Intraspecific and Intrageneric Genomic Variation across Three Sedum Species (Crassulaceae): A Plastomic Perspective.

Sedum is the largest succulent genus in Crassulaceae. Because of predominant maternal inheritance, little recombination, and slow evolution, plastomes can serve as powerful super barcodes for inter- or intra-species phylogenetic analyses. While previous research has focused on plastomes between Sedum species, intra-species studies are scarce. Here, we sequenced plastomes from three Sedum species (Sedum alfredii, Sedum plumbizincicola, and Sedum japonicum) to understand their evolutionary relationships and plastome structural evolution. Our analyses revealed minimal size and GC content variation across species. However, gene distribution at IR boundaries, repeat structures, and codon usage patterns showed diversity at both inter-specific and intra-specific levels. Notably, an rps19 gene expansion and a bias toward A/T-ending codons were observed. Codon aversion motifs also varied, potentially serving as markers for future studies. Phylogenetic analyses confirmed the non-monophyly of Sedum and divided the Acre clade into two groups. Individuals from the same species clustered together, with strong support for the relationships between S. alfredii, S. tricarpum, and S. plumbizincicola. Additionally, S. japonicum clearly affiliates with the Acre clade. This study provides valuable insights into both intra-specific and intra-generic plastome variation in Sedum, as well as overall plastome evolution within the genus.

Phylogenomics and plastomics offer new evolutionary perspectives on Kalanchoideae (Crassulaceae).

BACKGROUND AND AIMS: Kalanchoideae is one of three subfamilies within Crassulaceae and contains four genera. Despite previous efforts, the phylogeny of Kalanchoideae remains inadequately resolved with persistent issues including low support, unstructured topologies and polytomies. This study aimed to address two central objectives: (1) resolving the pending phylogenetic questions within Kalanchoideae by using organelle-scale 'barcodes' (plastomes) and nuclear data; and (2) investigating interspecific diversity patterns among Kalanchoideae plastomes. METHODS: To explore the plastome evolution in Kalanchoideae, we newly sequenced 38 plastomes representing all four constituent genera (Adromischus, Cotyledon, Kalanchoe and Tylecodon). We performed comparative analyses of plastomic features, including GC and gene contents, gene distributions at the IR (inverted repeat) boundaries, nucleotide divergence, plastomic tRNA (pttRNA) structures and codon aversions. Additionally, phylogenetic inferences were inferred using both the plastomic dataset (79 genes) and nuclear dataset (1054 genes). KEY RESULTS: Significant heterogeneities were observed in plastome lengths among Kalanchoideae, strongly correlated with LSC (large single copy) lengths. Informative diversities existed in the gene content at SSC/IRa (small single copy/inverted repeat a), with unique patterns individually identified in Adromischus leucophyllus and one major Kalanchoe clade. The ycf1 gene was assessed as a shared hypervariable region among all four genera, containing nine lineage-specific indels. Three pttRNAs exhibited unique structures specific to Kalanchoideae and the genera Adromischus and Kalanchoe. Moreover, 24 coding sequences revealed a total of 41 lineage-specific unused codons across all four constituent genera. The phyloplastomic inferences clearly depicted internal branching patterns in Kalanchoideae. Most notably, by both plastid- and nuclear-based phylogenies, our research offers the first evidence that Kalanchoe section Eukalanchoe is not monophyletic. CONCLUSIONS: This study conducted comprehensive analyses on 38 newly reported Kalanchoideae plastomes. Importantly, our results not only reconstructed well-resolved phylogenies within Kalanchoideae, but also identified highly informative unique markers at the subfamily, genus and species levels. These findings significantly enhance our understanding of the evolutionary history of Kalanchoideae.

Quantitative Determination of Aflatoxin B1 in Maize and Feed by ELISA and Time-Resolved Fluorescent Immunoassay Based on Monoclonal Antibodies.

In this study, a highly sensitive monoclonal antibody (mAb) was developed for the detection of aflatoxin B1 (AFB1) in maize and feed. Additionally, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluorescence immunoassay assay (TRFICA) were established. Firstly, the hapten AFB1-CMO was synthesized and conjugated with carrier proteins to prepare the immunogen for mouse immunization. Subsequently, mAb was generated using the classical hybridoma technique. The lowest half-maximal inhibitory concentration (IC50) of ic-ELISA was 38.6 ng/kg with a linear range of 6.25-100 ng/kg. The limits of detections (LODs) were 6.58 ng/kg and 5.54 ng/kg in maize and feed, respectively, with the recoveries ranging from 72% to 94%. The TRFICA was developed with a significantly reduced detection time of only 21 min, from sample processing to reading. Additionally, the limits of detection (LODs) for maize and feed were determined to be 62.7 ng/kg and 121 ng/kg, respectively. The linear ranges were 100-4000 ng/kg, with the recoveries ranging from 90% to 98%. In conclusion, the development of AFB1 mAb and the establishment of ic-ELISA for high-throughput sample detection, as well as TRFICA for rapid detection presented robust tools for versatile AFB1 detection in different scenarios.

Monospecific and ultrasensitive detection of ofloxacin: A computational chemistry-assisted hapten screening strategy and analysis of molecular recognition mechanism.

Contamination in food and the environment with fluoroquinolones (FQs) has become a serious threat to the global ecological balance and public health safety. Ofloxacin (OFL) is one of the most widely utilized sterilization agents in FQs. In the process of monitoring OFL, broad-spectrum monoclonal antibodies (mAb) cannot meet the demand for monospecific detection. Here, a computational chemistry-assisted hapten screening strategy was proposed in this study. Differences in the properties of antigenic epitopes were precisely extracted through a comprehensive comparative study of 16 common FQs molecules and a monospecific and ultrasensitive mAb-3B4 for OFL was successfully prepared. The screened fleroxacin (FLE) hapten was applied in a heterologous competition strategy resulting in a 20-fold improvement in the half inhibitory concentration (IC50) of mAb-3B4 to 0.0375 µg L-1 and cross-reacted only with marbofloxacin (MAR) in regulated FQs. In addition, a single-chain variable fragment (scFv) for OFL was constructed for the first time with an IC50 of 0.378 µg L-1. Molecular recognition mechanism studies validated the reliability of this strategy and revealed the key amino acid sites responsible for OFL specificity and sensitivity. Finally, ic-ELISA and GICA were established for OFL in real samples. This work provides new ideas for the preparation of monospecific mAb and improves the monitoring system of FQs.

Genome evolution of Buchnera aphidicola (Gammaproteobacteria): Insights into strand compositional asymmetry, codon usage bias, and phylogenetic implications.

Taxa of Buchnera aphidicola (hereafter "Buchnera") are mutualistic intracellular symbionts of aphids, known for their remarkable biological traits such as genome reduction, strand compositional asymmetry, and symbiont-host coevolution. With the growing availability of genomic data, we performed a comprehensive analysis of 103 genomes of Buchnera strains from 12 host subfamilies, focusing on the genomic characterizations, codon usage patterns, and phylogenetic implications. Our findings revealed consistent features among all genomes, including small genome sizes, low GC contents, and gene losses. We also identified strong strand compositional asymmetries in all strains at the genome level. Further investigation suggested that mutation pressure may have played a crucial role in shaping codon usage of Buchnera. Moreover, the genomic asymmetries were reflected in asymmetric codon usage preferences within chromosomal genes. Notably, the levels of these asymmetries were varied among strains and were significantly influenced by the degrees of genome shrinkages. Lastly, our phylogenetic analyses presented an alternative topology of Aphididae, based on the Buchnera symbionts, providing robust confirmation of the paraphylies of Eriosomatinae, and Macrosiphini. Our objectives are to further understand the strand compositional asymmetry and codon usage bias of Buchnera taxa, and provide new perspectives for phylogenetic studies of Aphididae.

Codon Usage Analyses Reveal the Evolutionary Patterns among Plastid Genes of Saxifragales at a Larger-Sampling Scale.

Saxifragales is a 15-family order of early-divergent Eudicots with a rich morphological diversity and an ancient rapid radiation. Codon usage bias (CUB) analyses have emerged as an essential tool for understanding the evolutionary dynamics in genes. Thus far, the codon utilization patterns had only been reported in four separate genera within Saxifragales. This study provides a comprehensive assessment of the codon manipulation based on 50 plastid genes, covering 11 constituent families at a larger sampling scale. Our results first showed a high preference for AT bases and AT-ending codons. We then used effective number of codons (ENC) to assess a range of codon bias levels in the plastid genes. We also detected high-informative intrafamilial differences of ENC in three families. Subsequently, parity rule 2 (PR2) plot analyses revealed both family-unique and order-shared bias patterns. Most importantly, the ENC plots and neutrality analyses collectively supported the dominant roles of selection in the CUB of Saxifragales plastid genes. Notably, the phylogenetic affinities inferred by both ML and BI methods were consistent with each other, and they all comprised two primary clades and four subclades. These findings significantly enhance our understanding of the evolutionary processes of the Saxifrage order, and could potentially inspire more CUB analyses at higher taxonomic levels.

A Sensitive and Specific Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Pretilachlor in Grains and the Environment.

Pretilachlor is a chloroacetamide herbicide mainly used for weed and broadleaf weed control in rice, that is widely utilized in China. In order to detect the residue of pretilachlor in the environment and food, a highly sensitive and specific monoclonal antibody (mAb) against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47 ± 2.35 µg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 µg/L was developed. The specificity of the antibody was explained by computer simulations and experimental validation. The mAb exhibited negligible cross-reactivity towards alachlor, acetochlor, propisochlor, butachlor, and metalaxyl, and the limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 µg/L. The recoveries of all samples were 78.3~91.3%. The reliability of the ic-ELISA method for residue detection of pretilachlor in the environment and grains was confirmed using high performance liquid chromatography.

Structural Diversities and Phylogenetic Signals in Plastomes of the Early-Divergent Angiosperms: A Case Study in Saxifragales.

As representative of the early-divergent groups of angiosperms, Saxifragales is extremely divergent in morphology, comprising 15 families. Within this order, our previous case studies observed significant structural diversities among the plastomes of several lineages, suggesting a possible role in elucidating their deep phylogenetic relationships. Here, we collected 208 available plastomes from 11 constituent families to explore the evolutionary patterns among Saxifragales. With thorough comparisons, the losses of two genes and three introns were found in several groups. Notably, 432 indel events have been observed from the introns of all 17 plastomic intron-containing genes, which could well play an important role in family barcoding. Moreover, numerous heterogeneities and strong intrafamilial phylogenetic implications were revealed in pttRNA (plastomic tRNA) structures, and the unique structural patterns were also determined for five families. Most importantly, based on the well-supported phylogenetic trees, evident phylogenetic signals were detected in combinations with the identified pttRNAs features and intron indels, demonstrating abundant lineage-specific characteristics for Saxifragales. Collectively, the results reported here could not only provide a deeper understanding into the evolutionary patterns of Saxifragales, but also provide a case study for exploring the plastome evolution at a high taxonomic level of angiosperms.

Ten Plastomes of Crassula (Crassulaceae) and Phylogenetic Implications.

The genus Crassula is the second-largest genus in the family Crassulaceae, with about 200 species. As an acknowledged super-barcode, plastomes have been extensively utilized for plant evolutionary studies. Here, we first report 10 new plastomes of Crassula. We further focused on the structural characterizations, codon usage, aversion patterns, and evolutionary rates of plastomes. The IR junction patterns-IRb had 110 bp expansion to rps19-were conservative among Crassula species. Interestingly, we found the codon usage patterns of matK gene in Crassula species are unique among Crassulaceae species with elevated ENC values. Furthermore, subgenus Crassula species have specific GC-biases in the matK gene. In addition, the codon aversion motifs from matK, pafI, and rpl22 contained phylogenetic implications within Crassula. The evolutionary rates analyses indicated all plastid genes of Crassulaceae were under the purifying selection. Among plastid genes, ycf1 and ycf2 were the most rapidly evolving genes, whereas psaC was the most conserved gene. Additionally, our phylogenetic analyses strongly supported that Crassula is sister to all other Crassulaceae species. Our findings will be useful for further evolutionary studies within the Crassula and Crassulaceae.

The Mitogenome of Sedum plumbizincicola (Crassulaceae): Insights into RNA Editing, Lateral Gene Transfer, and Phylogenetic Implications.

As the largest family within the order Saxifragales, Crassulaceae contains about 34 genera with 1400 species. Mitochondria play a critical role in cellular energy production. Since the first land plant mitogenome was reported in Arabidopsis, more than 400 mitogenomic sequences have been deposited in a public database. However, no entire mitogenome data have been available for species of Crassulaceae to date. To better understand the evolutionary history of the organelles of Crassulaceae, we sequenced and performed comprehensive analyses on the mitogenome of Sedum plumbizincicola. The master mitogenomic circle is 212,159 bp in length, including 31 protein-coding genes (PCGs), 14 tRNA genes, and 3 rRNA genes. We further identified totally 508 RNA editing sites in PCGs, and demonstrated that the second codon positions of mitochondrial genes are most prone to RNA editing events. Notably, by neutrality plot analyses, we observed that the mitochondrial RNA editing events have large effects on the driving forces of plant evolution. Additionally, 4 MTPTs and 686 NUMTs were detected in the mitochondrial and nuclear genomes of S. plumbizincicola, respectively. Additionally, we conducted further analyses on gene transfer, secondary structures of mitochondrial RNAs, and phylogenetic implications. Therefore, the findings presented here will be helpful for future investigations on plant mitogenomes.

Plastomes of Bletilla (Orchidaceae) and Phylogenetic Implications.

The genus Bletilla is a small genus of only five species distributed across Asia, including B. chartacea, B. foliosa, B. formosana, B. ochracea and B. striata, which is of great medicinal importance. Furthermore, this genus is a member of the key tribe Arethuseae (Orchidaceae), harboring an extremely complicated taxonomic history. Recently, the monophyletic status of Bletilla has been challenged, and the phylogenetic relationships within this genus are still unclear. The plastome, which is rich in both sequence and structural variation, has emerged as a powerful tool for understanding plant evolution. Along with four new plastomes, this work is committed to exploring plastomic markers to elucidate the phylogeny of Bletilla. Our results reveal considerable plastomic differences between B. sinensis and the other three taxa in many aspects. Most importantly, the specific features of the IR junction patterns, novel pttRNA structures and codon aversion motifs can serve as useful molecular markers for Bletilla phylogeny. Moreover, based on maximum likelihood and Bayesian inference methods, our phylogenetic analyses based on two datasets of Arethuseae strongly imply that Bletilla is non-monophyletic. Accordingly, our findings from this study provide novel potential markers for species identification, and shed light on the evolution of Bletilla and Arethuseae.

Plastome evolution of Aeonium and Monanthes (Crassulaceae): insights into the variation of plastomic tRNAs, and the patterns of codon usage and aversion.

MAIN CONCLUSION: This study reported 13 new plastomes from Aeonium and Monanthes, and observed new markers for phylogeny and DNA barcoding, such as novel tRNA structures and codon usage bias and aversion. The Macaronesian clade of Crassulaceae consists of three genera: Aichryson, with about 15 species; Monanthes, with about 10 species; Aeonium, with about 40 species. Within this clade, Aeonium, known as "the botanical equivalent of Darwin's finches", is regarded as an excellent model plant for researching adaptive evolution. Differing from the well-resolved relationships among three genera of the Macaronesian clade, the internal branching patterns within the genus Aeonium are largely unclear. In this study, we first reported 13 new plastomes from genus Aeonium and the closely related genus Monanthes. We further performed comprehensive analyses of the plastomes, with focuses on the secondary structures of pttRNAs and the patterns of codon usage and aversion. With a typical circular and quadripartite structure, the 13 plastomes ranged from 149,900 to 151,030 bp in size, and the unique pattern in IR junctions might become a family-specific marker for Crassulaceae species. Surprisingly, the π values of plastomes from Monanthes were almost twice those from Aeonium. Most importantly, we strongly recommend that highly polymorphic regions, novel putative pttRNA structures, patterns of codon usage bias and aversion derived from plastomes might have phylogenetic implications, and could act as new markers for DNA barcoding of plants. The results of phylogenetic analyses strongly supported a clear internal branching pattern in Macaronesian clade (represented by Aeonium and Monanthes), with higher nodal support values. The findings reported here will provide new insights into the variation of pttRNAs, and the patterns of codon usage and aversion of the family Crassulaceae.

Mitogenomic Codon Usage Patterns of Superfamily Certhioidea (Aves, Passeriformes): Insights into Asymmetrical Bias and Phylogenetic Implications.

The superfamily Certhioidea currently comprises five families. Due to the rapid diversification, the phylogeny of Certhioidea is still controversial. The advent of next generation sequencing provides a unique opportunity for a mitogenome-wide study. Here, we first provided six new complete mitogenomes of Certhioidea (Certhia americana, C. familiaris, Salpornis spilonota, Cantorchilus leucotis, Pheugopedius coraya, and Pheugopedius genibarbis). We further paid attention to the genomic characteristics, codon usages, evolutionary rates, and phylogeny of the Certhioidea mitogenomes. All mitogenomes we analyzed displayed typical ancestral avian gene order with 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and one control region (CR). Our study indicated the strand-biased compositional asymmetry might shape codon usage preferences in mitochondrial genes. In addition, natural selection might be the main factor in shaping the codon usages of genes. Additionally, evolutionary rate analyses indicated all mitochondrial genes were under purifying selection. Moreover, MT-ATP8 and MT-CO1 were the most rapidly evolving gene and conserved genes, respectively. According to our mitophylogenetic analyses, the monophylies of Troglodytidae and Sittidae were strongly supported. Importantly, we suggest that Salpornis should be separated from Certhiidae and put into Salpornithidae to maintain the monophyly of Certhiidae. Our findings are useful for further evolutionary studies within Certhioidea.

PRWHMDA: Human Microbe-Disease Association Prediction by Random Walk on the Heterogeneous Network with PSO.

Microorganisms resided in human body play a vital role in metabolism, immune defense, nutrition absorption, cancer control and protection against pathogen colonization. The changes of microbial communities can cause human diseases. Based on the known microbe-disease association, we presented a novel computational model employing Random Walking with Restart optimized by Particle Swarm Optimization (PSO) on the heterogeneous interlinked network of Human Microbe-Disease Associations (PRWHMDA) (see Figure 1). Based on the known human microbe-disease associations, we constructed the heterogeneous interlinked network with Cosine similarity. The extended random walk with restart (RWR) method was derived to get the potential microbe-disease associations. PSO was utilized to get the optimal parameters of RWR. To evaluate the prediction effectiveness, we performed leave one out cross validation (LOOCV) and 5-fold cross validation (CV), which got the AUC (The area under ROC curve) of 0.915 (LOOCV) and the average AUCs of 0.8875 ± 0.0046 (5-fold CV). Moreover, we carried out three case studies of asthma, inflammatory bowel disease (IBD) and type 1 diabetes (T1D) for the further evaluation. The result showed that 10, 10 and 9 of top-10 predicted microbes were verified by previously published experimental results, respectively. It is anticipated that PRWHMDA can be effective to identify the disease-related microbes and maybe helpful to disclose the relationship between microorganisms and their human host.

Simultaneous determination of fumonisins B1, B2 and B3 contaminants in maize by ultra high-performance liquid chromatography tandem mass spectrometry.

The present work developed an analytical method for simultaneous determination of fumonisins B(1), B(2) and B(3) residues in maize by ultra high-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UHPLC separation and MS/MS parameters. The method involves addition of fumonisins isotope internal standards, extraction with a mixture of acetonitrile and water and clean-up with solid-phase extraction (SPE) cartridges before UHPLC-MS/MS analysis. A single-laboratory method validation was conducted by testing three different spiking levels for repeatability and recovery according to International Union of Pure and Applied Chemistry (IUPAC) guidelines. The LOQ of FB(1), FB(2) and FB(3) were 1.50, 1.65 and 0.4 µg kg(-1), respectively, which were lower than the criteria of EU, USA and other countries regarding minimum residue limits of fumonisins in foods including baby foods and feedstuffs. Recoveries of three fumonisins ranged from 80.9% to 97.0% with RSD values of 2.4-11.1%.The advantages of this method include simple pretreatment, rapid determination and high sensitivity, and it fulfills the requirements for food analysis with respect to minimum residue limits of fumonisins in various countries.