A Simple, Rapid, and Practical Method for Distinguishing Lonicerae Japonicae Flos from Lonicerae Flos.

Lonicerae japonicae flos (LJF), the dried flower buds of Lonicera japonica Thunb., are often adulterated with Lonicerae. flos (LF), which is derived from the other four Lonicera species. Scholars at home and abroad have established several analytical methods to distinguish LJF from the four Lonicera species of LF; however, to date, no effective and practical method has been established for distinguishing LF from LJF. In our present study, the HPLC fingerprints of LJF and LF were compared, and differences in the content of one of the iridoids were found. Column chromatography combined with pre-HPLC was used for isolating and preparing the iridoid, and its structure was identified as secologanic acid. Then, a method for determining the content of secologanic acid was established using HPLC. The amounts of secologanic acid in 34 batches of LJF and 38 batches of LF were determined. The average amount of secologanic acid in 34 batches of LJF was 18.24 mg/g, with values ranging from 12.9 mg/g to 23.3 mg/g, whereas the average amount in 38 batches of LF was 1.76 mg/g, with values ranging from 0.2 mg/g to 7.2 mg/g. Therefore, secologanic acid can be considered as one of the characteristic components for distinguishing LJF and LF. Our study not only provides a rapid, simple, sensitive, and practical method for identifying LJF and LF but also establishes a method for discovering the characteristic components of other herb-medicines that are susceptible to adulteration.

Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua.

Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (ß-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.

The fertility research of "Huajin 6", a new variety of honeysuckle.

The aim of this study was to investigate the fertility of "Huajin 6" and the effect of exogenous methyl jasmonate on its fertility. In this study, "Huajin 6", "Huajin 6" treated with methyl jasmonate and "Damaohua" were used as the research objects, the stamen phenotypes and the shape of pollen grains were observed, pollen viability and stigma receptivity were measured. The results showed that the pistil structure and function were normal, and although the stamen anthers did not dehisce, they were still capable of producing pollen with a certain amount of vigor. Methyl jasmonate could promote the opening of the flowers of "Huajin 6" and improve the development of pollen grains to a certain extent, but it could not promote anthers dehiscence of "Huajin 6". This study can provide theoretical guidance for the cultivation of new honeysuckle varieties using "Huajin 6".

Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance.

Analysis of the HD-Zip I transcription factor family in Salvia miltiorrhiza and functional research of SmHD-Zip12 in tanshinone synthesis.

Background: The homeodomain-leucine zipper I (HD-Zip I) transcription factor is a plant-specific protein that plays an essential role in the abiotic stress response of plants. Research on the HD-Zip I family in Salvia miltiorrhiza is still lacking. Methods and Results: In this study, a total of 25 SmHD-Zip I proteins were identified. Their characterizations, phylogenetic relationships, conserved motifs, gene structures, and cis-elements were analyzed comprehensively using bioinformatics methods. Expression profiling revealed that SmHD-Zip I genes exhibited distinctive tissue-specific patterns and divergent responses to ABA, PEG, and NaCl stresses. SmHD-Zip12 responded the most strongly to ABA, PEG, and NaCl, so it was used for transgenic experiments. The overexpression of SmHD-Zip12 significantly increased the content of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by 2.89-fold, 1.85-fold, 2.14-fold, and 8.91-fold compared to the wild type, respectively. Moreover, in the tanshinone biosynthetic pathways, the overexpression of SmHD-Zip12 up-regulated the expression levels of SmAACT, SmDXS, SmIDS, SmGGPPS, SmCPS1, SmCPS2, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 compared with the wild type. Conclusions: This study provides information the possible functions of the HD-Zip I family and lays a theoretical foundation for clarifying the functional mechanism of the SmHD-Zip12 gene in regulating the synthesis of tanshinone in S. miltiorrhiza.

Genome-wide identification, molecular characterization, and gene expression analyses of honeysuckle NHX antiporters suggest their involvement in salt stress adaptation.

Background: Ion homeostasis is an essential process for the survival of plants under salt stress. Na+/H+ antiporters (NHXs) are secondary ion transporters that regulate Na+ compartmentalization or efflux reduce Na+ toxicity and play a critical role during plant development and stress responses. Methods and Results: To gain insight into the functional divergence of NHX genes in honeysuckle, a total of seven LjNHX genes were identified on the whole genome level and were renamed according to their chromosomal positions. All LjNHXs possessed the Na+/H+ exchanger domain and the amiloride-binding site was presented in all NHX proteins except LjNHX4. The phylogenetic analysis divided the seven NHX genes into Vac-clade (LjNHX1/2/3/4/5/7) and PM-clade (LjNHX6) based on their subcellular localization and validated by the distribution of conserved protein motifs and exon/intron organization analysis. The protein-protein interaction network showed that LjNHX4/5/6/7 shared the same putatively interactive proteins, including SOS2, SOS3, HKT1, and AVP1. Cis-acting elements and gene ontology (GO) analysis suggested that most LjNHXs involve in the response to salt stress through ion transmembrane transport. The expression profile analysis revealed that the expression levels of LjNHX3/7 were remarkably affected by salinity. These results suggested that LjNHXs play significant roles in honeysuckle development and response to salt stresses. Conclusions: The theoretical foundation was established in the present study for the further functional characterization of the NHX gene family in honeysuckle.

Artemisinin biosynthesis enhancement in transgenic Artemisia annua plants by downregulation of the ß-caryophyllene synthase gene.

Artemisinin is an effective antimalarial drug isolated from the medicinal plant Artemisia annua L. Due to its increasing market demand and the low yield in A. annua, there is a great interest in increasing its production. In this paper, in an attempt to increase artemisinin content of A. ANNUA by suppressing the expression of ß-caryophyllene synthase, a sesquiterpene synthase competing as a precursor of artemisinin, the antisense fragment (750 bp) of ß-caryophyllene synthase cDNA was inserted into the plant expression vector pBI121 and introduced into A. annua by Agrobacterium-mediated transformation. PCR and Southern hybridization confirmed the stable integration of multiple copies of the transgene in 5 different transgenic lines of A. annua. Reverse transcription PCR showed that the expression of endogenous CPS in the transgenic lines was significantly lower than that in the wild-type control A. annua plants, and ß-caryophyllene content decreased sharply in the transgenic lines in comparison to the control. The artemisinin content of one of the transgenic lines showed an increase of 54.9 % compared with the wild-type control. The present study demonstrated that the inhibition pathway in the precursor competition for artemisinin biosynthesis by anti-sense technology is an effective means of increasing the artemisinin content of A. annua plants.

Genome-Wide Identification of CBL-CIPK Gene Family in Honeysuckle (Lonicera japonica Thunb.) and Their Regulated Expression Under Salt Stress.

In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). CBL-CIPK gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. To gain insight into the functional divergence of CBL-CIPK genes in honeysuckle, a total of six LjCBL and 17 LjCIPK genes were identified. The phylogenetic analysis along with the gene structure analysis divided both CBL and CBL-interacting protein kinase genes into four subgroups and validated by the distribution of conserved protein motifs. The 3-D structure prediction of proteins shown that most LjCBLs shared the same Protein Data Bank hit 1uhnA and most LjCIPKs shared the 6c9Da. Analysis of cis-acting elements and gene ontology implied that both LjCBL and LjCIPK genes could be involved in hormone signal responsiveness and stress adaptation. Protein-protein interaction prediction suggested that LjCBL4 is hypothesized to interact with LjCIPK7/9/15/16 and SOS1/NHX1. Gene expression analysis in response to salinity stress revealed that LjCBL2/4, LjCIPK1/15/17 under all treatments gradually increased over time until peak expression at 72 h. These results demonstrated the conservation of salt overly sensitive pathway genes in honeysuckle and a model of Ca2+-LjCBL4/LjSOS3-LjCIPK16/LjSOS2 module-mediated salt stress signaling in honeysuckle is proposed. This study provides insight into the characteristics of the CBL-CIPK gene families involved in honeysuckle salt stress responses, which could serve as a foundation for gene transformation technology, to obtain highly salt-tolerant medicinal plants in the context of the global reduction of cultivated land.

Network Pharmacology-Based Identification of Potential Targets of Lonicerae japonicae Flos Acting on Anti-Inflammatory Effects.

Lonicerae japonicae flos (LJF) is widely used for the treatment of inflammation-related diseases in traditional Chinese medicine (TCM). To clarify the anti-inflammatory mechanism of LJF, 29 compounds with high content in LJF were selected for network pharmacology. Then, a comprehensive network pharmacology strategy was implemented, which involved compound-inflammation-target construction, protein-protein interaction (PPI) network analysis, and enrichment analysis. Finally, molecular docking and in vitro experiments were performed to verify the anti-inflammatory activity and targets of the key compound. As a result, 279 inflammation-associated proteins were identified, which are mainly involved in the AGE/RAGE signaling pathway in diabetic complications, the HIF-1 signaling pathway, the PI3K-AKT signaling pathway, and EGFR tyrosine kinase inhibitor resistance. A total of 12 compounds were linked to more than 35 targets, including apigenin, kaempferol, quercetin, luteolin, and ferulic acid. The results of molecular docking showed that AKT has the most binding activity, exhibiting certain binding activity with 10 compounds, including vanillic acid, protocatechuic acid, secologanic acid, quercetin, and luteolin; the results of qRT-PCR and WB confirmed that two key compounds, secologanic acid and luteolin, could significantly decrease the secretion of TNF-α and the AKT expression of RAW264.7 murine macrophages stimulated by LPS (lipopolysaccharide). These results demonstrate that the comprehensive strategy can serve as a universal method to illustrate the anti-inflammatory mechanisms of traditional Chinese medicine by identifying the pathways or targets.

Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis.

Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP (green fluorescent protein) reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Co-expression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS greatly activated expression of the GUS (beta-glucuronidase) gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

Salicylic acid activates artemisinin biosynthesis in Artemisia annua L.

This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.

Ameliorating effects of exogenous calcium on the photosynthetic physiology of honeysuckle (Lonicera japonica) under salt stress.

Calcium (Ca2+) plays pivotal roles in modulating plant growth, development and stress responses. This work was conducted to study the effects of 20 mM calcium on the biomass, malondialdehyde content, chlorophyll content, ion ratio, chlorophyll a fluorescence and gas-exchange parameters, gene expression of annual honeysuckle under 50, 100 and 200 mM NaCl. At the end of treatment, Na+ concentration was increased with the mounting salinity, but a higher ratio of K+/Na2+, Ca2+/Na+, Mg2+/Na+ were obtained after calcium addition. Salinity exerted an adverse effect on the dry weights and chlorophyll content, whereas CaCl2 played a positive role. Consistent with biomass reduction, the photosynthetic rate and stomatal conductance declined in leaves of honeysuckle exposed to elevated salinity. However, the extent of reduction was much less under CaCl2 combination treatments than one caused by NaCl treatments. Exogenous calcium also protects the photochemical activity of PSII by protecting reaction centre from inactivation and maintaining electron transport from QA- to QB-. Further, exogenous calcium promoted the overexpression of LHCB coding gene Cab and Rubisco large subunit coding gene rbcL under short-term stress. In conclusion, exogenous calcium was effective in improving the salt tolerance of honeysuckle in the photosynthetic base, thereby improving the growth of plants.

Artemisinin Biosynthesis in Non-glandular Trichome Cells of Artemisia annua.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

Abstract A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats

Co-delivery of baicalein and doxorubicin by hyaluronic acid decorated nanostructured lipid carriers for breast cancer therapy.

PURPOSE: Combination anticancer therapy is promising to generate synergistic anticancer effects, to maximize the treatment effect and to overcome multi-drug resistance. Nanostructured lipid carriers (NLCs), composed of solid and liquid lipids, and surfactants are potentially good colloidal drug carriers. The aim of this study is to construct a hyaluronic acid (HA) decorated NLCs as nanocarriers for co-delivery baicalein (BCL) and doxorubicin (DOX). METHODS: BCL- and DOX-loaded NLCs (BCL/DOX-NLCs) were prepared. HA ligands were used for the decoration of BCL/DOX-NLCs to form HA decorated BCL/DOX-NLCs (HA-BCL/DOX-NLCs). The in vitro cytotoxicity studies of different formulations were evaluated on DOX drug-resistant MCF-7 breast cancer cell line (MCF-7/ADR cells). In vivo anti-tumor effects were observed on the murine bearing MCF-7/ADR cells model. RESULTS: HA-BCL/DOX-NLCs showed highest cytotoxicity and synergistic effect of two drugs in tumor cells in vitro. The in vivo study revealed the greatest anti-tumor activity than all the other formulations in the murine breast cancer model. CONCLUSIONS: HA decorated NLCs could be used as a novel carrier to co-delivery BCL and DOX for breast cancer therapy. HA-BCL/DOX-NLCs could be a promising targeted and combinational therapy nanomedicine.

Biochemical characterization and identification of a cinnamyl alcohol dehydrogenase from Artemisia annua.

It is well known in the literature that cinnamyl alcohol dehydrogenase (CAD) reduces hydroxycinnamyl aldehydes, such as coumaryl, coniferyl, and sinapyl aldehydes, to their corresponding alcohols in the presence of NADPH, and these alcohols act as the precursors of lignin biosynthesis. Here, we report the isolation of a cDNA encoding an NADP(+)-dependent CAD, designated as AaCAD, from the cDNA library using glandular secretory trichomes of Artemisia annua as the source of mRNA. A phylogenetic analysis indicated that AaCAD was clustered with AtCAD4 and AtCAD5, which were involved in monolignol biosynthesis from Arabidopsis. Semi-quantitative RT-PCR showed that the AaCAD transcript was abundant mostly in leaf and root, followed by flower, and lowest in stem. Functional and enzymatic assays showed that the recombinant enzyme was able to reversibly reduce a variety of common CADs substrates, namely geranial, cinnamyl aldehyde, sinapyl aldehyde, coniferyl aldehyde, and a sesquiterpenoid artemisinic aldehyde, to geraniol, cinnamyl alcohol, sinapyl alcohol, coniferyl alcohol, and artemisinic alcohol respectively. Besides, considering that AaCAD was identified from the glandular secretory trichomes of A. annua, and that the recombinant enzyme exhibited reductase activity by using artemisinic aldehyde as substrate, some possible role of AaCAD in artemisinin biosynthesis is also discussed.

Isolation of the 5'-end of plant genes from genomic DNA by TATA-box-based degenerate primers.

We report a rapid and simple method for isolating the 5'-end of plant genes from genomic DNA by polymerase chain reaction (PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed according to the TATA box, which is conserved in the promoter region of most plant genes. The unknown 5'-ends of several genes in different plants were isolated by PCR with gene-specific primers of the known core fragment and the TDPs. Our method does not require the arduous RNA manipulations and expensive enzyme treatments of the popular rapid amplification of cDNA ends (RACE) and its variants, and so could be a cheap practical alternative.

A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum.

A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS "gatekeepers" Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.