The miRNA-mRNA Regulatory Modules of Pinus massoniana Lamb. in Response to Drought Stress.

Masson pine (Pinus massoniana Lamb.) is a major fast-growing woody tree species and pioneer species for afforestation in barren sites in southern China. However, the regulatory mechanism of gene expression in P. massoniana under drought remains unclear. To uncover candidate microRNAs, their expression profiles, and microRNA-mRNA interactions, small RNA-seq was used to investigate the transcriptome from seedling roots under drought and rewatering in P. massoniana. A total of 421 plant microRNAs were identified. Pairwise differential expression analysis between treatment and control groups unveiled 134, 156, and 96 differential expressed microRNAs at three stages. These constitute 248 unique microRNAs, which were subsequently categorized into six clusters based on their expression profiles. Degradome sequencing revealed that these 248 differentially expressed microRNAs targeted 2069 genes. Gene Ontology enrichment analysis suggested that these target genes were related to translational and posttranslational regulation, cell wall modification, and reactive oxygen species scavenging. miRNAs such as miR482, miR398, miR11571, miR396, miR166, miRN88, and miRN74, along with their target genes annotated as F-box/kelch-repeat protein, 60S ribosomal protein, copper-zinc superoxide dismutase, luminal-binding protein, S-adenosylmethionine synthase, and Early Responsive to Dehydration Stress may play critical roles in drought response. This study provides insights into microRNA responsive to drought and rewatering in Masson pine and advances the understanding of drought tolerance mechanisms in Pinus.

Association Mapping and Expression Analysis of the Genes Involved in the Wood Formation of Poplar.

Xylogenesis is a complex and sequential biosynthetic process controlled by polygenes. Deciphering the genetic architecture of this complex quantitative trait could provide valuable information for increasing wood biomass and improving its properties. Here, we performed genomic resequencing of 64 24-year-old trees (64 hybrids of section Aigeiros and their parents) grown in the same field and conducted full-sib family-based association analyses of two growth and six woody traits using GEMMA as a choice of association model selection. We identified 1342 significantly associated single nucleotide polymorphisms (SNPs), 673 located in the region upstream and downstream of 565 protein-encoding genes. The transcriptional regulation network of secondary cell wall (SCW) biosynthesis was further constructed based on the published data of poplar miRNA, transcriptome, and degradome. These provided a certain scientific basis for the in-depth understanding of the mechanism of poplar timber formation and the molecular-assisted breeding in the future.

Poplar coma morphogenesis and miRNA regulatory networks by combining ovary tissue sectioning and deep sequencing.

Poplar coma, commonly referred to as "seed hairs", is a tuft of trichomes attached to the seed coat that helps seed dispersal. However, they can also trigger health impacts for humans, including sneezing, shortness of breath, and skin irritation. Despite efforts to study the regulatory mechanism of herbaceous trichome formation, poplar coma remains poorly understood. In this study, we showed that the epidermal cells of the funiculus and placenta are the origin of poplar coma based on observations of paraffin sections. Small RNA (sRNA) and degradome libraries were also constructed at three stages of poplar coma development, including initiation and elongation stages. Based on 7,904 miRNA-target pairs identified by small RNA and degradome sequencing, we constructed a miRNA-transcript factor and a stage-specific miRNA regulatory network. By combining paraffin section observation and deep sequencing, our research will provide greater insight into the molecular mechanisms of poplar coma development.

Long Non-Coding RNA lncWOX11a Suppresses Adventitious Root Formation of Poplar by Regulating the Expression of PeWOX11a.

Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem-loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cinnamomum camphora ('Gantong 1').

Cinnamomum camphora is one of the most commonly used tree species in landscaping. Improving its ornamental traits, particularly bark and leaf colors, is one of the key breeding goals. The basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial in controlling anthocyanin biosynthesis in many plants. However, their role in C. camphora remains largely unknown. In this study, we identified 150 bHLH TFs (CcbHLHs) using natural mutant C. camphora 'Gantong 1', which has unusual bark and leaf colors. Phylogenetic analysis revealed that 150 CcbHLHs were divided into 26 subfamilies which shared similar gene structures and conserved motifs. According to the protein homology analysis, we identified four candidate CcbHLHs that were highly conserved compared to the TT8 protein in A. thaliana. These TFs are potentially involved in anthocyanin biosynthesis in C. camphora. RNA-seq analysis revealed specific expression patterns of CcbHLHs in different tissue types. Furthermore, we verified expression patterns of seven CcbHLHs (CcbHLH001, CcbHLH015, CcbHLH017, CcbHLH022, CcbHLH101, CcbHLH118, and CcbHLH134) in various tissue types at different growth stages using qRT-PCR. This study opens a new avenue for subsequent research on anthocyanin biosynthesis regulated by CcbHLH TFs in C. camphora.

SMRT and Illumina sequencing provide insights into mechanisms of lignin and terpenoids biosynthesis in Pinus massoniana Lamb.

Wood and oleoresin are important industrial raw materials with high economic value; however, their molecular formation and biosynthesis mechanisms in different tissues of Pinus massoniana remain unexplored. Therefore, we used single-molecule real-time sequencing technology (SMRT) and Illumina RNA sequencing to establish a transcriptome dataset and explore the expression pattern of genes related to secondary metabolites involved in wood formation and oleoresin biosynthesis in six different P. massoniana tissues. In total, 63.58 Gb of polymerase reads were obtained, including 41,407 isoforms with an average length of 1822 bp. We identified 3939 and 8785 isoforms and 161 and 481 transcription factors with tissue expression specificity and in the reproductive and vegetative organs, respectively. Eighty isoforms were annotated as cellulose synthases and 224 isoforms involved in lignin biosynthesis were enriched. Additionally, we identified 217 isoforms involved in the terpenoid biosynthesis pathway, with needles having the most tissue-specific genes for terpenoid biosynthesis. Some isoforms related to lignin biosynthesis were highly expressed in the xylem, according to the results of transcriptome sequencing and real-time quantitative reverse-transcription polymerase chain reaction. Our research confirmed the advantages of SMRT sequencing and provided valuable information for the transcriptional annotation of P. massoniana, which will be beneficial for producing better raw wood and oleoresin materials.

Exploring miRNA-mRNA regulatory modules responding to tannic acid stress in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) via small RNA sequencing.

MicroRNAs (miRNAs) are small noncoding RNAs (sRNAs) that regulate gene expression by inhibiting translation or degrading mRNA. Although the functions of miRNAs in many biological processes have been reported, there is currently no research on the possible roles of miRNAs in Micromelalopha troglodyta (Graeser) involved in the response of plant allelochemicals. In this article, six sRNA libraries (three treated with tanic acid and three control) from M. troglodyta were constructed using Illumina sequencing. From the results, 312 known and 43 novel miRNAs were differentially expressed. Notably, some of the most abundant miRNAs, such as miR-432, miR-541-3p, and miR-4448, involved in important physiological processes were also identified. To better understand the function of the targeted genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results indicated that differentially expressed miRNA targets were involved in metabolism, development, hormone biosynthesis, and immunity. Finally, we visualized a miRNA-mRNA regulatory module that supports the role of miRNAs in host-allelochemical interactions. To our knowledge, this is the first report on miRNAs responding to tannic acid in M. troglodyta. This study provides indispensable information for understanding the potential roles of miRNAs in M. troglodyta and the applications of these miRNAs in M. troglodyta management.

Genome-Scale Identification, Classification, and Expression Profiling of MYB Transcription Factor Genes in Cinnamomum camphora.

The camphor tree (Cinnamomum camphora (L.) Presl.) is the representative species of subtropical evergreen broadleaved forests in eastern Asia and an important raw material for essential oil production worldwide. Although MYBs have been comprehensively characterized and their functions have been partially resolved in many plants, it has not been explored in C. camphora. In this study, 121 CcMYBs were identified on 12 chromosomes in the whole genome of C. camphora and found that CcMYBs were mainly expanded by segmental duplication. They were divided into 28 subgroups based on phylogenetic analysis and gene structural characteristics. In the promoter regions, numerous cis-acting elements were related to biological processes. Analysis of RNA sequencing data from seven tissues showed that CcMYBs exhibited different expression profiles, suggesting that they have various roles in camphor tree development. In addition, combined with the correlation analysis of structural genes in the flavonoid synthesis pathway, we identified CcMYBs from three subgroups that might be related to the flavonoid biosynthesis pathway. This study systematically analyzed CcMYBs in C. camphora, which will set the stage for subsequent research on the functions of CcMYBs during their lifetime and provide valuable insights for the genetic improvement of camphor trees.

Integrated SMRT and Illumina Sequencing Provide New Insights into Crocin Biosynthesis of Gardenia jasminoides.

Crocins are valuable bioactive components of gardenia fruit, and their biosynthesis and accumulation have attracted widespread interest. Studies have investigated the biosynthesis and accumulation of crocin based on Illumina sequencing, but there is a lack of reports based on full-length transcriptome sequencing. Utilising SMRT sequencing and high-performance liquid chromatography (HPLC), we explored crocin biosynthesis and accumulation in the fruit of Gardenia jasminoides. HPLC analysis showed that crocins specifically exist in fruit and that the content of crocins increases gradually during fruit development. SMRT sequencing generated 46,715 high-quality full-length isoforms, including 5230 novel isoforms that are not present in the G. jasminoides genome. Furthermore, a total of 46 genes and 91 lncRNAs were involved in the biosynthesis and accumulation of crocin. The qRT-PCR indicated that genes involved in crocin biosynthesis reached a peak in the NOV stage. These findings contributed to our understanding of crocin biosynthesis and accumulation.

Integrated Degradome and Srna Sequencing Revealed miRNA-mRNA Regulatory Networks between the Phloem and Developing Xylem of Poplar.

Lignin and cellulose are the most abundant natural organic polymers in nature. MiRNAs are a class of regulatory RNAs discovered in mammals, plants, viruses, and bacteria. Studies have shown that miRNAs play a role in lignin and cellulose biosynthesis by targeting key enzymes. However, the specific miRNAs functioning in the phloem and developing xylem of Populus deltoides are still unknown. In this study, a total of 134 miRNAs were identified via high-throughput small RNA sequencing, including 132 known and two novel miRNAs, six of which were only expressed in the phloem. A total of 58 differentially expressed miRNAs (DEmiRNAs) were identified between the developing xylem and the phloem. Among these miRNAs, 21 were significantly upregulated in the developing xylem in contrast to the phloem and 37 were significantly downregulated. A total of 2431 target genes of 134 miRNAs were obtained via high-throughput degradome sequencing. Most target genes of these miRNAs were transcription factors, including AP2, ARF, bHLH, bZIP, GRAS, GRF, MYB, NAC, TCP, and WRKY genes. Furthermore, 13 and nine miRNAs were involved in lignin and cellulose biosynthesis, respectively, and we validated the miRNAs via qRT-PCR. Our study explores these miRNAs and their regulatory networks in the phloem and developing xylem of P.deltoides and provides new insight into wood formation.

The reference genome of camellia chekiangoleosa provides insights into camellia evolution and tea oil biosynthesis.

Camellia oil extracted from Camellia seeds is rich in unsaturated fatty acids (UFAs) and secondary metabolites beneficial to human health. However, no oil-tea tree genome has yet been published, which is a major obstacle to investigating the heredity improvement of oil-tea trees. Here, using both Illumina and PicBio sequencing technologies, we present the first chromosome-level genome sequence of the oil-tea tree species Camellia chekiangoleosa Hu. (CCH). The assembled genome consists of 15 pseudochromosomes with a genome size of 2.73 Gb and a scaffold N50 of 185.30 Mb. At least 2.16 Gb of the genome assembly consists of repetitive sequences, and the rest involves a high-confidence set of 64 608 protein-coding gene models. Comparative genomic analysis revealed that the CCH genome underwent a whole-genome duplication (WGD) event shared across the Camellia genus at ~57.48 MYA and a γ-WGT event shared across all core eudicot plants at ~120 MYA. Gene family clustering revealed that the genes involved in terpenoid biosynthesis have undergone rapid expansion. Furthermore, we determined the expression patterns of oleic acid accumulation- and terpenoid biosynthesis-associated genes in six tissues. We found that these genes tend to be highly expressed in leaves, pericarp tissues, roots, and seeds. The first chromosome-level genome of oil-tea trees will provide valuable resources for determining Camellia evolution and utilizing the germplasm of this taxon.

Protoplast isolation and transcriptome analysis of developing xylem in Pinus massoniana (Pinaceae).

BACKGROUND: With active physiological and biochemical activities, tissue-specific protoplasts from cambial derivatives, could serve as a specific source for information on xylogenesis for softwood species resistant to stable genetic transformation and lacking available mutants. METHODS AND RESULTS: In this study, protoplasts were isolated from developing xylem of the Chinese red pine, Pinus massoniana, by enzymolysis. High-quality RNAs were extracted from developing xylem and their protoplasts for constructing transcriptome libraries. Using Illumina HiSeq 2500 PE150 platform, a total of 362,328,426 clean paired-end reads (54.35G) were generated from multiple cDNA libraries and assembled into 146,422 unigenes. The transcriptome data were further analysed to identify 1567 differentially expressed genes (DEGs) between the isolated protoplasts and developing xylem of P. massoniana (Masson pine), 1126 DEGs were upregulated in protoplasts relative to developing xylem cells and 441 were downregulated. Most of the differentially expressed genes in biological process terms are related to plant response, which may be due to the response to cell wall removal. Further, the expression pattern of 71 unigenes involved in lignin biosynthesis was verified by RNA-seq. CONCLUSIONS: This study is the first to report the transcriptome profiles of the developing xylem and its protoplasts of coniferous trees, which provide a new perspective and valuable resource for tracking transcriptional regulatory events in wood formation of Masson pine.

Convolutional squeeze-and-excitation network for ECG arrhythmia detection.

Automatic detection of arrhythmia through an electrocardiogram (ECG) is of great significance for the prevention and treatment of cardiovascular diseases. In Convolutional neural network, the ECG signal is converted into multiple feature channels with equal weights through the convolution operation. Multiple feature channels can provide richer and more comprehensive information, but also contain redundant information, which will affect the diagnosis of arrhythmia, so feature channels that contain arrhythmia information should be paid attention to and given larger weight. In this paper, we introduced the Squeeze-and-Excitation (SE) block for the first time for the automatic detection of multiple types of arrhythmias with ECG. Our algorithm combines the residual convolutional module and the SE block to extract features from the original ECG signal. The SE block adaptively enhances the discriminative features and suppresses noise by explicitly modeling the interdependence between the channels, which can adaptively integrate information from different feature channels of ECG. The one-dimensional convolution operation over the time dimension is used to extract temporal information and the shortcut connection of the Se-Residual convolutional module in the proposed model makes the network easier to optimize. Thanks to the powerful feature extraction capabilities of the network, which can effectively extract discriminative arrhythmia features in multiple feature channels, so that no extra data preprocessing including denoising in other methods are need for our framework. It thus improves the working efficiency and keeps the collected biological information without loss. Experiments conducted with the 12-lead ECG dataset of the China Physiological Signal Challenge (CPSC) 2018 and the dataset of PhysioNet/Computing in Cardiology (CinC) Challenge 2017. The experiment results show that our model gains great performance and has great potential in clinical.

Uncovering miRNA-mRNA Regulatory Modules in Developing Xylem of Pinus massoniana via Small RNA and Degradome Sequencing.

Xylem is required for the growth and development of higher plants to provide water and mineral elements. The thickening of the xylem secondary cell wall (SCW) not only improves plant survival, but also provides raw materials for industrial production. Numerous studies have found that transcription factors and non-coding RNAs regulate the process of SCW thickening. Pinus massoniana is an important woody tree species in China and is widely used to produce materials for construction, furniture, and packaging. However, the target genes of microRNAs (miRNAs) in the developing xylem of P. massoniana are not known. In this study, a total of 25 conserved miRNAs and 173 novel miRNAs were identified via small RNA sequencing, and 58 differentially expressed miRNAs were identified between the developing xylem (PM_X) and protoplasts isolated from the developing xylem (PM_XP); 26 of these miRNAs were significantly up-regulated in PM_XP compared with PM_X, and 32 were significantly down-regulated. A total of 153 target genes of 20 conserved miRNAs and 712 target genes of 113 novel miRNAs were verified by degradome sequencing. There may be conserved miRNA-mRNA modules (miRNA-MYB, miRNA-ARF, and miRNA-LAC) involved in softwood and hardwood formation. The results of qRT-PCR-based parallel validation were in relatively high agreement. This study explored the potential regulatory network of miRNAs in the developing xylem of P. massoniana and provides new insights into wood formation in coniferous species.

Development of PDA Nanoparticles for H9N2 Avian Influenza BPP-V/BP-IV Epitope Peptide Vaccines: Immunogenicity and Delivery Efficiency Improvement.

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines.

Bursal peptide BP-IV as a novel immunoadjuvant enhances the protective efficacy of an epitope peptide vaccine containing T and B cell epitopes of the H9N2 avian influenza virus.

Short peptide antigens covering conserved T or B cell epitopes have been investigated in influenza vaccines. Bursal pentapeptide V (BPP-V) and bursal peptide IV (BP-IV) are small molecular peptides that were isolated and identified from the bursa of Fabricius (BF) and induce a strong immune response at both the humoural and cellular levels. To explore the molecular adjuvant potential of BPP-V and BP-IV with an epitope vaccine, an epitope peptide (HA284-298, GNCVVQCQTERGGLN) rich in T and B cell epitopes for the H9N2 avian influenza virus (AIV) haemagglutinin (HA) protein was selected. BPP-V and BP-IV were coupled with the epitope peptide sequence to form BPP-V and BP-IV-epitope vaccines, respectively. The immunoefficacy of BPP-V and BP-IV-epitope peptide vaccines was evaluated. The results showed that the epitope peptide had weak immunogenicity. The BPP-V-epitope peptide vaccine promoted only the secretion of anti-HA IgG and IgG1 antibodies. The BP-IV-epitope peptide vaccine not only promoted the production of anti-HA IgG and IgG1 antibodies but also significantly induced the production of the IgG2a antibody. The BP-IV-epitope peptide vaccine significantly promoted the production of interleukin (IL-4) and interferon-γ (IFN-γ) (the BPP-V epitope peptide vaccine promoted only the production of IL-4), enhanced the cytotoxic T lymphocyte (CTL) response, and increased the proportion of CD3+ T lymphocytes. Moreover, the BP-IV-epitope peptide vaccine promoted a cell-mediated immune response similar to that of the AIV vaccine group. Furthermore, BPP-V and BP-IV-epitope peptide vaccines could also accelerate the clearance of pulmonary virus and reduce pathological damage after the challenge with H9N2 AIV. This study demonstrates the potential of BP-IV as an effective adjuvant for the epitope peptide vaccine for the H9N2 AIV.

Molecular Cloning, Transcriptional Profiling, Subcellular Localization, and miRNA-Binding Site Analysis of Six SCL9 Genes in Poplar.

The SCL9 subfamily is a key member of the GRAS family that regulates plant development and stress responses. Nevertheless, the functional role of these genes in the growth and development of poplar still unclear. Here, we reported the six SCL9 genes, which were found to be differentially expressed during poplar adventitious root formation. The full-length sequences of PeSCL9 genes of 'Nanlin895' poplar (Populus deltoids × Populus euramericana) were cloned by the RACE technique All PeSCL9 genes lacked introns. RT-qPCR revealed that PeSCL9 genes displayed a dynamic expression pattern in the adventitious root of poplar, according to RT-qPCR data. A series of comprehensive genes characteristics analysis were carried out for six genes by bioinformation. Meanwhile, transient expression analysis of the Populus protoplasts showed that all the PeSCL9 proteins were localized in the nucleus. In addition, the degradome and sRNA of 'Nanlin895' poplar in combination were used to predict miRNAs that regulate PeSCL9. It was found that miR396a and miR396c may affect PeSCL9 expression via cleavage, which was further verified by a transient expression experiment in Populus protoplasts. Overall, the development of poplar adventitious root and other tissues was closely related to these six SCL9 genes, and they serve as a starting point for further research into the mechanisms regulating poplar growth and development.

Porcine Myeloid Antimicrobial Peptides: A Review of the Activity and Latest Advances.

Traditional antibiotics have made great contributions to human health and animal husbandry since the discovery of penicillin in 1928, but bacterial resistance and drug residues are growing threats to global public health due to the long-term uncontrolled application of antibiotics. There is a critical need to develop new antimicrobial drugs to replace antibiotics. Antimicrobial peptides (AMPs) are distributed in all kingdoms of life, presenting activity against pathogens as well as anticancer, anti-inflammatory, and immunomodulatory activities; consequently, they have prospects as new potential alternatives to antibiotics. Porcine myeloid antimicrobial peptides (PMAPs), the porcine cathelicidin family of AMPs, have been reported in the literature in recent years. PMAPs have become an important research topic due to their strong antibacterial activity. This review focuses on the universal trends in the biochemical parameters, structural characteristics and biological activities of PMAPs.

Analogs of the Cathelicidin-Derived Antimicrobial Peptide PMAP-23 Exhibit Improved Stability and Antibacterial Activity.

Antimicrobial peptides (AMPs) have gained interesting as a new type of antimicrobial agent. The cathelicidin-derived antimicrobial peptide PMAP-23 has broad-spectrum antibacterial activity, and to improve its antimicrobial activity, we used amino acid substitution at position 5 or 19 of PMAP-23 to design three analogs, named PMAP-23R (Leu5--Arg), PMAP-23I (Thr19--Ile), and PMAP-23RI (Leu5--Arg and Thr19--Ile). We found that the analog peptides exhibited higher stability and improved antibacterial activity compared with PMAP-23. Additionally, the analog peptides PMAP-23I and PMAP-23RI inhibited the growth of Shigella flexneri CICC 21534, whereas PMAP-23 and PMAP-23R exhibited no antibacterial activity against S. flexneri CICC 21534. Moreover, the peptide analogs showed negligible hemolysis and cytotoxicity. We also found that PMAP-23RI exerted impressive therapeutic effects on mice infected with Staphylococcus aureus ATCC 25923 and Salmonella enterica serovar Typhimurium SL1344. PMAP-23RI induced a greater reduction in pathological damage and a higher decrease in the bacterial gene copies in the lung and liver tissues and greatly reduced mouse mortality. In conclusion, the peptide analogs PMAP-23R, PMAP-23I, and PMAP-23RI enhanced the stability and antimicrobial activity of PMAP-23, but PMAP-23RI exhibits more promise as a new antimicrobial agent candidate for the treatment of bacterial infections.