Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Pathogen-induced biosynthetic pathways encode defense-related molecules in bread wheat.

Wheat is a widely grown food crop that suffers major yield losses due to attack by pests and pathogens. A better understanding of biotic stress responses in wheat is thus of major importance. The recently assembled bread wheat genome coupled with extensive transcriptomic resources provides unprecedented new opportunities to investigate responses to pathogen challenge. Here, we analyze gene coexpression networks to identify modules showing consistent induction in response to pathogen exposure. Within the top pathogen-induced modules, we identify multiple clusters of physically adjacent genes that correspond to six pathogen-induced biosynthetic pathways that share a common regulatory network. Functional analysis reveals that these pathways, all of which are encoded by biosynthetic gene clusters, produce various different classes of compounds­namely, flavonoids, diterpenes, and triterpenes, including the defense-related compound ellarinacin. Through comparative genomics, we also identify associations with the known rice phytoalexins momilactones, as well as with a defense-related gene cluster in the grass model plant Brachypodium distachyon. Our results significantly advance the understanding of chemical defenses in wheat and open up avenues for enhancing disease resistance in this agriculturally important crop. They also exemplify the power of transcriptional networks to discover the biosynthesis of chemical defenses in plants with large, complex genomes.

Biosynthesis of saponin defensive compounds in sea cucumbers.

Soft-bodied slow-moving sea creatures such as sea stars and sea cucumbers lack an adaptive immune system and have instead evolved the ability to make specialized protective chemicals (glycosylated steroids and triterpenes) as part of their innate immune system. This raises the intriguing question of how these biosynthetic pathways have evolved. Sea star saponins are steroidal, while those of the sea cucumber are triterpenoid. Sterol biosynthesis in animals involves cyclization of 2,3-oxidosqualene to lanosterol by the oxidosqualene cyclase (OSC) enzyme lanosterol synthase (LSS). Here we show that sea cucumbers lack LSS and instead have two divergent OSCs that produce triterpene saponins and that are likely to have evolved from an ancestral LSS by gene duplication and neofunctionalization. We further show that sea cucumbers make alternate sterols that confer protection against self-poisoning by their own saponins. Collectively, these events have enabled sea cucumbers to evolve the ability to produce saponins and saponin-resistant sterols concomitantly.

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.

Oxidosqualene cyclase and CYP716 enzymes contribute to triterpene structural diversity in the medicinal tree banaba.

Pentacyclic triterpenes (PCTs) represent a major class of bioactive metabolites in banaba (Lagerstroemia speciosa) leaves; however, biosynthetic enzymes and their involvement in the temporal accumulation of PCTs remain to be studied. We use an integrated approach involving transcriptomics, metabolomics and gene function analysis to identify oxidosqualene cyclases (OSCs) and cytochrome P450 monooxygenases (P450s) that catalyzed sequential cyclization and oxidative reactions towards PCT scaffold diversification. Four monofunctional OSCs (LsOSC1,3-5) converted the triterpene precursor 2,3-oxidosqualene to either lupeol, ß-amyrin or cycloartenol, and a multifunctional LsOSC2 formed α-amyrin as a major product along with ß-amyrin. Two CYP716 family P450s (CYP716A265, CYP716A266) catalyzed C-28 oxidation of α-amyrin, ß-amyrin and lupeol to form ursolic acid, oleanolic acid and betulinic acid, respectively. However, CYP716C55 catalyzed C-2α hydroxylation of ursolic acid and oleanolic acid to produce corosolic acid and maslinic acid, respectively. Besides, combined transcript and metabolite analysis suggested major roles for the LsOSC2, CYP716A265 and CYP716C55 in determining leaf ursane and oleanane profiles. Combinatorial expression of OSCs and CYP716s in Saccharomyces cerevisiae and Nicotiana benthamiana led to PCT pathway reconstruction, signifying the utility of banaba enzymes for bioactive PCT production in alternate plant/microbial hosts that are more easily tractable than the tree species.

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.

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.

Methyl jasmonate-elicited transcriptional responses and pentacyclic triterpene biosynthesis in sweet basil.

Sweet basil (Ocimum basilicum) is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments. Although a variety of secondary metabolites with potent biological activities are identified, our understanding of the biosynthetic pathways that produce them has remained largely incomplete. We studied transcriptional changes in sweet basil after methyl jasmonate (MeJA) treatment, which is considered an elicitor of secondary metabolites, and identified 388 candidate MeJA-responsive unique transcripts. Transcript analysis suggests that in addition to controlling its own biosynthesis and stress responses, MeJA up-regulates transcripts of the various secondary metabolic pathways, including terpenoids and phenylpropanoids/flavonoids. Furthermore, combined transcript and metabolite analysis revealed MeJA-induced biosynthesis of the medicinally important ursane-type and oleanane-type pentacyclic triterpenes. Two MeJA-responsive oxidosqualene cyclases (ObAS1 and ObAS2) that encode for 761- and 765-amino acid proteins, respectively, were identified and characterized. Functional expressions of ObAS1 and ObAS2 in Saccharomyces cerevisiae led to the production of ß-amyrin and α-amyrin, the direct precursors of oleanane-type and ursane-type pentacyclic triterpenes, respectively. ObAS1 was identified as a ß-amyrin synthase, whereas ObAS2 was a mixed amyrin synthase that produced both α-amyrin and ß-amyrin but had a product preference for α-amyrin. Moreover, transcript and metabolite analysis shed light on the spatiotemporal regulation of pentacyclic triterpene biosynthesis in sweet basil. Taken together, these results will be helpful in elucidating the secondary metabolic pathways of sweet basil and developing metabolic engineering strategies for enhanced production of pentacyclic triterpenes.

Effect of Moderate Aerobic Exercise Training on Autonomic Functions and its Correlation with the Antioxidant Status.

Aerobic exercise is a proven measure to enhance the cardio-respiratory efficiency. This improvement isprimarily due to improvement in autonomic function where in there is increase in the parasympathetic function and decrease in sympathetic function. Exercise also affects many metabolic activities in our body and may be one of the factors which reduce the oxidative stress. The aim of the present study was to see the effect of moderate aerobic exercise training on autonomic function and its correlation with antioxidant status. 30 healthy volunteers in the age group of 18-22 years were screened. Autonomic function tests included activity (tone) and reactivity parameters. Antioxidant status was assessed by the level of malondialdehyde in plasma. We observed a significant change in SBP, DBP, LF nu, HF nu, delta value of DBP in CPT and MDA. Our findings are consistent with earlier findings that short duration physical training is known to reduce blood pressure and that there is a relationship between HF (in HRV) and training response Physical exercise also provides a favorable change in the biochemical parameters such as MDA.

Effect of stress on academic performance in medical students--a cross sectional study.

INTRODUCTION: Stress, a universal phenomenon, affects an individual's productivity either by increasing it ('eustress') or decreasing it ('distress'). It is widely acknowledged that the medical fraternity is predisposed to enormous stress. The same may be true for the budding medicos- the undergraduate medical students. In our study we attempted to identify situations that predisposed the medical students to stress and their effects on academic performance and to suggest certain coping mechanisms. OBJECTIVES: firstly to explore common sources of stress in medical students, secondly to establish correlation of stress, gender, attendance, and academic performance if any. METHOD: 114 medical undergraduates were assessed for the common sources of stress and the level of stress using semi structured Performa and stress scale. The results were compared and correlated with various variables like attendance, demographic factors, average marks etc. Pearson correlation coefficient was used for statistical correlation amongst different variables. RESULTS & CONCLUSIONS: Stress shows beneficial effects in females when compared to males. High attendance and better day to day performance in female medical students was associated with more amount of stress when compared to male students. Thus, stress among medical students should be acknowledged and attempts should be made to alleviate it.

Correction: Altered Heart Rate Variability During Rest in Schizophrenia: A State Marker.

[This corrects the article DOI: 10.7759/cureus.44145.].

Comparative study of effect of anuloma-viloma (pranayam) and yogic asanas in premenstrual syndrome.

The present study was planned to investigate the effects of Anuloma-viloma and specific yogic asanas in Premenstrual syndrome (PMS). The study group comprised of 60 females suffering from PMS between the age group of 18- 40 years, having 28-34 days regular menstrual cycle, further subdivided into 3 groups having equal number (n = 20) of subjects-group A (no intervention), group B (Anuloma-viloma) and group C (yogic asanas). Age-matched 30 healthy female subjects were taken as control. In all the subjects, a baseline recording of the systolic (SBP) and diastolic blood pressure (DBP) from the right arm was taken using an automated sphygmomanometer. The heart rate (HR/min), electromyogram (EMG; mV), galvanic skin response (GSR; komega), respiratory rate (RR/min), peripheral temperature (T; degrees F), were recorded simultaneously, on an automated biofeedback apparatus Relax 701. The subjects of group A and group B performed yogic exercises, regularly for 7 days prior to the expected date of menstruation for 3 consecutive menstrual cycles. The parameters were recorded again at the end of 7 days in each menstrual cycle. We observed that, in the group A and group B, HR, SBP, DBP, EMG, GSR and RR showed a very significant reduction (P < 0.001) and T rose more significantly (P < 0.001) after the 3rd menstrual cycle, when compared with their basal levels. On computing the percentage difference between the baseline and post values in all the three groups and than comparing this percentage difference, we found a y significant difference (P < 0.05) between the groups. In the present study, the relaxation response in the females suffering from PMS showed a reduction in an abnormally high basal sympathetic activity and a heightened relaxation response in both the study groups (group B and Group C) in comparison with group A.

Altered Heart Rate Variability During Rest in Schizophrenia: A State Marker.

BACKGROUND: Autonomic nervous system (ANS) imbalance has been reported in a number of psychiatric disorders such as depression, schizophrenia, panic disorder, etc. Autonomic dysfunction in schizophrenia has been associated with the symptoms and manifestation of psychosis. Heart rate variability (HRV) as a tool has been widely used to assess ANS activity and the effect of disease on the sympathovagal balance. Therefore, in the present study, HRV derived from electrocardiogram (ECG) lead II at rest was investigated in order to understand the changes in frequency domain measures in patients with schizophrenia and their first-degree relatives compared to healthy controls. METHODS: Twenty-five patients with schizophrenia, 24 first-degree relatives of patients, and 24 healthy controls (Diagnostic and Statistical Manual of Mental Disorders (DSM)-5; 18-45 years) were included in the study. HRV of the subjects was measured after five minutes of rest. ECG lead II was recorded for five minutes and HRV was analysed in the frequency domain: low frequency (LF), high frequency (HF), total power, and LF/HF ratio. HRV parameters and heart rate were statistically analysed for group comparisons using general linear model multivariate analysis. RESULTS: Patients had significantly higher minimum heart rate and lower HF (normalized units (nu)) compared to their first-degree relatives. A trend was observed in HF (nu) with the lowest in patients followed by healthy controls and first-degree relatives and LF/HF ratio was the highest in patients followed by healthy controls and first-degree relatives, although not statistically significant. No significant difference was found between first-degree relatives and healthy controls. CONCLUSION: The alteration of HRV in schizophrenia could be attributed to reduction in vagal tone and sympathetic dominance, which in turn could serve as state markers of schizophrenia.

Discovery of isoflavone phytoalexins in wheat reveals an alternative route to isoflavonoid biosynthesis.

Isoflavones are a group of phenolic compounds mostly restricted to plants of the legume family, where they mediate important interactions with plant-associated microbes, including in defense from pathogens and in nodulation. Their well-studied health promoting attributes have made them a prime target for metabolic engineering, both for bioproduction of isoflavones as high-value molecules, and in biofortification of food crops. A key gene in their biosynthesis, isoflavone synthase, was identified in legumes over two decades ago, but little is known about formation of isoflavones outside of this family. Here we identify a specialized wheat-specific isoflavone synthase, TaCYP71F53, which catalyzes a different reaction from the leguminous isoflavone synthases, thus revealing an alternative path to isoflavonoid biosynthesis and providing a non-transgenic route for engineering isoflavone production in wheat. TaCYP71F53 forms part of a biosynthetic gene cluster that produces a naringenin-derived O-methylated isoflavone, 5-hydroxy-2',4',7-trimethoxyisoflavone, triticein. Pathogen-induced production and in vitro antimicrobial activity of triticein suggest a defense-related role for this molecule in wheat. Genomic and metabolic analyses of wheat ancestral grasses further show that the triticein gene cluster was introduced into domesticated emmer wheat through natural hybridization ~9000 years ago, and encodes a pathogen-responsive metabolic pathway that is conserved in modern bread wheat varieties.

Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

Comparison of cardiac autonomic activity between malnourished and healthy children.

Severe malnutrition can significantly compromise autonomic nervous system. However, less is known about the cardiac autonomic activity in mild and moderate grades of malnutrition in children. Therefore, the objective of this study was to assess the effect of mild/moderate malnutrition on heart rate variability (HRV), a non invasive tool to estimate the cardiac autonomic activity. A cross sectional, community based study was conducted in which 35 malnourished children (mean age: 6.06 +/- 2.04 yrs), on the basis of anthropometric parameters, were enrolled in the study group by random samplings, who were the children of urban slum dwellers and 35 age and sex matched healthy children, were taken as controls. Grading of malnutrition was done according to Indian Academy of Pediatrics (IAP) classification. Anthropometry, basal heart rate (BHR), blood pressure were determined. Time domain and frequency domain indices of HRV were assessed using RMS Polyrite D (version 2.4). Weight, height, mid arm circumference (MAC) and body mass index (BMI) were found to be statistically lower in the study group. There was a strong negative correlation between MAC and LF component (P < 0.01). BHR was found to be increased in the malnourished group (P = 0.027). Low frequency (LFnu) & LF-HF ratio were found to be increased (P = 0.000 & P = 0.001 respectively) while high frequency (HFnu) component was decreased (P = 0.000) in malnourished group. Our results suggested that impaired cardiac autonomic nerve function characterized by sympathetic over activity may occur in malnourished children. This study also enables us to compare, in future works, HRV in pediatric subjects with different grades of malnutrition.

Correlation of anthropometric parameters with QTc interval in malnourished children.

Various studies have reported the effect of severe malnutrition on electrolyte levels and electrocardiographic parameters. However, only a few have reported these findings in mild and moderate grades of malnutrition in children. Therefore, the objective of this study was to assess the effect of malnutrition (mainly mild and moderate grades) on corrected QT interval (QTc) and QT dispersion (QTcd) and electrolyte changes. A total of 20 malnourished children in the age group of 2-11 yrs were enrolled in the study group and 20 age and sex matched healthy children were taken as controls. Anthropometry, serum levels of albumin & electrolytes were determined. QTc and QTcd (difference between maximum & minimum corrected QT interval) were measured with the help of RMS Polyrite D. Our results have shown that body weight, height, body mass index (BMI), serum levels of albumin, potassium & calcium were lower (P<0.01) in malnourished children. QTc (P<0.01) & QTcd (P<0.01) were significantly greater in malnourished children than controls. We concluded that increase in QTc and QTcd intervals is associated with electrolyte disturbances in malnourished children. Electrolyte disturbance correction and appropriate nutrition therapy followed by further cardiac evaluation must be taken into account in the management of these patients.

Prospective Randomized Control Trial Comparing Effect of Dexamethasone Versus Fentanyl as Adjuvants to Intrathecal Bupivacaine for Orthopedic Surgery.

Introduction Spinal anesthesia is the most consistent block for lower limb orthopedic surgeries. We conducted this randomized prospective study to evaluate comparative efficacy of intrathecal dexamethasone with fentanyl and normal saline as adjuvants to hyperbaric bupivacaine in spinal anesthesia administered to patients scheduled for lower limb orthopedic surgery. Materials and methods 105 patients scheduled for lower limb orthopedic surgeries under spinal anesthesia were included in this clinical trial. After randomization, patients received an intrathecal injection of hyperbaric bupivacaine (12.5 mg) with 4 mg of dexamethasone in group I, hyperbaric bupivacaine (12.5 mg) with 25 ug fentanyl with 0.5 ml of normal saline in group II and hyperbaric bupivacaine (12.5 mg) with normal saline (1 ml) in group III, so as to make volume of drug equal in all three groups. The observer evaluated the sensory and motor blocks and other parameters like time to self-void, stay in post-anesthesia care unit (PACU) and complications.  Results The total duration of sensory blockade was found to be 311.43, 197.86 and 115.29 minutes and motor blockade of 223.43, 163.86 and 83.0 minutes in groups I, II and III respectively. The PACU stay was 233.14, 173.86 and 93.00 minutes in groups I, II and III, respectively. The average time to self-void was 400.00, 315.29 and 203.00 in three groups, respectively. Conclusion Intrathecal dexamethasone seemed to be an effective adjuvant to spinal bupivacaine as it prolongs the duration of analgesia, stable hemodynamic profile with minimal side effects. Further studies are required to evaluate the optimum dose and long-term safety of intrathecal dexamethasone.

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.