MicrobiomeCensus estimates human population sizes from wastewater samples based on inter-individual variability in gut microbiomes.

The metagenome embedded in urban sewage is an attractive new data source to understand urban ecology and assess human health status at scales beyond a single host. Analyzing the viral fraction of wastewater in the ongoing COVID-19 pandemic has shown the potential of wastewater as aggregated samples for early detection, prevalence monitoring, and variant identification of human diseases in large populations. However, using census-based population size instead of real-time population estimates can mislead the interpretation of data acquired from sewage, hindering assessment of representativeness, inference of prevalence, or comparisons of taxa across sites. Here, we show that taxon abundance and sub-species diversisty in gut-associated microbiomes are new feature space to utilize for human population estimation. Using a population-scale human gut microbiome sample of over 1,100 people, we found that taxon-abundance distributions of gut-associated multi-person microbiomes exhibited generalizable relationships with respect to human population size. Here and throughout this paper, the human population size is essentially the sample size from the wastewater sample. We present a new algorithm, MicrobiomeCensus, for estimating human population size from sewage samples. MicrobiomeCensus harnesses the inter-individual variability in human gut microbiomes and performs maximum likelihood estimation based on simultaneous deviation of multiple taxa's relative abundances from their population means. MicrobiomeCensus outperformed generic algorithms in data-driven simulation benchmarks and detected population size differences in field data. New theorems are provided to justify our approach. This research provides a mathematical framework for inferring population sizes in real time from sewage samples, paving the way for more accurate ecological and public health studies utilizing the sewage metagenome.

PcENO3 interacts with patchoulol synthase to positively affect the enzymatic activity and patchoulol biosynthesis in Pogostemon cablin.

Patchouli alcohol, a significant bioactive component of the herbal plant Pogostemon cablin, has considerable medicinal and commercial potential. Several genes and transcription factors involved in the biosynthesis pathway of patchouli alcohol have been identified. However, so far, regulatory factors directly interacting with patchouli synthase (PTS) have not been reported. This study was conducted to analyze the interaction between PcENO3 and PcPTS to explore the molecular regulation effect of PcENO3 on patchouli alcohol biosynthesis. PcENO3, a homologous protein of Arabidopsis ENO3 belonging to the enolase family, was identified and characterized. Subcellular localization experiments in Arabidopsis protoplast cells indicated that the PcENO3 protein was localized in both the cytoplasm and nucleus. The physical interaction between PcENO3 and PcPTS was confirmed through yeast two-hybrid (Y2H), GST pull-down, and bimolecular fluorescence complementation assays. Furthermore, the Y2H assay demonstrated that PcENO3 could also interact with JAZ proteins in the JA pathway. Enzymatic assays showed that the interaction with PcENO3 increased the catalytic activity of patchoulol synthase. Additionally, suppression of PcENO3 expression with VIGS (virus-induced gene silencing) decreased patchouli alcohol content compared to the control. These findings suggest that PcENO3 interacts with patchoulol synthase and modulates patchoulol biosynthesis by enhancing the enzymatic activity of PcPTS.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Full-length transcriptome sequencing and methyl jasmonate-induced expression profile analysis of genes related to patchoulol biosynthesis and regulation in Pogostemon cablin.

BACKGROUND: Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. RESULTS: In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. 'Zhanxiang', and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. CONCLUSIONS: The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. 'Zhanxiang', these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.

PatJAZ6 Acts as a Repressor Regulating JA-Induced Biosynthesis of Patchouli Alcohol in Pogostemon Cablin.

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.

Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3.

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability.

Integrating RNA-seq and ChIP-seq data to characterize long non-coding RNAs in Drosophila melanogaster.

BACKGROUND: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs. RESULTS: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not. CONCLUSIONS: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

Chromosome mapping, molecular cloning and expression analysis of a novel gene response for leaf width in rice.

Genetic analysis revealed that narrow leaf, small panicle, thin and slender stems as well as low fertility rate of an Indica rice variety were recessive traits and controlled by a single gene. Applying map-based cloning strategy, a novel narrow leaf gene, which was named nal11 was delimited to an interval of 58.3 kb between the InDel markers N10 and InD5016. There are 9 genes in the mapping interval, and only a heat shock DNAJ protein encode gene (Os07g09450) has a specific G to T SNP, which was occurred at the last base of the second exon of Os07g09450 in ZYX. 5' and 3' RACE result shown that there were two transcripts in NAL11, and the SNP in nal11 leads to a variable shear of mRNA. In addition, this type of mRNA alternative splicing together with a stop codon closely followed the SNP which caused termination of translation destroyed the DNAJ domain of nal11's product. These results suggested that the heat shock DNAJ gene was most likely to be the candidate gene of nal11. The results of RT-PCR and real-time PCR further verified that the SNP in the ZYX-nal11 gene affects mRNA splicing pattern. Phenotype of ZYX may be caused by a statistically significant reduction in the total number of small veins in leaf, size and number of small vascular bundles and cells in stems, similar to several previous reported mutations. The basic molecular information we provide here will be useful for further investigations of the physiological function of the heat shock DNAJ gene, which will be helpful in better understanding the role of the DNAJ family in regulation of plant type traits such as leaf width of rice.

Rapid Osseointegration of Titanium Implant With Innovative Nanoporous Surface Modification: Animal Model and Clinical Trial.

OBJECTIVES: SLAffinity is the hybrid topography consisting of micropits and nanoporous TiO2 layers through electrochemical oxidation to mimic the natural bony environment. The aim of this study was to examine the rate of osseointegration in animal models and to further investigate the stability for implants with SLAffinity-treated surface in the clinical trial. MATERIALS AND METHODS: Implants were installed in the mandibular canine-premolar area of 12 miniature pigs. Each pig received 2 implants with the same shapes but with different chemical surfaces. In the clinical trial, 25 patients were included. Each patient received 1 SLAffinity-treated implant on the posterior area of either arch. Resonance frequency analysis and computed tomography were assessed weekly over the first 12 weeks after implant placement. RESULTS: The results found that surface treatment did affect the bone-to-implant contact (BIC) significantly. Comparison of BIC at 3 weeks in animal study showed that the SLAffinity-treated implants presented significantly higher values than machine surface implants. SLAffinity-treated implants also proved clinically successful through 12 months, ready for prosthodontic restoration. CONCLUSION: The effect of SLAffinity treatments enhanced osseointegration significantly, especially at early stages of bone healing. Clinical trial finding, furthermore, ensured that the SLAffinity treatment was a reliable surface modification alternative.

Clinical treatment outcomes for 40 patients with bisphosphonates-related osteonecrosis of the jaws.

BACKGROUND/PURPOSE: Bisphosphonates (BPs) are used to treat osteoporosis and bone metastases from malignancy. They may result in BPs-related osteonecrosis of the jaws (BRONJ) in a subset of patients receiving BPs. This study examined whether conservative or aggressive surgical approach could result in successful treatment of BRONJ lesions and assessed whether concomitant steroid administration or tobacco smoking habit might hinder the remission of BRONJ lesions. METHODS: The 40 BRONJ patients were evenly divided into four different groups. Group 1 contained 10 patients with concomitant corticosteroid medication but without smoking habit. Group 2 contained 10 patients with smoking habit but without concomitant corticosteroid medication. Groups 3 and 4 each consisted of 10 patients without concomitant corticosteroid medication and smoking habit. To avoid bias, each group contained equal number of patients with different stages of BRONJ. Patients in Groups 1, 2, and 3 received conservative treatment, including antibiotic coverage, antibacterial solution irrigation, and minor surgical debridement. Patients in Group 4 were treated with aggressive surgical excision of necrotic bone segment. RESULTS: The mean duration to achieve complete remission of BRONJ lesion was 19.7±0.6, 18.2±0.5, 13.0±1.0, and 7.6±1.1 months for patients in Groups 1, 2, 3 and 4, respectively. Student's t-test showed significant differences in the mean duration to achieve complete remission of BRONJ lesion between Groups 1 and 3, between Groups 2 and 3, between Groups 3 and 4, between Groups 1 and 4, and between Groups 2 and 4 (all p values < 0.001). CONCLUSION: Although both conservative and aggressive surgical approaches can result in successful treatment of BRONJ lesions, aggressive surgical treatment needs a shorter mean duration to achieve complete remission of BRONJ lesion than conservative treatment. Concomitant corticosteroid administration or tobacco smoking may prolong the duration for complete remission of BRONJ lesion.

Volatilome and flavor analyses based on e-nose combined with HS-GC-MS provide new insights into ploidy germplasm diversity in Platostoma palustre.

Platostoma palustre (Mesona chinensis Benth or Hsian-tsao, also known as "Xiancao" in China), is an edible and medicinal plant native to India, Myanmar, and Indo-China. It is the main ingredient in the popular desserts Hsian-tsao tea, herbal jelly, and sweet herbal jelly soup. P. palustre is found abundantly in nutrient-rich substances and possesses unique aroma compounds. Variations in the contents of volatile compounds among different germplasms significantly affect the quality and flavor of P. palustre, causing contradiction in demand. This study investigates the variation in the volatile compound profiles of distinct ploidy germplasms of P. palustre by utilising headspace gas chromatography-mass spectrometry (HS-GC-MS) and an electronic nose (e-nose). The results showed significant differences in the aroma characteristics of stem and leaf samples in diverse P. palustre germplasms. A total of sixty-seven volatile compounds have been identified and divided into ten classes. Six volatile compounds (caryophyllene, α-bisabolol, benzaldehyde, ß-selinene, ß-elemene and acetic acid) were screened as potential marker volatile compounds to discriminate stems and leaves of P. palustre. In this study, leaves of P. palustre showed one odor pattern and stems showed two odor patterns under the influence of α-bisabolol, acetic acid, and butyrolactone. In addition, a correlation analysis was conducted on the main volatile compounds identified by HS-GC-MS and e-nose. This analysis provided additional insight into the variations among samples resulting from diverse germplasms. The present study provides a valuable volatilome, and flavor, and quality evaluation for P. palustre, as well as new insights and scientific basis for the development and use of P. palustre germplasm resources.

Antibacterial nanostructured composite films for biomedical applications: microstructural characteristics, biocompatibility, and antibacterial mechanisms.

Hydrogenated Cu-incorporated diamond-like carbon (a-C:H/Cu) films were prepared in the present study using a radio-frequency plasma magnetron sputtering system at various CH4/Ar gas ratios. The a-C:H/Cu films were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, transmission electron microscopy, nano-indentation and a contact angle goniometer. The antibacterial properties and cell cytotoxicity of a-C:H/Cu films were evaluated as per JIS Z2801:2010 and ISO 10993-5 specifications, respectively. The analytical results revealed that the production of a-C:H/Cu films varied with the CH4/Ar ratio, and the phase transformation (amorphous-like → nano-polycrystalline structure) was induced by Cu doping/ion bombardment and radical reactions. Moreover, it was found that the microhardness of the a-C:H/Cu films decreased with increasing Ar fraction in the gas ratio. The a-C:H/Cu films exhibited a high hydrophobic surface feature. The film which contained 77.3 ± 4.4 at.% Cu did not influence cell adhesion and proliferation behaviors. Antibacterial tests also demonstrated that a-C:H/Cu films possessed excellent antibacterial properties. Therefore, a-C:H/Cu films could be developed as promising antibacterial coatings for biomedical applications.

GC-MS and UHPLC-QTOFMS-assisted identification of the differential metabolites and metabolic pathways in key tissues of Pogostemon cablin.

Pogostemon cablin is an important aromatic medicinal herb widely used in the pharmaceutical and perfume industries. However, our understanding of the phytochemical compounds and metabolites within P. cablin remains limited. To our knowledge, no integrated studies have hitherto been conducted on the metabolites of the aerial parts of P. cablin. In this study, twenty-three volatile compounds from the aerial parts of P. cablin were identified by GC-MS, predominantly sesquiterpenes. Quantitative analysis showed the highest level of patchouli alcohol in leaves (24.89 mg/g), which was 9.12 and 6.69-fold higher than in stems and flowers. UHPLC-QTOFMS was used to analyze the non-volatile compounds of leaf, stem and flower tissues. The differences in metabolites between flower and leaf tissues were the largest. Based on 112, 77 and 83 differential metabolites between flower-leaf, flower-stem and leaf-stem, three tissue-specific biomarkers of metabolites were identified, and the differential metabolites were enriched in several KEGG pathways. Furthermore, labeling differential metabolites in the primary and secondary metabolic pathways showed that flowers accumulated more lipids and amino acids, including proline, lysine and tryptophan; the leaves accumulated higher levels of terpenoids, vitamins and flavonoids, and stems contained higher levels of carbohydrate compounds. Based on the role of acetyl coenzyme A, the distribution and possible exchange mechanism of metabolites in leaves, stems and flowers of P. cablin were mapped for the first time, laying the groundwork for future research on the metabolites in P. cablin and their regulatory role.

Silicate ions as soluble form of bioactive ceramics alleviate aortic aneurysm and dissection.

Aortic aneurysm and dissection (AAD) are leading causes of death in the elderly. Recent studies have demonstrated that silicate ions can manipulate multiple cells, especially vascular-related cells. We demonstrated in this study that silicate ions as soluble form of bioactive ceramics effectively alleviated aortic aneurysm and dissection in both Ang II and ß-BAPN induced AAD models. Different from the single targeting therapeutic drug approaches, the bioactive ceramic derived approach attributes to the effect of bioactive silicate ions on the inhibition of the AAD progression through regulating the local vascular microenvironment of aorta systematically in a multi-functional way. The in vitro experiments revealed that silicate ions did not only alleviate senescence and inflammation of the mouse aortic endothelial cells, enhance M2 polarization of mouse bone marrow-derived macrophages, and reduce apoptosis of mouse aortic smooth muscle cells, but also regulate their interactions. The in vivo studies further confirm that silicate ions could effectively alleviate senescence, inflammation, and cell apoptosis of aortas, accomplished with reduced aortic dilation, collagen deposition, and elastin laminae degradation. This bioactive ceramic derived therapy provides a potential new treatment strategy in attenuating AAD progression.

Blood responses to titanium surface with TiO2 nano-mesh structure.

OBJECTIVES: The goal of this study was to enhance the blood responses to titanium (Ti) surfaces used for dental implant application through the formation of a TiO2 nano-mesh surface layer produced by a fast electrochemical anodization treatment. MATERIAL AND METHODS: Electrochemical anodization treatments with different anodization currents and temperatures in an alkaline solution were used to create a nano-mesh oxide layer on polished Ti surface. Surface characterizations of the mesh structure were carried out using thin-film X-ray diffractometer, field-emission scanning electron microscope, and atomic force microscope. The blood responses, including the blood-clotting ability and platelet adhesion morphology, to the test Ti surfaces were evaluated. The blood-clotting ability, in terms of optical density of blood, was statistically analyzed using a nonparametric method, Kruskal-Wallis test, for the factor of anodization treatment. RESULTS: A multilayer TiO2 nano-mesh structure was rapidly formed on the polished Ti surface using a simple electrochemical anodization treatment in an alkaline solution. The TiO2 nano-mesh had an average mesh size between 34 and 93 nm, depending on the anodization current and temperature. The features on the TiO2 nano-mesh structure on the anodized Ti surface were of a similar size scale as blood proteins, giving the material better blood clot ability (P<0.05) and improved platelet activation and aggregation as compared with an untreated polished Ti surface. CONCLUSIONS: The formation of TiO2 nano-mesh on the Ti surfaces was shown to enhance blood responses, which we expect to promote cell growth in the application of dental implants.

Lycopene attenuates oxidative stress-induced hepatic dysfunction of insulin signal transduction: involvement of FGF21 and mitochondria.

Lycopene (LYC) has been regarded as a nutraceutical that has powerful antioxidant and hepatoprotective bioactivities. In the present study, we aimed to investigate the beneficial effects of LYC on hepatic insulin signal transduction under oxidative stress conditions and the possible involvement of FGF21 and mitochondria pathways. Two-month-old CD-1 mice were treated by intraperitoneal injection of D-galactose (D-gal) 150 mg/kg/day for 8 weeks and received 0.03% LYC (w/w, mixed into diet). The results showed that LYC increased the expression of FGF21, alleviated mitochondrial dysfunction and improved hepatic insulin signal transduction in D-gal-treated mice. Furthermore, knockdown of FGF21 by small interfering RNA notably suppressed mitochondrial function and blunted LYC-stimulated insulin signal transduction in H2O2-treated HepG2 cells. Moreover, suppressed mitochondrial function via oligomycin also inhibited insulin signal transduction, indicating that LYC supplementation ameliorated oxidative stress-induced hepatic dysfunction of insulin signal transduction by up-regulating FGF21 and enhancing mitochondrial function.

PatDREB Transcription Factor Activates Patchoulol Synthase Gene Promoter and Positively Regulates Jasmonate-Induced Patchoulol Biosynthesis.

The production of patchoulol in the patchouli (Pogostemon cablin) plant determines its application value, as it is the principal active sesquiterpene of essential oil extracted from this plant. Here, the promoter of patchoulol synthase gene (PatPTSpro) was isolated and found to be methyl jasmonate (MeJA)-induced. A nucleus-localized AP2/ERF transcription factor PatDREB was identified as a transcription activator binding to PatPTSpro, regulating patchoulol biosynthesis through modulating the gene expression. PatDREB also interacts with jasmonate ZIM-domain 4 (JAZ4). Furthermore, PatDREB could physically interact with the MYB-related transcription factor PatSWC4 and synergistically facilitate patchoulol biosynthesis. However, the transcriptional activation activity of the PatDREB-PatSWC4 complex could be inhibited by PatJAZ4, and JA could reverse this interference. Overall, we demonstrated the positive roles of PatDREB and the PatDREB-PatSWC4 complex in regulating patchoulol production, which advance our understanding of the regulatory network of patchoulol biosynthesis.

Self-organization of zinc ions with a photosensitizer in vivo for enhanced antibiofilm and infected wound healing.

Antimicrobial materials have been developed to combat bacteria more effectively and promote infected wound healing. However, it is widely recognized that the potential toxic effects and complexity of the synthesis process hinder their practical applications. In this work, we introduced a strategy for fighting bacteria and promoting wound healing caused by Staphylococcus epidermidis (S. epidermidis) infection by the self-combination of Zn2+ and clinically applied 5-aminolevulinic acid hydrochloride (ALA) in the microbes. The clinical ALA could target and accumulate in the biofilm as well as contribute to the low-dose Zn2+ penetrating the biofilm due to the self-organized formation of Zn protoporphyrin IX in situ. Upon exposing to a 635 nm laser, the self-combination of ALA and Zn2+ significantly inhibited and eliminated the S. epidermidis biofilm via a synergistic biofilm eradication mechanism that enhanced photodynamic inactivation and aggravated cell wall/membrane disruption. In addition, the combination of ALA and Zn2+ could accelerate wound repair and reduce inflammatory response without causing cytotoxicity. The proposed strategy in this study illustrates the clinical prospects of eradicating biofilms and repairing infected wounds and demonstrates good biocompatibility towards infectious diseases.

Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol.

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin. In this study, the full-length cDNA of the FPP synthase gene from P. cablin (PcFPPS) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene (PcPTS), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.