Nitrogen dioxide exposure, health outcomes, and associated demographic disparities due to gas and propane combustion by U.S. stoves.

Gas and propane stoves emit nitrogen dioxide (NO2) pollution indoors, but the exposures of different U.S. demographic groups are unknown. We estimate NO2 exposure and health consequences using emissions and concentration measurements from >100 homes, a room-specific indoor air quality model, epidemiological risk parameters, and statistical sampling of housing characteristics and occupant behavior. Gas and propane stoves increase long-term NO2 exposure 4.0 parts per billion volume on average across the United States, 75% of the World Health Organization's exposure guideline. This increased exposure likely causes ~50,000 cases of current pediatric asthma from long-term NO2 exposure alone. Short-term NO2 exposure from typical gas stove use frequently exceeds both World Health Organization and U.S. Environmental Protection Agency benchmarks. People living in residences <800 ft2 in size incur four times more long-term NO2 exposure than people in residences >3000 ft2 in size; American Indian/Alaska Native and Black and Hispanic/Latino households incur 60 and 20% more NO2 exposure, respectively, than the national average.

ApCPS2 contributes to medicinal diterpenoid biosynthesis and defense against insect herbivore in Andrographis paniculata.

Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent-labdane-related diterpenoids (ent-LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent-copalyl pyrophosphate (ent-CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent-CPP precursor supply for ent-LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent-CPP-forming diTPS. Further, we elucidated in planta function of the ent-CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent-LRD contents revealed a strong correlation of ApCPS2 expression and ent-LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated ß-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2-silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent-LRD contents and herbivore defense in ApCPS1 or ApCPS4-silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent-CPP for medicinal ent-LRD biosynthesis and defense against insect herbivore.

Investigating behavioral patterns of procrastinators in a Wiki-based activity.

Procrastination is one of the issues affecting more than half of the student population and is known to impact them negatively. It is also one of the major reasons for failure and dropout. Therefore, several studies have been conducted in this domain to understand when and why students procrastinate. The existing studies use self-reported procrastination scales and/or digital traces of student interactions recorded in learning environments to identify procrastination behavior. The majority of the extant studies leverage individual tasks such as assignments submission, quizzes attempted, course materials assessed by a student, etc., to study such behavior. This paper uses group-based collaborative wiki activity to explore the procrastination behavior among the students. This study will help us explore student behavior in a group activity. The results would help us investigate if the student's behavior changes when it comes to a group activity. The results would be beneficial for instructors, practitioners, and educational researchers to know if group activity could be utilized to overcome procrastination behavior.

The Relationship between Mindfulness and Readiness to Change in Alcohol Drinkers.

Mindfulness is a multi-faceted construct that involves paying attention to thoughts and emotions without automatically reacting and being critical of them. Recent research has suggested that mindfulness might play an important role in reducing problematic alcohol use. Further, Readiness to Change (RTC) is related to motivation to change drinking behaviours. The RTC scale identifies motivation to change drinking behaviours including Precontemplation, Contemplation, and Action stages. The current study investigated, for the first time, the relationship between mindfulness (and its facets) and RTC in relation to drinking behaviours. Undergraduate students from Western Sydney University (N = 279) were screened for drinking levels using the Alcohol Use Disorder Identification Test (AUDIT) and then completed the Readiness to Change Questionnaire (RCT) and the Five Facets Mindfulness Questionnaire (FFMQ), which includes the following facets: Acting with Awareness, Non-Judging of Inner Experience, Non-Reactivity to Inner Experience, Describing, and Observing. Results show that overall, mindfulness and its facets negatively correlated with RTC. Multiple regression analysis further showed that Awareness and Non-Judgement facets negatively predicted RTC. These findings provide insight into how the facets of mindfulness interact with the drinking motives of individuals and their intentions to change drinking behaviours. Based on these findings, we recommend the incorporation of mindfulness techniques in interventions targeting problematic drinking.

Mathlete: an adaptive assistive technology tool for children with dyscalculia.

PURPOSE: The use of technology in teaching and learning process for children is gaining importance. Children with specific learning disabilities have shown positive results with assistive technology tools. Dyscalculia is a specific learning disability in which an individual faces problem in mathematical skills. Around 3-7% of the world population is affected with it. The aim of the current research study is to determine the effectiveness of an assistive technology tool, Mathlete on children with dyscalculia's early numeracy skills. Such assistive technology tools can serve as a boon for these children. METHOD: A total of 40 children with dyscalculia (Mage = 6.8 years) were a part of the study. The group of 40 children were randomly divided into two groups: experiment group using mathlete for improving their learning (30 children) and a control group using only traditional teaching for learning (10 children). The children were screened for dyscalculia using the screening module of mathlete. RESULTS: The two groups showed no significant difference in terms of their socio-economic status, gender and parents education level. Children in the experiment group showed improvement from pre-test to post-test after using mathlete. Also, there was significant difference in learning of both the groups, children in experiment group could understand the concepts better than the ones in control group. CONCLUSION: Mathlete helped improve the mathematical learning of children with dyscalculia from pre-test to post-test.

Despite a huge prevalence of dyscalculia globally, this specific learning disability has always been overlooked due to lack of awareness and evident research.Assistive technology in form of computer aided instructions are the need of hour as children with special needs are usually neglected by many EdTech platforms, dyscalculia being a prominent one.An adaptive tool can give the child a customised experience thus developing interest in using the tool.Mathlete, focuses on early numeracy skills among children and helps them work on their deficit areas in their initial stages.

Oxidative Stress: Meeting Multiple Targets in Pathogenesis of Vascular Endothelial Dysfunction.

The vascular endothelium is the innermost lining of blood vessels, which maintains vasoconstriction and vasodilation. Loss of vascular tone is a hallmark for cardiovascular disorders. Numerous factors, such as over-activation of the renin-angiotensin-aldosterone system, kinases, growth factors, etc., play a crucial role in the induction and progression of vascular abrasion. Interestingly, dysregulation of these pathways either enhances the intensity of oxidative stress, or these pathways are affected by oxidative stress. Thus, oxidative stress has been considered a key culprit in the progression of vascular endothelial dysfunction. Oxidative stress induced by reactive oxygen and nitrogen species causes abnormal gene expression, alteration in signal transduction, and the activation of pathways, leading to induction and progression of vascular injury. In addition, numerous antioxidants have been noted to possess promising therapeutic potential in preventing the development of vascular endothelial dysfunction. Therefore, we have focused on current perspectives in oxidative stress signalling to evaluate common biological processes whereby oxidative stress plays a crucial role in the progression of vascular endothelial dysfunction.

UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis.

Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.

Application of virus-induced gene silencing in Andrographis paniculata, an economically important medicinal plant.

Kalmegh [Andrographis paniculata (Burm.f.) Wall. ex Nees] is one of the most studied medicinal plants for pharmaceutical properties and phytochemistry. However, functional genomics studies in kalmegh are so far limited due to the unavailability of a robust tool for gene silencing. Here, we tested the application of virus-induced gene silencing (VIGS) in kalmegh using the well-known Tobacco rattle virus (TRV)-based vectors and achieved targeted silencing of phytoene desaturase (ApPDS) which is essential in plants for carotenoid biosynthesis that protects chlorophyll from photooxidation. ApPDS silencing in kalmegh leaves developed a typical photobleaching phenotype. The silencing of ApPDS was confirmed by analysing ApPDS transcript level and determining chlorophyll content in the leaves of VIGS seedlings. The analysis revealed ~30% reduction in chlorophyll content, and 40 to 60% reduction in ApPDS transcript level in the leaves of VIGS seedlings. These findings clearly demonstrated the applicability of VIGS in kalmegh using TRV-based vectors. The VIGS protocol presented in this study might be useful for studying gene function related to medicinal and agricultural traits in kalmegh.

Comprehensive study on impact assessment of lockdown on overall ambient air quality amid COVID-19 in Delhi and its NCR, India.

Indian government announced the complete lockdown from 25 March, 2020 for all outdoor activities across the country due to containment of COVID-19. This study is an attempt to assess the impacts of lockdown on ambient air quality in five cities of Indian National Capital Region including Delhi, Gurugram, Noida, Ghaziabad and Faridabad. In this context, the data of air pollutants (PM10, PM2.5, NOx, NO, NO2, SO2, NH3, SO2, CO, and C6H6) from 36 locations of the study area were analyzed from 1st March to 1st May, 2020. The results showed that PM10 and PM2.5 level decreased upto 55-65 %. NOx and NO have shown maximum reduction (∼ 50-78 %). Similarly, consistent and significant reduction in other air pollutants such as SO2 (∼33 %), CO (∼45 %), NH3 (∼27 %) and C6H6 (∼53 %) has been observed. During lockdown Air Quality Index (AQI) shows improvement as its value significantly decreased (∼ 45 %-68 %). An interesting feature observed that during first week of lockdown O3 decreased but later it increased by ∼19-27%. The study suggests that this pandemic gives lessons for interventions for urban air pollution mitigation in controlling the health impact due to urban air pollution.

The Great Smog Month and Spatial and Monthly Variation in Air Quality in Ambient Air in Delhi, India.

BACKGROUND: In recent years, poor urban air quality in Delhi, India has gained significant attention. Episodic events including crop stubble burning and Diwali celebrations are considered major factors in the worsening quality of ambient air. OBJECTIVE: This study aimed to investigate spatial and monthly variation as well as the role of episodic events in ambient air quality in Delhi, including the 'Great Smog' month of November 2017. METHODS: Monitoring of air pollutants (particulate matter (PM10, PM2.5, PM1) and nitrogen dioxide (NO2)) was carried out at three distinct locations of Delhi from April 2017-February 2018. The concentration of NO2 was measured using a modified Jacob and Hochheiser method and PM was measured using a GRIMM aerosol spectrometer. Air quality index was also determined to identify the effects of air pollution on human health. RESULTS: Overall, the levels of air pollution were found to be approximately 2.1-3.2 times higher along a traffic intersection and about 1.4-2.0 times higher in a commercial area compared with an institutional area. The highest average monthly concentrations of PM10, PM2.5, PM1 and NO2 were 768, 374, 298 and 149 µg/m3, respectively, during the Great Smog month of November 2017. November and August were recorded as the most polluted and cleanest months, respectively, in the city. Generally, poor to severe categories of the air quality index (AQI) were obtained from October to February. Higher concentrations during November were attributed to stubble burning in the nearby states of Delhi with the additive effect of fireworks during Diwali celebrations. CONCLUSIONS: Severe ambient air quality as observed in the present study is a serious matter of concern for the health of Delhi's population. To control spikes in poor air quality during episodic events, it is imperative to raise awareness among farmers regarding the severe health hazards of stubble burning. COMPETING INTERESTS: The authors declare no competing financial interests.

Virus-Induced Gene Silencing in Sweet Basil (Ocimum basilicum).

Virus-induced gene silencing (VIGS) is a powerful reverse genetic tool for rapid functional analysis of plant genes. Over the last decade, VIGS has been widely used for conducting rapid gene knockdown experiment in plants and played a crucial role in advancing applied and basic research in plant science. VIGS was studied extensively in model plants Arabidopsis and tobacco. Moreover, several non-model plants such as Papaver (Hileman et al., Plant J 44:334-341, 2005), Aquilegia (Gould and Kramer, Plant Methods 3:6, 2007), Catharanthus (Liscombe and O'Connor, Phytochemistry 72:1969-1977, 2011), Withania (Singh et al., Plant Biol J 13:1287-1299, 2015), and Ocimum (Misra et al., New Phytol 214:706-720, 2017) were also successfully explored. We have recently developed a robust protocol for VIGS in sweet basil (Ocimum basilicum). Sweet basil, a popular medicinal/aromatic herb, is being studied for the diversity of specialized metabolites produced in it.

Ability of children to perform touchscreen gestures and follow prompting techniques when using mobile apps.

BACKGROUND: Children today get access to smartphones at an early age. However, their ability to use mobile apps has not yet been studied in detail. PURPOSE: This study aimed to assess the ability of children aged 2-8 years to perform touchscreen gestures and follow prompting techniques, i.e., ways apps provide instructions on how to use them. METHODS: We developed one mobile app to test the ability of children to perform various touchscreen gestures and another mobile app to test their ability to follow various prompting techniques. We used these apps in this study of 90 children in a kindergarten and a primary school in New Delhi in July 2019. We noted the touchscreen gestures that the children could perform and the most sophisticated prompting technique that they could follow. RESULTS: Two- and 3-year-old children could not follow any prompting technique and only a minority (27%) could tap the touchscreen at an intended place. Four- to 6-year-old children could perform simple gestures like a tap and slide (57%) and follow instructions provided through animation (63%). Sevenand 8-year-old children could perform more sophisticated gestures like dragging and dropping (30%) and follow instructions provided in audio and video formats (34%). We observed a significant difference between the number of touchscreen gestures that the children could perform and the number of prompting techniques that they could follow (F=544.0407, P<0.05). No significant difference was observed in the performance of female versus male children (P>0.05). CONCLUSION: Children gradually learn to use mobile apps beginning at 2 years of age. They become comfortable performing single-finger gestures and following nontextual prompting techniques by 8 years of age. We recommend that these results be considered in the development of mobile apps for children.

Transcriptome analysis and functional characterization of oxidosqualene cyclases of the arjuna triterpene saponin pathway.

Arjuna (Terminalia arjuna) tree has been popular in Indian traditional medicine to treat cardiovascular ailments. The tree accumulates bioactive triterpene glycosides (saponins) and aglycones (sapogenins), in a tissue-preferential manner. Oleanane triterpenes/saponins (derived from ß-amyrin) with potential cardioprotective function predominantly accumulate in the bark. However, arjuna triterpene saponin pathway enzymes remain to be identified and biochemically characterized. Here, we employed a combined transcriptomics, metabolomics and biochemical approach to functionally define a suite of oxidosqualene cyclases (OSCs) that catalyzed key reactions towards triterpene scaffold diversification. De novo assembly of 131 millions Illumina NextSeq500 sequencing reads obtained from leaf and stem bark samples led to a total of 156,650 reference transcripts. Four distinct OSCs (TaOSC1-4) with 54-71 % sequence identities were identified and functionally characterized. TaOSC1, TaOSC3 and TaOSC4 were biochemically characterized as ß-amyrin synthase, cycloartenol synthase and lupeol synthase, respectively. However, TaOSC2 was found to be a multifunctional OSC producing both α-amyrin and ß-amyrin, but showed a preference for α-amyrin product. Both TaOSC1 and TaOSC2 produced ß-amyrin, the direct precursor for oleanane triterpene/saponin biosynthesis; but, TaOSC1 transcript expressed preferentially in bark, suggesting a major role of TaOSC1 in the biosynthesis of oleanane triterpenes/saponins in bark.

A comprehensive study on spatio-temporal distribution, health risk assessment and ozone formation potential of BTEX emissions in ambient air of Delhi, India.

The hazardous air pollutants like benzene, toluene, ethylbenzene and xylene (BTEX) are considered as toxic because of their role in ozone formation and adverse effects on human health. Owing to this, the present study was carried out at six spatially distributed sites in Delhi from November 2017- June 2018. Activated charcoal tubes were used to collect samples of BTEX and were further analyzed using GC-FID. The minimum BTEX concentration was found at institutional site (9.94 µg/m3) and maximum at roadside site (103.12 µg/m3) with the average of 46.66 µg/m3. Also, the levels of BTEX were 1.18-1.74 times higher during rush hours as compared to non-rush hours. The high T/B ratio (2.26-3.41) observed is the indication of the traffic-originated sources of emission. The cancer risks calculated for benzene at probability 0.50 ranged as 1.29E-06 - 1.80E-05, whereas 4.09E-06 - 3.40E-05 at probability 0.95, which were higher than the acceptable value of 1.0E-06. The non-cancer health risks in terms of hazard index were observed less than unity i.e. within acceptable limit. The total ozone formation potential (OFP) was obtained as 207.51 ±â€¯123.40 µg/m3 with maximum potential by toluene. Such high levels of BTEX, cancer risks and OFP obtained in the study especially at roadside and connectivity hub are harmful for people residing near these areas, and also to large commuters, who are exposed to such emissions during travelling.

Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis.

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and ß-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

BACKGROUND: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. RESULTS: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs. CONCLUSIONS: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.