Urinary metabolomics reveals potential biomarkers for early detection of pregnancy in Mithun (Bos frontalis) cows.

The present study investigated the urinary metabolic profiles of early pregnant and non-pregnant Mithun to identify potential pregnancy detection biomarkers. Urine samples were collected on days 0, 10, 18, 35 and 45 of gestation from pregnant (n = 6) and on days 0, 10 and 18 from non-pregnant (n = 6) Mithun. Urinary metabolites were assessed using proton nuclear magnetic resonance (1H NMR) spectroscopy and identified 270 metabolites. Statistical analyses demonstrated pronounced distinctions in metabolite profiles between pregnant and non-pregnant samples. Twenty-five metabolites that could discriminate between pregnant and non-pregnant Mithun based on Variable Importance in Projection (VIP) scores >1 were identified. Upon further examination of six metabolites (kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine and leucine) identified with high VIP scores, ROC curve analyses demonstrated their significant predictive potential, with AUC values ranging between 0.50 and 0.85. Additionally, a combined panel of top 25 metabolites yielded an AUC value of 0.85. Pathway analysis identified seven potential metabolic pathway modulations during early gestation, with particular emphasis on phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan pathway and pathways involved in the metabolism of various amino acids. In conclusion, kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine, and leucine show promise as non-invasive urinary biomarkers for early pregnancy detection in Mithun. SIGNIFICANCE: This study presents the first report on the metabolic profile of urine from early pregnant and non-pregnant Mithun (Bos frontalis). The metabolites like kynurenine and its derivatives (kynurenate, 3-hydroxykynurenine and quinolinate), tyrosine and leucine were documented signature urinary metabolites associated with early pregnancy in Mithun. The identified combination of metabolites holds promise as predictive biomarkers for non-invasive urinary-based early pregnancy diagnostics in Mithun. In addition, this study identified changes in metabolic pathways that involve phenylalanine, tyrosine, tryptophan and related amino acids and biomarkers identified were either precursors or products within these metabolic pathways.

Sublobar Resection, Stereotactic Body Radiation Therapy, and Percutaneous Ablation Provide Comparable Outcomes for Lung Metastasis-Directed Therapy.

BACKGROUND: Prolonged survival of patients with metastatic disease has furthered interest in metastasis-directed therapy (MDT). RESEARCH QUESTION: There is a paucity of data comparing lung MDT modalities. Do outcomes among sublobar resection (SLR), stereotactic body radiation therapy (SBRT), and percutaneous ablation (PA) for lung metastases vary in terms of local control and survival? STUDY DESIGN AND METHODS: Medical records of patients undergoing lung MDT at a single cancer center between January 2015 and December 2020 were reviewed. Overall survival, local progression, and toxicity outcomes were collected. Patient and lesion characteristics were used to generate multivariable models with propensity weighted analysis. RESULTS: Lung MDT courses (644 total: 243 SLR, 274 SBRT, 127 PA) delivered to 511 patients were included with a median follow-up of 22 months. There were 47 local progression events in 45 patients, and 159 patients died. Two-year overall survival and local progression were 80.3% and 63.3%, 83.8% and 9.6%, and 4.1% and 11.7% for SLR, SBRT, and PA, respectively. Lesion size per 1 cm was associated with worse overall survival (hazard ratio, 1.24; P = .003) and LP (hazard ratio, 1.50; P < .001). There was no difference in overall survival by modality. Relative to SLR, there was no difference in risk of local progression with PA; however, SBRT was associated with a decreased risk (hazard ratio, 0.26; P = .023). Rates of severe toxicity were low (2.1%-2.6%) and not different among groups. INTERPRETATION: This study performs a propensity weighted analysis of SLR, SBRT, and PA and shows no impact of lung MDT modality on overall survival. Given excellent local control across MDT options, a multidisciplinary approach is beneficial for patient triage and longitudinal management.

Exploring the knowledge, attitudes, and perceptions of women of reproductive age towards fertility and elective oocyte cryopreservation for age-related fertility decline in the UK: a cross-sectional survey.

STUDY QUESTION: What are the knowledge, perceptions and attitudes towards fertility and elective oocyte cryopreservation (OC) for age-related fertility decline (ARFD) in women in the UK? SUMMARY ANSWER: Awareness of OC for ARFD has reportedly improved compared to studies carried out almost a decade ago, but inconsistencies in knowledge remain regarding the rate of miscarriage amongst specific age groups, the financial costs and optimal age to undergo OC for ARFD. WHAT IS KNOWN ALREADY: The age of first-time motherhood has increased amongst western societies, with many women of reproductive age underestimating the impact of age on fertility. Further understanding of women's awareness of their fertility, the options available to preserve it and the barriers for seeking treatment earlier are required in order to prevent the risk of involuntary childlessness. STUDY DESIGN, SIZE, DURATION: A hyperlink to a cross-sectional survey was posted on social media (Instagram) between 25 February 2021 and 11 March 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women from the general population aged 18-50 years were invited to complete the survey. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 5482 women fulfilled the inclusion criteria and completed the survey. The mean age of participants was 35.0 years (SD 10.25; range 16-52). Three quarters (74.1%; n = 4055) disagreed or strongly disagreed they felt well informed regarding the options available to preserve their fertility, in case of a health-related problem or ARFD. The majority overestimated the risk of miscarriage in women aged ≥30 years old, with 14.5% correctly answering 20%, but underestimated the risks in women ≥40, as 20.1% correctly answered 40-50%. Three quarters (73.2%; n = 4007) reported an awareness of OC for ARFD and 65.8% (n = 3605) reported that they would consider undergoing the procedure. The number of women who considered OC for ARFD across age groups were as follows: 18-25 (8.3%; n = 300), 26-30 (35.8%; n = 1289), 31-35 (45.9%; n = 1654), 36-40 (9.6%; n = 347), 41-45 (0.3%; n = 13), and 46-50 (0.1%; n = 2). The majority of women (81.3%; n = 4443) underestimated the cost of a single cycle of OC for ARFD (<£5000). Furthermore, 10.4% (n = 566) believed a single cycle would be adequate enough to retrieve sufficient oocytes for cryopreservation. Approximately 11.0% (n = 599) believed OC for ARFD may pose significant health risks and affect future fertility. Less than half agreed or strongly agreed that the lack of awareness regarding OC for ARFD has impacted the likelihood of pursuing this method of fertility preservation further (41.4%; n = 2259). LIMITATIONS, REASONS FOR CAUTION: Results from cross-sectional studies are limited as interpretations made are merely associations and not of causal relationships. The online nature of participant recruitment is subject to selection bias, considering women with access to social media are often from higher socioeconomic and education backgrounds, thus limiting generalizability of the findings. WIDER IMPLICATIONS OF THE FINDINGS: Further education regarding the financial costs and optimal age to undergo elective OC to increase the chances of successful livebirth are required. Clinicians should encourage earlier fertility counselling to ensure that OC is deemed a preventative measure of ARFD, rather than an ultimate recourse to saving declining fertility. STUDY FUNDING/COMPETING INTEREST(S): No funding was required for this article. There are no conflicts of interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Linking emotional valence and anxiety in a mouse insula-amygdala circuit.

Responses of the insular cortex (IC) and amygdala to stimuli of positive and negative valence are altered in patients with anxiety disorders. However, neural coding of both anxiety and valence by IC neurons remains unknown. Using fiber photometry recordings in mice, we uncover a selective increase of activity in IC projection neurons of the anterior (aIC), but not posterior (pIC) section, when animals are exploring anxiogenic spaces, and this activity is proportional to the level of anxiety of mice. Neurons in aIC also respond to stimuli of positive and negative valence, and the strength of response to strong negative stimuli is proportional to mice levels of anxiety. Using ex vivo electrophysiology, we characterized the IC connection to the basolateral amygdala (BLA), and employed projection-specific optogenetics to reveal anxiogenic properties of aIC-BLA neurons. Finally, we identified that aIC-BLA neurons are activated in anxiogenic spaces, as well as in response to aversive stimuli, and that both activities are positively correlated. Altogether, we identified a common neurobiological substrate linking negative valence with anxiety-related information and behaviors, which provides a starting point to understand how alterations of these neural populations contribute to psychiatric disorders.

Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Introduction of Flipped Classroom Approach for Teaching Embryology to Phase-I MBBS Students.

Embryology topics are conceptual and difficult to understand. In a flipped class set up, the student participates with basic understanding of topic with intent to engage in interactive discussion. The study intends to analyze the impact of the flipped approach in teaching conceptual embryology topics. As the flipped classroom approach for teaching embryology matures, it may fully replace the traditional model of teaching embryology to Phase-I MBBS students. Two hundred forty seven (247) Phase-I MBBS students (Batch 2021) at Government Medical College, Amritsar, Punjab, India were exposed to the flipped class module. Six lectures of embryology were taken in three months using the flipped classroom approach. At the end of each lecture the students in the flipped classroom were tested by MCQ's. All Phase-I MBBS students and 16 Anatomy faculty was provided a feedback form with items based on a five point likert scale at the end of six lectures. The mean rating was determined for each item in feedback form and a qualitative feedback from faculty was obtained by 'interview'. The study with results compiled, was completed in nine months. More than 80.0% students (likert scale response strongly agree and agree) and the entire anatomy teaching faculty provided a favorable feedback. 43.75% from faculty however gave a neutral response regarding acceptability for both slow and fast learners. This probably felt that slow learners were not intrinsically motivated for the flipped class experiment. The faculty interview yielded valuable comments and suggestions. The student and faculty responses indicate that the flipped class approach stimulates a deeper learning process for conceptual embryology topics. This approach facilitates in becoming a 'self directed adult learner' because the students come prepared for an interactive teaching session. The faculty acceptance of this teaching methodology indicates that the flipped approach provides better learning outcomes in teaching embryology.

Optimising the use of physiotherapy resources after manipulation under anaesthetic for frozen shoulder.

INTRODUCTION: Manipulation under anaesthetic (MUA) is a successful treatment for frozen shoulder (FS), and the recovery period and recurrence rates may be reduced by postoperative physiotherapy. This study evaluates two physiotherapy pathways for patients undergoing MUA for FS. METHODS: Between 2016 and 2018, 248 age- and sex-matched patients presented to either a NHS secondary care upper limb service or the lead author's independent practice with a diagnosis of FS. The patients had differential access to postprocedure physiotherapy based on which service they presented to. In Group 1, physiotherapy advice only was given to the patient. In Group 2, supervised hydrotherapy and physiotherapy occurred postoperatively. Pre- and postprocedure Oxford Shoulder Scores (OSS) were collected for each group. Analysis of covariance (ANCOVA) was used to measure the effect of physiotherapy on postoperative OSS. RESULTS: Group 2 showed a significantly greater improvement in postprocedure OSS when compared with Group 1 (18.2 vs 16.7) p<0.001). The estimated maximum effect of physiotherapy on postoperative OSS was an increase of 3.2. CONCLUSION: Following MUA for FS, a statistically significant increase in OSS was detected in patients receiving postprocedure physiotherapy compared with advice alone. There was no difference in recurrence rates. The increase in OSS (3.2) is below the minimal clinically important difference, raising questions regarding the relative importance of postprocedure physiotherapy in a resource-limited environment.

Comparative transcriptome analysis reveals an insight into the candidate genes involved in anthocyanin and scent volatiles biosynthesis in colour changing flowers of Combretum indicum.

Combretum indicum is a widely cultivated ornamental species displaying the distinct phenomenon of floral colour change. Flowers display a gradual colour change from white to red, attributed to increased cyanidin 3-O glucoside in petal tissues. The differently coloured flowers also emanate a complex blend of VOCs with trans-linalool oxide (furanoid) as the major compound in the emission profile. To understand molecular mechanisms regulating floral colour shifts and scent biosynthesis, we performed Illumina transcriptome sequencing, including de novo assembly and functional annotation, for the two stages of floral maturation (white and red). Homology analysis with functional classification identified 84 and 42 candidate genes associated with pigment and scent biosynthesis, respectively. Genes encoding transcription factors, such as MYB, ERF, WD40, WRKY, NAC, bHLH and bZIP, that play critical roles in regulating specialized metabolism were also identified in the transcriptome data. Differences in expression of genes were consistent with accumulation patterns of anthocyanins in the two different flower colours. A clear upregulation of flavonoid biosynthesis genes in red flower tissue is associated with increased pigment content. RT-qPCR-based expression analyses gave results consistent with the RNA-Seq data, suggesting the sequencing data are consistent and reliable. This study presents the first report of genetic information for C. indicum. Gene sequences generated from RNA-Seq, along with candidate genes identified by pathway mapping and their expression profiles, provide a valuable resource for subsequent studies towards molecular understanding of specialized metabolism in C. indicum flowers.

Combinatorial topology and geometry of fracture networks.

A map is proposed from the space of planar surface fracture networks to a four-parameter mathematical space, summarizing the average topological connectivity and geometrical properties of a network idealized as a convex polygonal mesh. The four parameters are identified as the average number of nodes and edges, the angular defect with respect to regular polygons, and the isoperimetric ratio. The map serves as a low-dimensional signature of the fracture network and is visually presented as a pair of three-dimensional graphs. A systematic study is made of a wide collection of real crack networks for various materials, collected from different sources. To identify the characteristics of the real materials, several well-known mathematical models of convex polygonal networks are presented and worked out. These geometric models may correspond to different physical fracturing processes. The proposed map is shown to be discriminative, and the points corresponding to materials of similar properties are found to form closely spaced groups in the parameter space. Results for the real and simulated systems are compared in an attempt to identify crack networks of unknown materials.

Causal discovery of drivers of surface ozone variability in Antarctica using a deep learning algorithm.

The discovery of causal structures behind a phenomenon under investigation has been at the heart of scientific inquiry since the beginning. Randomized control trials, the gold standard for causal analysis, may not always be feasible, such as in the domain of climate sciences. In the absence of interventional data, we are forced to depend only on observational data. This study demonstrates the application of one such causal discovery algorithm using a neural network for identifying the drivers of surface ozone variability in Antarctica. The analyses reveal the overarching influence of the stratosphere on the surface ozone variability in Antarctica, buttressed by the southern annular mode and tropospheric wave forcing in mid-latitudes. We find no significant and robust evidence for the influence of tropical teleconnection on the ground-level ozone in Antarctica. As the field of atmospheric science is now replete with a massive stock of observational data, both satellite and ground-based, this tool for automated causal structure discovery might prove to be invaluable for scientific investigation and flawless decision making.

Comparative Effectiveness Analyses of Salvage Prostatectomy and Salvage Radiotherapy Outcomes Following Focal or Whole-Gland Ablative Therapy (High-Intensity Focused Ultrasound, Cryotherapy or Electroporation) for Localised Prostate Cancer.

AIMS: Ablative therapy, such as focal therapy, cryotherapy or electroporation, aims to treat clinically significant prostate cancer with reduced treatment-related toxicity. Up to a third of patients may require further local salvage treatment after ablative therapy failure. Limited descriptive, but no comparative, evidence exists between different salvage treatment outcomes. The aim of this study was to compare oncological and functional outcomes after salvage robot-assisted radical prostatectomy (SRARP) and salvage radiotherapy (SRT). MATERIALS AND METHODS: Data were collected prospectively and retrospectively on 100 consecutive SRARP cases and 100 consecutive SRT cases after ablative therapy failure in a high-volume tertiary centre. RESULTS: High-risk patients were over-represented in the SRARP group (66.0%) compared with the SRT group (48.0%) (P = 0.013). The median (interquartile range) follow-up after SRARP was 16.5 (10.0-30.0) months and 37.0 (18.5-64.0) months after SRT. SRT appeared to confer greater biochemical recurrence-free survival at 1, 2 and 3 years compared with SRARP in high-risk patients (year 3: 86.3% versus 66.0%), but biochemical recurrence-free survival was similar for intermediate-risk patients (year 3: 90.0% versus 75.6%). There was no statistical difference in pad-free continence at 12 and 24 months between SRARP (77.2 and 84.7%) and SRT (75.0 and 74.0%) (P = 0.724, 0.114). Erectile function was more likely to be preserved in men who underwent SRT. After SRT, cumulative bowel and urinary Radiation Therapy Oncology Group toxicity grade I were 25.0 and 45.0%, grade II were 11.0 and 11.0% and grade III or IV complications were 4.0 and 5.0%, respectively. CONCLUSION: We report the first comparative analyses of salvage prostatectomy and radiotherapy following ablative therapy. Men with high-risk disease appear to have superior oncological outcomes after SRT; however, treatment allocation does not appear to influence oncological outcomes for men with intermediate-risk disease. Treatment allocation was associated with a different spectrum of toxicity profile. Our data may inform shared decision-making when considering salvage treatment following focal or whole-gland ablative therapy.

Dietary flaxseed oil improve boar semen quality, antioxidant status and in-vivo fertility in humid sub-tropical region of North East India.

Environmental stress in the form of high temperature humidity index (THI) in tropical and sub-tropical region negatively affects semen quality and fertility of boar. Therefore, the present study was done to evaluate the effect of supplementing flaxseed oil (FLO) to boar's diet on its semen quality, antioxidant status, fatty acid composition of seminal plasma and fertility under sub-tropical climate. For this purpose, six Hampshire crossbreed (50% Hampshire and 50% Gunghroo) boars were divided into two groups i.e control (CON) and treatment (FLO). In FLO and CON group, flaxseed and vegetable oil, respectively, was top dressed at the rate of 3% in basal diets for each boar on daily basis for 16 weeks during monsoon season. A total of 60 ejaculates, comprising 30 ejaculates from each group (ten ejaculates from each boar) were collected. Semen samples were evaluated for sperm quality parameters (SQPs: motility, viability, abnormality, acrosomal integrity and Hypo-osmotic swelling test) and velocity attributes by computer assisted semen analysis (CASA) at fresh and after 72 h of preservation at 17 °C. Antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC and malondialdehyde; MDA) were analyzed in seminal plasma and serum. Fatty acid compositions of seminal plasma were analyzed by gas chromatography-mass spectrometry (GC-MS). In-vivo fertility study was also conducted. Reaction time and false mounts were significantly (p < 0.05) reduced in FLO group as compared to CON group. Semen quality parameters were significantly (p < 0.05) improved at fresh stage and after 72 h of liquid storage in FLO group as compared to CON group. Velocity attributes (VAP, VSL, VCL, ALH, BCF and LIN) were significantly (p < 0.05) higher in FLO group. Flaxseed oil supplementation significantly (p < 0.01) enhanced serum GPx and CAT concentration. Serum and seminal plasma MDA concentration decreased significantly (p < 0.01) in FLO group. Similarly, GPx, TAC and CAT were significantly (p < 0.01) elevated in seminal plasma of FLO group. The study revealed that feeding of flaxseed oil altered the fatty acid composition of seminal plasma and significantly (p < 0.05) improved the farrowing rate. In summary, flaxseed oil supplementation improved the semen quality parameters and fertility of boars in sub-tropical climate by improving the antioxidant capacity and altering the fatty acid composition of seminal plasma.

A Morphometric Study of the Internal Thoracic Artery and Its Branches.

The clinical importance of the internal thoracic arteries in procedures such as coronary artery bypass grafting, transverse rectus abdominis myocutaneous flaps and minimal incision direct coronary artery bypass surgery is well appreciated. The broad clinical utilization of the internal thoracic artery including the role of its branches in supplying blood to the sternum requires explicit anatomical knowledge of the vessel. The internal thoracic artery and its branches are known to exhibit morphological variations for different ethnic groups. Few studies have provided information regarding structural details and patterns of artery and its branches. The present morphometric study was conducted during year 2019. The internal thoracic artery and its branches were dissected and observed on 100 embalmed adult human cadavers of either sex (200 thoracic halves). The origin, diameter at origin and length of the artery were noted. The branching pattern and variations in branching blueprint were documented in this pioneer study. Mean length of branches of artery and mean distance between anterior intercostal arteries was quantified in this pioneer study. Sternal branches were observed in 87% instances on right side. Incidence of pericardiophrenic branches was 90% cases on left side. The origin of pericardiophrenic artery was found to be in 3rd or 4th intercostal space. The incidence of anterior intercostal arteries arising from musculophrenic artery was 73%. A detailed realization of this branching pattern would definitely allow prevention of sternal necrosis and improve prognosis in internal thoracic artery surgical mobilizations. The quantification of diameter at origin in North Indians would aid in determining central venous pressure, introducing pacemaker and administrating drugs in emergency.

Intraprostatic Cancer Recurrence following Radical Radiotherapy on Transperineal Template Mapping Biopsy: Implications for Focal Ablative Salvage Therapy.

PURPOSE: Men in whom external beam radiotherapy fails are usually placed on delayed hormone therapy. Some of these men have localized recurrence that might be suitable for further local therapy. We describe patterns of recurrence and suitability for focal ablative therapy in those undergoing transperineal template prostate mapping biopsies. MATERIALS AND METHODS: The study included 145 consecutive patients (December 2007 to May 2014) referred with suspicion of recurrence due to rising prostate specific antigen after external beam radiotherapy or brachytherapy who underwent transperineal template prostate mapping biopsies. Suitability for focal ablative therapy required the cancer to be unifocal or unilateral, or bilateral/multifocal with 1 dominant index lesion and secondary lesions with Gleason score 3+3=6 with no more than 3 mm cancer core involvement. RESULTS: Mean patient age was 70.7 (SD 5.8) years. Median prostate specific antigen at time of transperineal template prostate mapping biopsy was 4.5 ng/ml (IQR 2.5-7.7). Overall 75.9% (110) were suitable for a form of focal salvage treatment, 40.7% (59) were suitable for quadrant ablation, 14.5% (21) hemiablation, 14.5% (21) bilateral focal ablation and 6.2% (9) for index lesion ablation. CONCLUSIONS: Three-quarters of patients who have localized radiorecurrent prostate cancer may be suitable for focal ablative therapy to the prostate based on transperineal template prostate mapping biopsies.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

Dynamic trajectories of volatile and non-volatile specialised metabolites in 'overnight' fragrant flowers of Murraya paniculata.

Ephemeral flowers, especially nocturnal ones, usually emit characteristic scent profiles within their post-anthesis lifespans of a few hours. Whether these flowers exhibit temporal variability in the composition and profile of volatile and non-volatile specialised metabolites has received little attention. Flowers of Murraya paniculata bloom in the evenings during the summer and monsoon, and their sweet, intense fragrance enhances the plant's value as an ornamental. We aimed to investigate profiles of both volatile and non-volatile endogenous specialised metabolites (ESM) in nocturnal ephemeral flowers of M. paniculata to examine whether any biochemically diverse groups of ESM follow distinct patterns of accumulation while maintaining synchrony with defensive physiological functions. Targeted ESM contents of M. paniculata flowers were profiled at ten time points at 2-h intervals, starting from late bud stage (afternoon) up to the start of petal senescence (mid-morning). Emitted volatiles were monitored continuously within the whole 20-h period using headspace sampling. The ESM contents were mapped by time point to obtain a highly dynamic and biochemically diverse profile. Relative temporal patterns of ESM accumulation indicated that the active fragrance-emitting period might be divided into 'early bloom', 'mid-bloom' and 'late bloom' phases. Early and late bloom phases were characterised by high free radical generation, with immediate enhancement of antioxidant enzymes and phenolic compounds. The mid-bloom phase was relatively stable and dedicated to maximum fragrance emission, with provision for strong terpenoid-mediated defence against herbivores. The late bloom phase merged into senescence with the start of daylight; however, even the senescent petals continued to emit fragrance to attract diurnal pollinators. Our study suggests that dynamic relations between the different ESM groups regulate the short-term requirements of floral advertisement and phytochemical defence in this ephemeral flower. This study also provided fundamental information on the temporal occurrence of emitted volatiles and internal pools of specialised metabolites in M. paniculata flowers, which could serve as an important model for pollination biology of Rutaceae, which includes many important fruit crops.