De Novo Shoot Bud Induction from the Catharanthus roseus Leaf Explants and Agrobacterium tumefaciens-Mediated Technique to Raise Transgenic Plants.

The regeneration of a whole plant from a single cell or organ explant was a valuable task for plant biotechnology. However, important medicinal plants such as Catharanthus roseus have shown recalcitrance to regeneration protocols, thus limiting investigations on MIA metabolism and metabolic engineering in this plant system. In this chapter, successful regeneration protocols were detailed for Catharanthus roseus, either by direct shoot bud induction from leaf explants and Agrobacterium-mediated genetic transformation.

Molecular marker-based characterization of cytoplasm and restorer of male sterility (Ms) locus in commercially grown onions in India.

BACKGROUND: The cytoplasmic-genic male-sterility system has been extensively employed for the production of onion hybrids. Molecular marker-assisted characterization of the cytotypes and genotyping at the restorer-of male-fertility (Ms) locus is important for the accelerated breeding of onion hybrids. Indian onion breeding has focussed more on open-pollinated varieties than hybrids. To accelerate the breeding efforts, marker-assisted selection (MAS) plays a pivotal role. METHODS AND RESULTS: This study aimed to characterize the Indian breeding lines, varieties, hybrids, and exotic accessions for cytotype and Ms locus. For cytoplasm, cytotype markers, accD, and MKFR and for Ms locus identification, PCR markers AcPMS1 and AcSKP1 were employed. Bulk strategy to identify cytoplasm and Ms locus was tested. Sequencing of PCR products amplified by accD was also tried. Both the accD and MKFR were synonymous in cytoplasm identification except in T821 where T cytoplasm was identified. AcPMS1 was more reliable than AcSKP1 for Ms locus identification. Sequencing proved that N and T cytoplasm are identical. Bulking strategy can be used for cytotype identification but not for Ms locus. CONCLUSIONS: Indian onions have a predominance of normal (N) cytoplasm and homozygous recessive (msms) locus. This might be beneficial for hybrid development. S cytoplasm was identified in exotic varieties. For the first time, T cytoplasm has been reported from India. These findings will assist Indian onion breeders to develop MAS strategies for accelerating hybrid development programs. And for the release of onion hybrids with high productivity and uniformity.

68 Ga-DOTATATE PET/CT imaging in endogenous hyperinsulinemic hypoglycemia: A tertiary endocrine centre experience.

OBJECTIVE: Literature regarding utility of 68 Ga-DOTATATE PET/CT in insulinoma localization across various subgroups [benign/malignant/multiple endocrine neoplasia-1 (MEN-1) syndrome associated] remains scarce. In this study, the performance of 68 Ga-DOTATATE PET/CT was compared with contrast-enhanced computed tomography (CECT) and 68 Ga-NODAGA-Exendin-4 PET/CT (whenever available) in an endogenous hyperinsulinemic hypoglycemia (EHH) cohort. DESIGN: Retrospective audit. PATIENTS: EHH patients [N = 36, lesions (n) = 49, final diagnosis: benign sporadic insulinoma (BSI) (N = 20), malignant insulinoma (N = 4, n = 14), MEN-1 syndrome associated insulinoma (N = 9, n = 15), Munchausen syndrome (N = 2) and drug-induced hypoglycemia (N = 1)] having both preoperative imaging modalities (CECT and 68 Ga-DOTATATE PET/CT). MEASUREMENTS: Per-lesion sensitivity (Sn) and positive predictive value (PPV) for histopathological diagnosis of insulinoma. RESULTS: Sn and PPV of 68 Ga-DOTATATE PET/CT were 67.3% and 89.2%; 55% and 100%; 85.7% and 100%; and 66.7% and 77% for overall EHH, BSI, malignant, and MEN-1 syndrome associated insulinoma cohorts respectively. Despite having comparatively lower sensitivity in BSI cohort, 68 Ga-DOTATATE PET/CT localized a pancreatic tail lesion missed by other modalities. 68 Ga-DOTATATE PET/CT had comparatively higher sensitivity in malignant insulinoma than BSI cohort. 68 Ga-DOTATATE PET/CT also paved the way for successful response to 177 Lu-based peptide receptor radionuclide therapy (PRRT). In MEN-1 cases, lower PPV as compared with BSI was due to uptake in non-insulinoma pancreatic neuroendocrine tumours (Pan-NET). CONCLUSIONS: 68 Ga-DOTATATE PET/CT has supplemental role in selected cases of BSI with negative and/or discordant results with CECT and 68 Ga-NODAGA-Exendin-4 PET/CT. In malignant insulinoma, 68 Ga-DOTATATE-PET/CT has an additional theranostic potential. Interference due to uptake in non-insulinoma Pan-NET in MEN-1 syndrome may hinder insulinoma localization with 68 Ga-DOTATATE-PET/CT.

TIAs pathway genes and associated miRNA identification in Vinca minor: supporting aspidosperma and eburnamine alkaloids linkage via transcriptomic analysis.

V. minor contains monomeric eburnamine-type of indole alkaloids having utilization as a neuro-medicinal plant. The biosynthetic pathway studies using miRNAs has been the focal point for plant genomic research in recent years and this technique is utilized to get an insight into a possible pathway level study in V. minor as understanding of genes in this prized medicinal plant is meagrely understood. The de novo transcriptomic analysis using Illumina Next gen sequencing has been performed in glasshouse shifted plant and transformed roots to elucidate the possible non confirmed steps of terpenoid indole alkaloids (TIAs) pathway in V. minor. A putative TIA pathway is elucidated in the study including twelve possible TIAs biosynthetic genes. The specific miRNA associated with TIAs pathway were identified and their roles were discussed for the first time in V. minor. The comparative analysis of transcriptomic data of glasshouse shifted plant and transformed roots showed that the raw reads of transformed roots were higher (83,740,316) compared to glasshouse shifted plant (67,733,538). The EST-SSR prediction showed the maximum common repeats among glasshouse shifted plant and transformed roots, although small variation was found in trinucleotide repeats restricted to glasshouse shifted plant. The study reveals overall 37 miRNAs which were observed to be true and can have a role in pathway as they can regulate the growth and alkaloid production. The identification of putative pathway genes plays an important role in establishing linkage between Aspidosperma and Eburnamine alkaloids.

Differential Iron Status and Trafficking in Blood and Placenta of Anemic and Non-anemic Primigravida Supplemented with Daily and Weekly Iron Folic Acid Tablets.

ABSTRACT: The molecular mechanism of iron transfer across placenta in response to maternal anemic status/ iron supplementation is not clear. We hypothesized that maternal iron/ anemia status during early trimesters can be utilized as a biomarker tool to get estimates of placental iron status. Early interventions can be envisaged to maintain optimum placental/ foetal iron levels for healthy pregnancy outcomes. One hundred twenty primigravida were recruited and divided into non-anemic and anemic group on the basis of hemoglobin levels. The groups were randomly allocated to receive daily and weekly iron folic acid (IFA) tablets till six weeks postpartum. Hematological and iron status markers in blood and placenta were studied along with the delivery notes. Weekly IFA supplementation in anemic primigravidas resulted in significantly reduced levels of hematological markers (p < 0.01); whereas non-anemic primigravidas showed lower ferritin and iron levels, and higher soluble transferrin receptor levels (p < 0.05). At baseline, C-reactive protein and cortisol hormone levels were also significantly lower in non-anemic primigravidas (p < 0.05). A significantly decreased placental ferritin expression (p < 0.05); and an increased placental transferrin expression was seen in anemic primigravidas supplemented with weekly IFA tablets. A significant positive correlation was observed between serum and placental ferritin expression in anemic pregnant women (r = 0.80; p < 0.007). Infant weight, gestational length and placental weight were comparable in both the supplementation groups. To conclude, mother's serum iron / anemia status switches the modulation in placental iron transporter expression for delivering the optimum iron to the foetus for healthy pregnancy outcomes. TRIAL REGISTRATION: Clinical Trial Registry-India: CTRI/2014/10/005135.

Differential Uptake of 68Ga-PSMA-HBED-CC (PSMA-11) in Low-Grade Versus High-Grade Gliomas in Treatment-Naive Patients.

PURPOSE OF THE REPORT: Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein, which is not only overexpressed in prostate cancers but also in variety of solid tumors including glioblastoma multiforme. The aim of the present study was to demonstrate PSMA expression in gliomas using Ga-PSMA-HBED-CC(PSMA 11) PET/CT. PATIENTS AND METHODS: Ten patients with initially MRI suspected and eventually histopathologically proven gliomas [8 males (age range 30-73 yr; mean age 51.8 yr); 2 females aged 39 and 55 years] were subjected preoperatively to regional brain PET scan with Ga-PSMA-11 and F-FDG PET/CT. Final histopathology of brain lesions, their MIB-1 proliferation index (MIB-1 PI) were compared with PSMA and FDG PET findings. RESULTS: FDG PET/CT showed distinct FDG uptake in high-grade gliomas, whereas low-grade gliomas were non-FDG-avid amidst physiological tracer uptake. In vivo PSMA expression was seen in all patients with glioma. Of these, the 7 patients of glioblastoma harboring 8 lesions showed significantly higher PSMA expression than those with low-grade gliomas, average SUVmax being 16.93 and 2.93, respectively. Similarly, average tumor-to-background ratios (13.95 and 3.42, respectively) and MIB-1 PI (17.31 and 3.3, respectively) were substantially more in high-grade versus low-grade gliomas. CONCLUSIONS: The results of this pilot study show that Ga-HBED-CC-PSMA PET/CT can be used to characterize the PSMA expression in gliomas, high-grade ones demonstrating higher SUVmax, MIB-1 PI tumor-to-background ratio than the low-grade ones. With these results as basis, certain patients may benefit from potential PSMA-targeted radionuclide therapy.

The Yin and Yang of Myeloid Derived Suppressor Cells.

In recent years, most of our knowledge about myeloid derived suppressor cells (MDSCs) has come from cancer studies, which depicts Yin side of MDSCs. In cancer, inherent immunosuppressive action of MDSCs favors tumor progression by inhibiting antitumor immune response. However, recently Yang side of MDSCs has also been worked out and suggests the role in maintenance of homeostasis during non-cancer situations like pregnancy, obesity, diabetes, and autoimmune disorders. Continued work in this area has armored the biological importance of these cells as master regulators of immune system and prompted scientists all over the world to look from a different perspective. Therefore, explicating Yin and Yang arms of MDSCs is obligatory to use it as a double edged sword in a much smarter way. This review is an attempt toward presenting a synergistic coalition of all the facts and controversies that exist in understanding MDSCs, bring them on the same platform and approach their "Yin and Yang" nature in a more comprehensive and coherent manner.

Role of Trace Elements, Oxidative Stress and Immune System: a Triad in Premature Ovarian Failure.

The risk of premature ovarian failure (POF) increases in association with alteration in immunological parameters and oxidative stress (OS). Adequate intake of trace elements is required for antioxidant property and immune defense mechanism. The aim of this study was to explore the involvement of trace elements, OS, and immunological parameters in POF. This was a cross-sectional, case-control study, involving 65 participants divided into the POF (n = 35) and control (n = 30) groups. Serum levels of Se, Zn, and Cu were determined along with hormonal, OS, and immunological markers. POF group had significantly lower levels of Zn, Cu, Se, and Zn:Cu ratio. However, Se:Cu ratio was not significant between the groups. FSH and LH levels were negatively correlated with Zn and Cu levels and positively correlated with Se levels. Estrogen levels were negatively correlated with all the studied trace elements. Inter-element association between Zn and Se was significant in POF (r = - 0.39, p = 0.02) compared to control group (r = - 0.078, p = 0.65). In all the POF patients, SOD and GPx activities were significantly (p < 0.05) lower and MDA level was higher (p > 0.05) than control group. B cell marker CD19 was significantly (p < 0.0001) high in POF group. There are involvement of trace elements in hormonal regulation and antioxidant defense mechanism, which once gets altered leads to high ROS generation and affect functions of the immune system. Exaggereative immune system causing higher expression of B cell associated markers (CD19) leading to autoimmune condition in POF.

Genetic engineering approach using early Vinca alkaloid biosynthesis genes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus.

Catharanthus roseus today occupies the central position in ongoing metabolic engineering efforts in medicinal plants. The entire multi-step biogenetic pathway of its very expensive anticancerous alkaloids vinblastine and vincristine is fairly very well dissected at biochemical and gene levels except the pathway steps leading to biosynthesis of monomeric alkaloid catharanthine and tabersonine. In order to enhance the plant-based productivity of these pharma molecules for the drug industry, cell and tissue cultures of C. roseus are being increasingly tested to provide their alternate production platforms. However, a rigid developmental regulation and involvement of different cell, tissues, and organelles in the synthesis of these alkaloids have restricted the utility of these cultures. Therefore, the present study was carried out with pushing the terpenoid indole alkaloid pathway metabolic flux towards dimeric alkaloids vinblastine and vincristine production by over-expressing the two upstream pathway genes tryptophan decarboxylase and strictosidine synthase at two different levels of cellular organization viz. callus and leaf tissues. The transformation experiments were carried out using Agrobacterium tumefaciens LBA1119 strain having tryptophan decarboxylase and strictosidine synthase gene cassette. The callus transformation reported a maximum of 0.027% dry wt vindoline and 0.053% dry wt catharanthine production, whereas, the transiently transformed leaves reported a maximum of 0.30% dry wt vindoline, 0.10% catharanthine, and 0.0027% dry wt vinblastine content.

Differential rubisco content and photosynthetic efficiency of rol gene integrated Vinca minor transgenic plant: Correlating factors associated with morpho-anatomical changes, gene expression and alkaloid productivity.

Transgenic plants obtained from a hairy root line (PVG) of Vinca minor were characterized in relation to terpenoid indole alkaloids (TIAs) pathway gene expression and vincamine production. The hairy roots formed callus with green nodular protuberances when transferred onto agar-gelled MS medium containing 3.0mg/l zeatin. These meristematic zones developed into shoot buds on medium with 1.0mg/l 2, 4-dichlorophenoxyacetic acid and 40mg/l ascorbic acid. These shoot buds subsequently formed rooted plants when shifted onto a hormone-free MS medium with 6% sucrose. Transgenic nature of the plants was confirmed by the presence of rol genes of the Ri plasmid in them. The transgenic plants (TP) had elongated internodes and a highly proliferating root system. During glass house cultivation TP consistently exhibited slower growth rate, low chlorophyll content (1.02±0.08mg/gm fr. wt.), reduced carbon exchange rate (2.67±0.16µmolm-2s-1), less transpiration rate (2.30±0.20mmolm-2 s-1) and poor stomatal conductance (2.21±0.04mmolm-2 s-1) when compared with non-transgenic population. The activity of rubisco enzyme in the leaves of TP was nearly two folds less in comparison to non-transgenic controls (1.80milliunitsml-1mgprotein-1 against 3.61milliunits ml-1mgprotein-1, respectively). Anatomically, the TP had a distinct tetarch arrangement of vascular bundles in their stem and roots against a typical ployarched pattern in the non-transgenic plants. Significantly, the transgenic plants accumulated 35% higher amount of total TIAs (3.10±0.21% dry wt.) along with a 0.03% dry wt. content of its vasodilatory and nootropic alkaloid vincamine in their leaves. Higher productivity of alkaloids in TP was corroborated with more than four (RQ=4.60±0.30) and five (RQ=5.20±0.70) times over-expression of TIAs pathway genes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) that are responsible for pushing the metabolic flux towards TIAs synthesis in this medicinal herb.

Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor.

Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 µM shikimate. This was also supported by real-time (RT) (qPCR), where 8-9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 µM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-µM shikimate was done on the 6th day of bioreactor run.

Shelf-life evaluation of Rasayana Churna: A preliminary study.

BACKGROUND: Concept of Saviryta Avadhi (shelf-life) of Ayurvedic dosage forms is well-defined in classics of Ayurveda. Information on this is scattered in initial classics of Ayurveda like Charaka Samhita, but focused well after 13(th) Century AD in texts such as Vangasena Samhita, Sharangadhara Samhita and Yogaratnakara. Though the concepts have a strong background; considering the pharmaceutical development, a need is felt to re-evaluate the age old concepts by following current norms. AIM: To evaluate shelf-life of two samples of Rasayana Churna. MATERIALS AND METHODS: Raw material was procured from The Pharmacy, Gujarat Ayurved University and authenticated at Pharmacognosy Laboratory. Individual drugs were powdered and blended in specified ratio to obtain Rasayana Churna (RC). RC was levigated with decoction of Amalaki, Guduchi and Gokshura to prepare Bhavita Rasayana Churna (BRC). Both the samples were subjected to accelerated stability studies by following standard guidelines. RESULTS: Both the samples are bitter and astringent with characteristic odor. RC is creamish brown in colour, while BRC is dark blackish brown. These Organoleptic characters were unchanged till the 6(th) month of study. On comparision, BRC is found to be more stable than RC. CONCLUSION: This signifies that, Bhavana not only incorporates additional therapeutic attributes, but also helpful in increasing shelf-life.

In vitro conservation of twenty-three overexploited medicinal plants belonging to the Indian sub continent.

Twenty-three pharmaceutically important plants, namely, Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions.

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng--Panax quinquefolium L.

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47-49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.