by SDS-PAGE and MALDI-TOF-MS. Their complete coding sequences were isolated by AS-PCR. 1Dx3(t) and 1Dx4(t) genes consist of 2535 bp and 2508 bp and encode 845 and 836 amino acid residues, respectively. The deduced molecular masses of 1Dx3(t) and 1Dx4(t) gene products are 87655.26 Da and 86664.24 Da, respectively, well corresponding to the molecular masses measured by MALDI-TOF-MS. A total of 18 SNPs were identified between 1Dx3(t) and 1Dx4(t). Comparing with 1Dx5 subunit, 1Dx3(t) had a six amino acid insertion at 146-151 while the 1Dx4(t) had a nine amino acid deletion when compared with 1Dx3(t) subunit. The authenticity of the cloned 1Dx3(t) and 1Dx4(t) genes were confirmed by successful expression of their ORFs in E. coli. Comparison and phylogenetic tree based on the amino acid and nucleotide sequences confirmed that 1Dx3(t) was most closely related to 1Dx5 subunit that is widely accepted as a superior subunit for bread-making property. The secondary structure prediction demonstrated that 1Dx3(t) subunit has significantly high α-helix and ß-strand contents, suggesting it might have positive effects on dough quality.

[Establishment and preliminary clinical application of human intestinal fluid transplantation].

Objective: To explore and establish the preparation system of human intestinal fluid transplantation (HIFT) and HIFT capsule, and to preliminarily apply it to clinic. Methods: Strict standards for donor screening and management were established. The nasojejunal tube was catheterized into the distal jejunum, and then it was connected with an improved disposable sterile negative pressure collection device for the collection of human intestinal fluid. After that, it was prepared into capsules by filtering, adding 10% glycerin protectant and freeze-drying method. The amount of living bacteria was used as the standard of therapeutic dose. The living bacteria amount in fluid is ≥ 5.0×108 /mL and the living bacteria proportion is ≥ 83%; the living bacteria amount in powder is ≥ 2.0×106 /g and the living bacteria proportion is ≥ 81%; The observational indicators included: (1) the basic information of the donor, the amount of living bacteria in the HIF and powder. (2) Preliminary analysis of the treatment for ASD, which combined HIFT capsule with standard FMT capsule, from February to December 2021 (Clinical trial Registration Number: ChiCTR2100043929). Evaluation criteria: Trypan blue staining method was used to detect the living bacteria amount in fluid and powder. The Autism Behavior Checklist (ABC) and Childhood Autism Rating Scale (CARS) were used to evaluate the efficacy. Results: Compared with the parent donor, the standard donor was younger [(25.4±0.9) y vs. (30.7±3.2) y, t=-19.097, P=0.001] and had a lower body mass index [(19.7±0.5) kg/m2 vs. (20.8±1.3) kg/m2, t=-8.726, P=0.001], more in the living bacteria amount in powder [(7.47±1.52)×106/g vs. (5.03±1.38)×106/g, t=11.331, P=0.031], Chao index (205.4±6.8 vs. 194.2±7.2, t=10.415, P=0.001), and Shannon index (3.25±0.14 vs 2.72±0.27, t=19.465, P=0.001). The differences were statistically significant (all P<0.05). However, there were no significant differences in gender, drainage volume and total number of bacterial liquid colonies between the two groups (all P>0.05). Both the standard donor and the parent donor met the donor screening criteria, and the preparation fluid and powder met the treatment criteria. Eight patients received the treatment of HIFT combined with fecal microbiota transplantation (FMT). Preliminary statistical results showed that HIFT combined with FMT improved ABC and CARS at the 1st, 2nd, 3rd and 4th months. The differences were statistically significant (all P<0.05). No severe adverse reaction occurred. Conclusion: Based on the previous research on FMT preparation system and the clinical technology in our center, this study developed a high standard HIFT preparation system, and explored the clinical study of HIFT combined with FMT, in order to provide an innovative therapy for the treatment of diseases.

Molecular characterization and comparative transcriptional analysis of LMW-m-type genes from wheat (Triticum aestivum L.) and Aegilops species.

Twelve new LMW-GS genes were characterized from bread wheat (Triticum aestivum L.) cultivar Zhongyou 9507 and five Aegilops species by AS-PCR. These genes belong to the LMW-m type and can be classified into two subclasses designated as 1 and 2, with the latter predominant in both wheat and related wild species. Genes in the two subclasses were significantly different from each other in SNPs and InDels variations. In comparison to subclass 1, the structural features of subclass 2 differs in possessing 21 amino acid residue substitutions, two fragment deletions (each with 7 amino acid residues), and a double-residue deletion and two fragment insertions (12 and 2-5 residues). Phylogenetic analysis revealed that the two subclasses were divergent at about 6.8 MYA, earlier than the divergence of C, M, N, S(s) and U genomes. The S(s) and B genomes displayed a very close relationship, whereas the C, M, N and U genomes appeared to be related to the D genome of bread wheat. The presently characterized genes ZyLMW-m1 and ZyLMW-m2 from Zhongyou 9507 were assigned to the D genome. Moreover, these genes were expressed successfully in Escherichia coli. Their transcriptional levels during grain developmental stages detected by quantitative real-time PCR (qRT-PCR) showed that both genes started to express at 5 days post-anthesis (DPA), reaching the maximum at 14 DPA after which their expressions decreased. Furthermore, the expression level of ZyLMW-m2 genes was much higher than that of ZyLMW-m1 during all grain developmental stages, suggesting that the expression efficiency of LMW-GS genes between the two subclasses was highly discrepant.

Effect of dietary supplementation of Ligustrum lucidum on performance, egg quality and blood biochemical parameters of Hy-Line Brown hens during the late laying period.

The fruit of Ligustrum lucidum (FLL, Nuzhenzi in Chinese) is an important traditional medicine, and have attracted significant research attention because of their various biological activities. However, there are few research reports available on the use of FLL as a feed additive in livestock nutrition, particularly in layers. This study was conducted to determine the effects of supplementation of the diet of laying hens with FLL on laying performance, egg quality and blood metabolites. A total of 360 72-week-old hens were allocated to three dietary treatments (eight replications of 15 hens/treatment group) and were fed either a control diet or a diet supplemented with an inclusion level of 0.25% or 0.50% of FLL powder in the final feed, until 78 weeks of age. Hens were housed in a three-tier cage system. Feed and water were provided ad libitum. Blood samples and eggs were collected at the end of the experiment. The results showed that dietary supplementation with FLL did not affect egg weight, feed conversion ratio, eggshell thickness, albumen height, egg yolk color, eggshell breaking strength or egg shape index. However, FLL supplementation significantly decreased (P<0.001) mortality, cracked-egg rate and blood serum levels of cholesterol, low-density lipoprotein cholesterol, triglycerides and alanine aminotransferase, and increased (P<0.001) blood serum levels of high-density lipoprotein cholesterol. No differences in serum levels of total protein, albumin, glucose, calcium, aspartate aminotransferase or alkaline phosphatase were observed in hens fed FLL compared with the control group. It can be concluded that FLL, at a supplementation level of 0.25% final feed, can be used as an effective feed additive to improve the performance of laying hens during the late laying period.

Developmental expression of neural cell adhesion molecules in the mouse neocortex and olfactory bulb.

Polyclonal antibodies to N-CAM and L1 and monoclonal antibodies to epitopes of N-CAM (designated 12F11, 8A2, and 12F8) were used to investigate the spatial and temporal distribution of these neural cell adhesion molecules during the development of mouse cortex and olfactory bulb. The aim of the study was to correlate developmental events such as cell migration, dendritic and axonal outgrowth, and synaptogenesis with the appearance and disappearance of specific molecules involved in cell-cell interactions. Western transfer studies indicated that 12F8 antibody recognized polysialic acid found on embryonic N-CAM; 8A2 antibody primarily recognized the 140 kD component of N-CAM while the 12F11 antibody recognized the 180 and the 140 kD forms. The study demonstrates a high degree of cell surface molecular specialization of different compartments in developing neocortex and olfactory bulb. L1 is found on a variety of unmyelinated fiber tracts including thalamocortical fibers, olfactory nerve, and inner plexiform layer of the olfactory bulb. In contrast, N-CAM epitope recognized by 12F11 antibody is present on olfactory nerve fibers but appears later and is much weaker than L1 on thalamocortical fibers and is absent from the olfactory lobe inner plexiform layer. Dendritic regions are best labeled by 12F8 antibody; the epitope becomes faint in adult cortex but remains strongly expressed in olfactory bulb. This study reveals that widespread N-CAM expression in the central nervous system is constituted by a diversity of local expression of different molecular forms of N-CAM; their different anatomical distributions suggest they may each have unique roles.

Cloning, expression, and evolutionary analysis of α-gliadin genes from Triticum and Aegilops genomes.

Fifteen novel α-gliadin genes were cloned and sequenced from Triticum and related Aegilops genomes by allele-specific polymerase chain reaction (AS-PCR). Sequence comparison displayed high diversities in the α-gliadin gene family. Four toxic epitopes and glutamine residues in the two polyglutamine domains facilitated these α-gliadins to be assigned to specific chromosomes. Five representative α-gliadin genes were successfully expressed in Escherichia coli, and their amount reached a maximum after 4 h induced by isopropyl-ß-D-thiogalactoside (IPTG), indicating a high level of expression under the control of T7 promoter. The transcriptional expression of α-gliadin genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR) showed a similar up-down regulation pattern in different genotypes. A neighbor-joining tree constructed with both full-open reading frame (ORF) α-gliadin genes and pseudogenes further revealed the origin and phylogenetic relationships among Triticum and related Aegilops genomes. The evolutionary analysis demonstrated that α-gliadin genes evolved mainly by synonymous substitutions under strong purifying selection during the evolutionary process.

Identification and molecular characterisation of HMW glutenin subunit 1By16* in wild emmer.

In this study, a novel y-type high molecular weight glutenin subunit (HMW-GS) in wild emmer wheat Triticum turgidum L. var. dicoccoides (Körn.) accession KU1952 was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE) and matrix-assisted laser desorption ionisation/time-of-flight/mass spectrometry (MALDI-TOF-MS). Its electrophoretic mobility and molecular weight were similar to those of 1By16 and was designated as 1By16*. The complete coding sequence of the 1By16* gene isolated by allelic-specific polymerase chain reaction (AS-PCR) consists of 2,157 bp, encoding 729 amino acid residues. The real presence and authenticity of the 1By16* gene in KU1952 were further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), heterologous expression and Western blotting. The molecular structure as well as phylogenetic analysis revealed that 1By16* had 21 single-nucleotide polymorphism (SNP) variations and possessed greater similarity with superior quality subunits 1By15 and 1By16 of common wheat. Secondary structure prediction displayed higher α-helix and ß-strand contents in the 1By16* subunit, which could form a superior gluten structure and, consequently, might have positive effects on dough quality. Our results suggest that 1By16* is expected to be a new potential gene for wheat quality improvement.

Matrix-assisted laser desorption/ionization time-of-flight based wheat gliadin protein peaks are useful molecular markers for wheat genetic study.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation has been used to analyze wheat seed gliadins as an alternative to other established methods, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), etc. The MALDI-TOF approach has shown to have many advantages such as high resolution, cost effectiveness and high throughput. MALDI-TOF-based gliadin profiles have been used for fast wheat cultivar identification. However, the genetic information represented by individual gliadin peaks has not been utilized. In this study a wheat doubled haploid population with a genetic linkage map of good coverage was used to assay individual gliadin peaks from MALDI-TOF profiles as molecular markers. Eight segregating peaks in the population were scored as polymorphic across the population. The 1 to 1 segregating ratios validated the scoring of the peaks and all peaks were mapped to the expected chromosomes or linkage groups on the available linkage map: 1 peak on chromosome 1A, 1 on 6A, 4 on 6B and 2 on 6D.