Efficient exon skipping by base-editor-mediated abrogation of exonic splicing enhancers.

Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.

Characteristics of plant trait network and its influencing factors in impounded lakes and channel rivers of South-to-North Water Transfer Project, China.

Trait-based approaches have been widely used to evaluate the effects of variable environments on submerged macrophytes communities. However, little research focused on the response of submerged macrophytes to variable environmental factors in impounded lakes and channel rivers of water transfer project, especially from a whole plant trait network (PTN) perspective. Here, we conducted a field survey designed to clarify the characteristic of PTN topology among impounded lakes and channel rivers of the East Route of South-to-North Water Transfer Project (ERSNWTP) and to unravel the effects of determining factors on the PTN topology structure. Overall, our results showed that leaf-related traits and organ mass allocation traits were the hub traits of PTNs in impounded lakes and channel rivers of the ERSNWTP, which traits with high variability were more likely to be the hub traits. Moreover, PTNs showed different structures among impounded lakes and channel rivers, and PTNs topologies were related to the mean functional variation coefficients of lakes and channel rivers. Specially, higher mean functional variation coefficients represented tight PTN, and lower mean functional variation coefficients indicated loose PTN. The PTN structure was significantly affected by water total phosphorus and dissolved oxygen. Edge density increased, while average path length decreased with increasing total phosphorus. Edge density and average clustering coefficient showed significant decreases with increasing dissolved oxygen, while average path length and modularity exhibited significant increases with increasing dissolved oxygen. This study explores the changing patterns and determinants of trait networks along environmental gradients to improve our understanding of ecological rules regulating trait correlations.

Small population of the largest water strider after the late Pleistocene and the implications for its conservation.

Climate oscillation and its synergistic impacts on habitat fragmentation have been identified as threatening the survival of some extant species. However, the mechanisms by which semi-aquatic insects impacted by such events remain poorly understood. Herein, we studied the largest water strider in the world, Gigantometra gigas, to explore the effect of these two factors on its evolutionary history. The sequences of mitogenomic and nrDNA cluster were utilized to reconstruct phylogenetic relationship among G. gigas populations and its demographic history. Mitochondrial genes were separately reconstructed topologies of that populations and detected remarkable differences. We found that G. gigas populations conform to the isolation-by-distance model, and decline occurred at about 120 ka, which was probably influenced by the climate change during the late Pleistocene and eventually maintained a small effective population size (Ne) around 85,717. The populations in Guangdong Province of China are worthy of note in that they exhibit low genetic diversity, a small Ne around 18,899 individuals, and occupy an area with little suitable future habitat for G. gigas. This work recommends that conservation efforts are implemented to ensure the long-term survival of small G. gigas populations, and notes that further evaluation of their extinction risk under the impacts of human activities is required.

Bone marrow mesenchymal stem cells combined with estrogen synergistically promote endometrial regeneration and reverse EMT via Wnt/ß-catenin signaling pathway.

BACKGROUND: Intrauterine adhesion (IUA) is a clinical disease characterized by the uterine cavity occlusion caused by the damage of the endometrial basal layer. Bone marrow mesenchymal stem cells (BMSCs) transplantation have the potential to promote endometrial regeneration mainly through paracrine ability. Estrogen is an indispensable and important factor in the repair of endometrial damage, which has been reported as a promising and adjunctive therapeutic application for stem cell transplantation therapy. This study aims to investigate the synergistic effect of BMSCs and estrogen on improving the endometrial regeneration and restoring the endometrium morphology in a dual damage model of IUA in rabbits and the underlying molecular mechanisms. METHODS: BMSCs were isolated and identified by adipogenic and osteogenic differentiation and flow cytometry assays. The rabbit IUA animal model was established by a dual damage method of mechanical curettage and lipopolysaccharide infection. Additionally, we investigated the therapeutic impact of both BMSCs and estrogen either separately or in combination in a rabbit model. The retention of PKH26-labeled BMSCs was observed by vivo fluorescence imaging.The number of endometrial glands and the degree of fibrosis were observed by H&E and Masson staining respectively. Western blotting, Immunohistochemistry and immunofluorescence staining were performed to detect biomarkers related to endometrial epithelium, endometrial fibrosis and EMT. Finally, the protein expression of core molecules of Wnt/ß-catenin pathway was detected by Western blotting. RESULTS: PKH26-labeled fluorescence results revealed that BMSCs appeared and located in the endometrial glands and extracellular matrix area when orthotopic transplanted into the uterine cavity. Histological assays showed that remarkably increasing the number of endometrial glands and decreasing the area of endometrial fibrosis in the BMSCs combined with estrogen treatment group. Moreover, downregulated expression of fibrosis markers (fibronectin, CollagenI, a-SMA) and interstitial markers (ZEB1, Vimentin, N-cadherin), as well as upregulated E-cadherin expression were found in the combined group. Further study of in vivo staining revealed that fluorescence intensity of CK7 was stronger in the combined group than that of direct BMSCs intrauterine transplantation, while vimentin showed the opposite results. Moreover, the protein levels of ß-catenin, Axin2, C-myc, CycinE of Wnt/ß-catenin signaling pathway increased in the BMSCs combined with estrogen group than in the other treatment groups. CONCLUSION: BMSCs combined with estrogen can promote the differentiation of stem cells into endometrial epithelial cells to facilitate the regeneration of damaged endometrium. The potential mechanism of the synergistic effect may inhibit the occurrence of EMT by activating the Wnt/ß-catenin signaling pathway.

Organoid Transplantation Can Improve Reproductive Prognosis by Promoting Endometrial Repair in Mice.

OBJECTIVE: Intrauterine adhesion (IUA) is one of the major causes of refractory secondary infertility, especially in regions and countries with high abortion rates. In this study, we used the mouse IUA model to evaluate the feasibility of the organoids, a 3D cell structure derived from endometrial tissue, as grafts for the treatment of post-traumatic endometrial regeneration disorders. METHODS: The isolated and cultured endometrial organoid was transplanted into the model IUA uterus by the hydrogel scaffold method. RESULTS: The cultured endometrial organoids were transplanted into the basal layer of the damaged endometrium for 28 days. They were completely implanted and grew normally. They not only reconstructed the structural integrity of the endometrial epithelium but also realized the functional repair of the endometrium through differentiation cultures and secretory functions. CONCLUSION: For severe IUA, this method may be better than stem cell transplantation. These findings provide useful insights into the use of endometrial organoid regeneration in the treatment of injury repair.

Climate Warming Since the Holocene Accelerates West-East Communication for the Eurasian Temperate Water Strider Species Aquarius paludum.

Holocene climate warming has dramatically altered biological diversity and distributions. Recent human-induced emissions of greenhouse gases will exacerbate global warming and thus induce threats to cold-adapted taxa. However, the impacts of this major climate change on transcontinental temperate species are still poorly understood. Here, we generated extensive genomic datasets for a water strider, Aquarius paludum, which was sampled across its entire distribution in Eurasia and used these datasets in combination with ecological niche modeling (ENM) to elucidate the influence of the Holocene and future climate warming on its population structure and demographic history. We found that A. paludum consisted of two phylogeographic lineages that diverged in the middle Pleistocene, which resulted in a "west-east component" genetic pattern that was probably triggered by Central Asia-Mongoxin aridification and Pleistocene glaciations. The diverged western and eastern lineages had a second contact in the Holocene, which shaped a temporary hybrid zone located at the boundary of the arid-semiarid regions of China. Future predictions detected a potentially novel northern corridor to connect the western and eastern populations, indicating west-east gene flow would possibly continue to intensify under future warming climate conditions. Further integrating phylogeographic and ENM analyses of multiple Eurasian temperate taxa based on published studies reinforced our findings on the "west-east component" genetic pattern and the predicted future northern corridor for A. paludum. Our study provided a detailed paradigm from a phylogeographic perspective of how transcontinental temperate species differ from cold-adapted taxa in their response to climate warming.

Analysis of Immune Landscape Reveals Prognostic Significance of Cytotoxic CD4+ T Cells in the Central Region of pMMR CRC.

BACKGROUND: Mismatch repair proficient colorectal cancer (pMMR CRC) lacks effective treatments and has a poor prognosis, which can be attributed to the complexity of tumor microenvironment. The coordinated function of immune cells is vital to anti-tumor immunity. However, the spatial characteristics of immune cells in the pMMR CRC immune microenvironment and their relationship with clinical prognosis are not fully understood. Meanwhile, the immune modulatory effect of neoadjuvant chemotherapy (NCT), which is the first-line treatment of pMMR CRC, needs further investigation. Therefore, this study aims to explore the spatial dynamics of immune cells and its prognostic value in pMMR CRC. METHODS: We analyzed the various immune cells in formalin-fixed, paraffin-embedded tumor tissues which were collected from 77 patients with stage II/III of pMMR CRC, including 39 non-NCT treated and 38 NCT treated patients. We used the optimized multiplex immunohistochemistry (mIHC) to identify and quantify the density, type and location of immune cells in pMMR CRC. Multivariate survival analysis was performed to assess the relationship of immune profiles and clinical prognosis of pMMR CRC patients. RESULTS: The densities of most T cell subsets, B cells and macrophages were higher in the central region of the pMMR CRC than in the invasion margin. Tumor infiltrating lymphocytes (TILs), especially the infiltration of CD4+ GzmB+ T cells in the central region of the tumor was identified to be positively correlated with the prognosis of the patients. Multivariate analysis confirmed that CD4+ GzmB+ T cells population was an independent predictor of disease-free survival (DFS) in non-NCT group. Meanwhile, NCT enhanced the infiltration of CD4+ GzmB+ T cells in the central region of the pMMR CRC, which was also identified as an independent protective factor of overall survival (OS) and DFS in NCT group. CONCLUSION: We demonstrated that the level of CD4+ GzmB+ T cells located in the center of tumor could provide great prognostic value for pMMR CRC patients. And the application of neoadjuvant chemotherapy further improves the infiltration of CD4+ GzmB+ T cells in the central compartment. Further studies into the application of CD4+ GzmB+ T cells in tumor immunotherapy are needed.

Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo.

BACKGROUND: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model. METHODS: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as DmdE4* mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal DmdE4* mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles. RESULTS: DmdE4* mice recapitulated many aspects of human DMD, including shortened life span (by ≈50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the Dmd transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the DmdE4* mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year. CONCLUSIONS: Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).

Lipid Metabolism in Tumor-Infiltrating T Cells.

T cells recognize "foreign" antigens and induce durable humoral and cellular immune responses, which are indispensable for defending pathogens, as well as maintaining the integrity and homeostasis of tissues and organs. T cells are the major immune cell population in the tumor microenvironment which play a critical role in the antitumor immune response and cancer immune surveillance. Defective immune response of tumor-infiltrating T cells is the main cause of cancer immune evasion. The antitumor response of T cells is affected by multiple factors in the tumor microenvironment, including immunosuppressive cells, immune inhibitory cytokines, tumor-derived suppressive signals like PD-L1, immnuogenicity of tumor cells, as well as metabolic factors like hypoxia and nutrient deprivation. Abundant studies in past decades have proved the metabolic regulations of the immune response of T cells and the tumor-infiltrating T cells. In this chapter, we will discuss the regulations of the antitumor response of tumor-infiltrating T cells by lipid metabolism, which is one of the main components of metabolic regulation.

Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling.

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8+ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Are population isolations and declines a threat to island endemic water striders? A lesson from demographic and niche modelling of Metrocoris esakii (Hemiptera: Gerridae).

Genetic stochasticity and bottlenecking in the course of Pleistocene glaciations have been identified as threatening the survival of local endemics. However, the mechanisms by which local endemic species balance the influences of these two events remain poorly understood. Here, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) data set, mined mitochondrial sequences and constructed ecological niche models for the island endemic water strider Metrocoris esakii (Hemiptera: Gerridae). We found that M. esakii comprised three divergent lineages (i.e., north, central and south) isolated by geographical barriers and generally experienced population declines with the constriction of suitable areas during the Last Glacial Maximum (LGM). Further demographic model testing and stairway plots revealed a history of recent gene flow among the neighbouring lineages and rapid recovery at the end of the LGM, indicating that M. esakii at least had the potential for an adaptive response to population fragmentation and bottlenecking. The northern lineage did not show genetic bottlenecking during the LGM, which was probably due to its large effective population size (Ne ) from migration, which improved its adaptive potential. Relative to the ddRAD-seq data set, the demographic results based on mitochondrial sequences were less conclusive, showing weak differentiation and oversimplified demographic trajectories for the three genetic lineages. Overall, this study provides some degree of optimism for the survival of island endemic water striders from a demographic perspective, but further evaluation of their extinction risk under the impacts of human activities is required.

Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.

This protocol uses lipofectamine to deliver base editors (i.e., dCas9 and AIDx fusion protein) and sgRNA expression vectors into Duchenne Muscular Dystrophy (DMD) patient-derived human induced pluripotent stem cells (hiPSCs). This protocol details mutation of the 5' splice site of DMD exon50 with TAM (targeted AID-induced mutagenesis) followed by amplicon-based NGS library preparation for high-throughput sequencing analysis. This protocol can be generalized for base editing in other hIPSCs and for correcting aberrant splicing associated with other genetic diseases. For complete information on the generation and use of this protocol, please refer to Yuan et al. (2018).

The complete mitochondrial genome of Tetraphleps aterrimus (Hemiptera: Anthocoridae): Genomic comparisons and phylogenetic analysis of Cimicomorpha.

The mitochondrial genome (mitogenome) provides important information for better understanding the phylogenetic relationships within heteropteran infraorder Cimicomorpha (Hemiptera: Heteroptera), but there are still limited representations at the family level of Anthocoridae. Here we sequenced the complete mitogenome of Tetraphleps aterrimus. It is 15,803 bp in size, and contains the expected 37 genes (13 PCGs, 22 tRNAs and 2 rRNAs) and control region. Gene order is identical to that of typical cimicomorphans. In comparison with other cimicomorphans, the ratios of Ka/Ks are increasing from 0.17 for COI to 0.85 for ATP8, which demonstrates COI shows the lowest evolutionary rate, while ATP8 appears to be the highest. The ratios of conserved sites of COI is the highest, while ATP8 is the lowest, suggesting that the evolutionary rate of ATP8 is higher than COI. The phylogenetic relationships based on mitogenomes using Bayesian inference (BI) and Maximum likelihood (ML) methods show that Tetraphleps aterrimus is sister to (Orius niger + Orius sauteri), suggesting that Tetraphleps aterrimus belongs to Anthocoridae. The monophyly of each superfamily is generally well supported and Reduvioidea is placed as basal branch in Cimicomorpha. The results support the remaining superfamily groupings (Miroidea + (Cimicoidea + (Velocipedoidea + Nabioidea))).

Phylogeny and historical biogeography of Gondwanan moss-bugs (Insecta: Hemiptera: Coleorrhyncha: Peloridiidae).

The moss bugs of the Peloridiidae, a small group of cryptic and mostly flightless insects, is the only living family in Coleorrhyncha (Insecta: Hemiptera). Today 37 species in 17 genera are known from eastern Australia, New Zealand, New Caledonia and Patagonia, and the peloridiids are thereby a group with a classical southern Gondwanan distribution. To explicitly test whether the present-day distribution of the Peloridiidae actually results from the sequential breakup of southern Gondwana, we provide the first total-evidence phylogenetic study based on morphological and molecular characters sampled from about 75% of recognized species representing 13 genera. The results largely confirm the established morphological phylogenetic context except that South American Peloridium hammoniorum constitutes the sister group to the remaining peloridiids. A timescale analysis indicates that the Peloridiidae began to diversify in the land mass that is today's Patagonia in the late Jurassic (153 Ma, 95% highest posterior density: 78-231 Ma), and that splitting into the three extant well-supported biogeographical clades (i.e. Australia, Patagonia and New Zealand/New Caledonia) is consistent with the sequential breakup of southern Gondwana in the late Cretaceous, indicating that the current transoceanic disjunct distributions of the Peloridiidae are best explained by a Gondwanan vicariance hypothesis.

Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

A mitochondrial genome of Micronectidae and implications for its phylogenetic position.

The mitochondrial genome (mitogenome) has been extensively used to better understand the phylogenetic relationships within the heteropteran infraorder Nepomorpha (Hemiptera), but no mitogenome in Micronectidae has been sequenced to date. Here we describe the first complete mitogenome of Micronecta sahlbergii (Jakovlev, 1881). The mitogenome is 15,005 bp in size, containing 13 typical PCGs, 22 tRNAs, two rRNAs and a control region (CR). All genes are arranged in the same gene order as the most other known heteropteran mitogenome. The phylogenetic relationships based on mitogenomes using Bayesian inference and Maximum likelihood methods showed that Micronecta sahlbergii was sister to Sigara septemlineata, suggesting that Micronecta sahlbergii belongs to Corixoidea. Corixoidea was basal within Nepomorpha. The PCG12 and PCG12RT matrices of BI and ML analyses yielded the consistent topology, respectively. Whereas there was no consistent conclusions in PCG123 and PCG123RT matrices. Saturation tests showed that PCG12 and PCG12RT were free of saturation in evaluation of transition and transversion substitution, while PCG123 and PCG123RT exhibited a plateau revealing saturation of transition suggesting that the third codon positions of PCGs were not suitable for addressing relationships at the superfamily level in Nepomorpha. So our results supported the phylogenetic analysis of PCG12 and PCG12RT in Nepomorpha.

The Prognostic Value of Nanog Overexpression in Lung Cancer: A Meta-Analysis.

BACKGROUND: Recent several studies have showed that the nanog overexpression leads to poor prognosis in some kinds of cancer including hepatocellular carcinoma and gastrointestinal luminal cancer. However, the correlations between prognosis and clinic-pathological features and nanog overexpression in lung cancer are still not well-known. Thus, we performed a meta-analysis to evaluate the role of nanog in lung cancer. METHODS: An electronic retrieval for related studies was conducted in PubMed, Cochrane Library, Web of Science, EMBASE databases, Chinese CNKI, and the Chinese Wan Fang database up to May 2018. The relationships between nanog overexpression and overall survival (OS) and disease-free survival (DFS) as well as clinic-pathological features in lung cancer were investigated. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by STATA12. RESULTS: 11 studies containing 1422 patients were identified in our meta-analysis. The overexpression of nanog showed decreased OS (HR = 1.83, 95% CI = 1.49-2.25, P ≤ 0.001) and DFS (HR = 1.86, 95% CI = 1.2-2.9, P = 0.006). Moreover, overexpression of nanog was significantly related to differentiation (OR = 4.17, 95% CI = 2.17-6.43, P ≤ 0.001), lymph node metastasis (OR = 1.76, 95% CI = 1.06-2.91, P = 0.028) and tumor size (OR = 1.93, 95% CI = 1.17-3.20, P = 0.010), and no correlation with T stage, TNM, stage, and gender. CONCLUSIONS: Our results suggested that nanog overexpression, a hazard factor of differentiation, lymph node metastasis, and tumor size, may predicate decreased OS and DFS for lung cancer.

Composition and antimicrobial activity of the essential oil from the branches of Jacaranda cuspidifolia Mart. growing in Sichuan, China.

This study represents the first report on the chemical composition and antimicrobial activity of the essential oil from the branches of Jacaranda cuspidifolia Mart. Thirty-three compounds were identified by Gas Chromatography-Mass Spectrometry (GC-MS) and the major constituents of the essential oil were Palmitic acid (31.36%), (Z) - 9,17-Octadecadienal (12.06%), Ethyl palmitate (3.81%), Perhydrofarnesyl acetone (2.07%), γ-Maaliene (1.88%), and Cedro (1.42%) and 9,12-Octadecadienoic acid ethyl ester (1.42%). The in vitro antimicrobial activities of the essential oil were evaluated by the disc diffusion method, and the inhibition zones against Escherichia coli, Staphylococcus aureus and Candida albicans were 7.10, 8.20 and 7.25 mm, respectively. The oil showed moderate activities against E. coli, S. aureus and C. albicans with minimum inhibition concentration (MIC) values of 17.3 mg/mL, 12.9 mg/mL and 16.0 mg/mL, respectively.