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Introduction: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases caused by a deficiency of enzymes responsible for the steroidogenesis. There are three forms of CAH due to 21-hydroxylase deficiency: the classic form with salt loss, the classic virilizing and the non-classic form. The aim of the paper was to analyze the changes in the diagnosis and treatment of children with CAH during previous 15 years. Material and methods: This retrospective cohort study includes patients who were diagnosed with CAH due to 21-hydroxylase deficiency in the period from 2007 to 2021 in endocrinology department of the Institute for Mother and Child Health Care Institute of Serbia "Dr Vukan Cupic". Respondents were divided into two groups - a group whose diagnosis was made in the period between 2007 and 2014 and another group of those whose diagnosis was made in the period between 2015 and 2021. Statistical analysis using Hi-square and Mann Whitney U test was conducted using the software IBM SPSS ver. 22, and p values

Introduction. Patients affected with Allan-Herndon-Dudley syndrome (AHDS) have a deficiency of monocarboxylate transporter 8 (MCT8), a protein primarily responsible for the transport of triiodothyronine (T3) into the brain. This X-linked disorder affects almost exclusively males with clinical presentation encompassing developmental delay, axial hypotonia, dystonia, poor head control, quadriplegia and absence of speech. Case reports. Patient 1 is a male child referred to a hospital investigation at 11 months due to severe developmental delay and elevated blood ammonia level (163 mcmol/L). Hypotonia and dystonic movements were noted at admission, with facial dysmorphic features. Laboratory findings revealed increased blood lactate (17.2 mmol/L), alanine (533 mcmol/L) and ammonia (391 mcmol/L) concentrations. Serum creatine-kinase levels showed substantial increase over the course of hospitalization up to 6,855 IU/L. Clinical exome sequencing detected a novel hemizygous frameshift insertion c.1456insC in gene SLC16A2, predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. Segregation genetic testing of the family members revealed that mother, maternal uncle and maternal grandmother carry the same mutation in SLC16A2. The boy`s mother experienced learning difficulties through childhood while maternal uncle is severely affected by AHDS. Patient 2 is a boy referred to clinical geneticist due to severe psychomotor delay of unknown etiology. Moderate serum lactate elevation was the only laboratory abnormality during initial investigations. Diagnosis of AHDS was established by clinical exome sequencing, and subsequent hormonal evaluation revealed increased triiodothyronine (T3) level which corresponds well to genetic diagnosis. Conclusion. Presence of lactic acidosis and/or hyperammonemia in children with severe developmental delay is not specific for inborn disorders of energy production, such as mitochondrial disease. Clinicians should consider thyroid hormones profiling in cases of unexplained severe developmental delay in male children, especially if associated with axial hypotonia and dystonic movements.

Objecive. Previous research has shown that CRP is elevated in patients with metabolic syndrome (MS) and that low-intensity inflammation is a characteristic of MS. The aim of this study was to determine the concentration of CRP and to examine the significance of elevated CRP in MS patients. Methods. The study included 149 outpatients, 67 patients with MS (68.9 ± 8.7 years) and 82 subjects who were in the control group of the appropriate age (68.9 ± 8.6 years) (p> 0.05). CRP was determined by turbidimetry and the reference range was up to 5.0 mg / L. Results. The average CRP concentration for MS patients was 5.33 ± 3.60 mg / L, while in the control group the CRP concentration value was 4.60 ± 2.03 mg / L (p 0.05). Almost a third of patients (29.8%) and only 6.1% of subjects in the control group had a CRP concentration greater than 5 mg / L (p

Introduction/aim: Disorders of sex development (DSD) comprise a heterogeneous group of congenital conditions with a difference between chromosomal, gonadal sex and the appearance of the external genitalia. The frequency of DSD is 1: 4,500-5,500 newborns per year. Congenital adrenal hyperplasia (CAH) due to the deficiency of the 21-hydroxylase enzyme is one of the most common and best-known causes of DSD. Other forms of CAH, as well as other causes of DSD, occur with significantly lower individual frequencies and are thus more challenging to diagnose and treat. The aim of the study was to analyse the etiology and clinical characteristics of DSD, as well as changes in the diagnostic and therapeutic approach to DSD in the tertiary center during the previous 13 years. Methods: The study was conducted in the form of a retrospective cohort study which included all patients investigated for DSD at the Department of Endocrinology of Mother and Child Health Care Institute of Serbia "Dr Vukan Cupic" during the period from December 2007 until November 2020. Children with DSD caused by CAH due to 21-hydroxylase deficiency were not included in this study. Results: The study included a total of 31 children with DSD: 24 children (77%) had 46XY DSD, 3 (10%) had 46XX DSD, and 4 children (13%) had chromosomal DSD. A definitive diagnosis of specific etiology has been made in 25 children (81%), and the most common etiology of DSD was gonadal dysgenesis (55%), followed by the androgen insensitivity syndrome (10%) and atypical forms of CAH (7%). During the period 2 (2016-2020) more children with DSD (n = 18) were examined compared to the period 1 (2007-2015) and the specific etiological diagnosis was established in a larger number of children with DSD (89%) compared to the period 1 (69%). Also, during period 2 (2016-2020) genital surgery was performed in a significantly lesser number of children (11%) and at a later age (average age 6.8 years) than in period 1 (64%, average age 4.8 years; p

Introduction/Aim: Spondylodysplastic Ehlers-Danlos Syndrome (sdEDS) is a rare genetic disorder of collagen synthesis, caused by a mutation in the B4GALT7, B3GALT6, or SLC39A13 gene. Features of this very rare disorder are short stature, hypotonia, hyperflexible joints, soft, thin, and overly stretchable skin, sparse hair and eyebrows, elderly face, wide forehead and prolonged wound healing. Molecular genetic analysis is needed for definite confirmation of the diagnosis. So far, only three case reports describing growth hormone treatment response in patients with sdEDS have been published. All of these patients had growth hormone (GH) deficiency. We present the first case report regarding growth hormone treatment response in a patient with sdEDS and normal GH secretion (without GH deficiency). Case report: Patient was a girl with short stature and normal GH secretion. Having in mind that the girl was born small for the gestational age, due to her short stature, she started using HR, before the diagnosis of sdEDS was made. Based on the lack of improvement in growth velocity as well as the girl's phenotype, genetic analyses were performed and the diagnosis of sdEDS due to biallelic mutations of the B4GALT7 gene was established. After the diagnosis of sdEDS was made and due to suboptimal response in growth velocity to the GH treatment, the GH therapy was stopped at the age of 11 years. Conclusion: This is a first case report regarding GH treatment in a child with sdEDS and normal GH secretion, demonstrating a very limited therapeutic effect of GH on linear growth in the presented patient.

We aimed to collect data on all paediatric patients who were diagnosed with type 1 diabetes mellitus (T1DM) between the years 2000 and 2019 in Serbia and estimate for the first time its prevalence. Also, the trends of diabetes ketoacidosis (DKA) occurrence at the time of diagnosis are monitored. We collected and retrospectively analysed the data of patients

Gibberellins (GAs) play a pivotal role in the induction of somatic embryogenesis from in vitro root apices of spinach plants. With the aim to understand the role of GAs in this process and to improve somatic embryo (SE) regeneration efficiency, the impact of light and GAs on SE initiation from the in vitro root apices was studied. The root sections were isolated from in vitro-grown SE-derived plants and placed on medium containing 20 µM α-naphthaleneacetic acid (NAA) and 0–10 µM GA3 or GA1, and cultivated under light conditions or in darkness. The most efficient SE regeneration response (100% regenerating SEs and 40.73 SEs per root apices) was achieved only in the presence of both light and GAs, with GA3 always exhibiting much stronger effect than GA1. Considering that light enhances GAs biosynthesis and the necessity of GAs for SE initiation, the expression levels of genes encoding the key enzymes involved in the final steps of GAs synthesis (SoGA20-ox1 and SoGA3-ox1) and deactivation (SoGA2-ox1, SoGA2-ox2 and SoGA2-ox3) were analyzed. Light enhanced the expression of all five GA-ox genes, while exogenously supplied NAA + GA3 provoked downregulation of SoGA20-ox1 and SoGA3-ox1 and upregulation of SoGA2ox-2 and SoGA2ox-3 expression. The expression of SoGA2ox-1 only slightly decreased. The results indicated the capability of isolated spinach roots to perceive the light and autonomously produce GAs. The expression levels of genes encoding key enzymes involved in GA biosynthesis suggest that lower levels of GAs favor SE initiation. Light and gibberellins (GA) synergistically promote somatic embryogenesis in spinach. Expression levels of genes encoding key enzymes for GA metabolism suggest that lower levels of GAs may favor somatic embryogenesis.

Centaurium erythraea (centaury) is a medicinal plant with exceptional developmental plasticity in vitro and vigorous, often spontaneous, regeneration via shoot organogenesis and somatic embryogenesis, during which arabinogalactan proteins (AGPs) play an important role. AGPs are highly glycosylated proteins belonging to the super family of O-glycosylated plant cell surface hydroxyproline-rich glycoproteins (HRGPs). HRGPs/AGPs are intrinsically disordered and not well conserved, making their homology-based mining ineffective. We have applied a recently developed pipeline for HRGP/AGP mining, ragp, which is based on machine learning prediction of proline hydroxylation, to identify HRGP sequences in centaury transcriptome and to classify them into motif and amino acid bias (MAAB) classes. AGP sequences with low AG glycomotif representation were also identified. Six members of each of the three AGP subclasses, fasciclin-like AGPs, receptor kinase-like AGPs and AG peptides, were selected for phylogenetic and expression analyses. The expression of these 18 genes was recorded over 48 h following leaf mechanical wounding, as well as in 16 tissue samples representing plants from nature, plants cultivated in vitro, and developmental stages during shoot organogenesis and somatic embryogenesis. None of the selected genes were upregulated during both wounding recovery and regeneration. Possible functions of AGPs with the most interesting expression profiles are discussed.

Cistus creticus subsp. creticus is a shrubby Mediterranean plant used since ancient times in folk medicine for the treatment of different diseases. C. creticus extracts and resin contain different types of secondary metabolites, such as terpenoids (predominantly labdane type diterpenoids), and phenylpropanoids. Growth conditions seem to influence the content of labdane-type diterpenes and flavan-3-ols in leaves of C. creticus subsp. creticus. Histochemical staining of leaves’ trichomes and comprehensive phytochemical characterization of resin, leaves and their exudates, indicated that long-stalked capitate trichomes of C. creticus subsp. creticus, grown both in vitro (IV) and in greenhouse (GH), are capable of producing bioactive oleoresin-related terpenoids and phenylpropanoids compounds. Bioactivity-guided approach was implemented in search for the major antibacterial compound in C. creticus resin against two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus cereus and Micrococcus flavus). Bioautographic assay on TLC plates with separated components of Cistus resin extract, revealed a pronounced zone of microbial growth inhibition, corresponded to a highly active compound with Rf values of 0.45, structurally characterized and identified as ent-3β-acetoxy-13-epi-manoyl oxide. This finding opens the route for focusing on isolation and functional characterization of genes involved in the biosynthesis of ent-3β-acetoxy-13-epi-manoyl oxide and its precursor ent-3β-hydroxy-13-epi-manoyl oxide, with the aim to establish sustainable in vitro biotechnological protocols for its large-scale production in homologous and heterologous plant and microbial hosts.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N&prime, phenylurea (CPPU). A medium containing 0.1 mgL&minus, 1 2,4-D and 0.25 mgL&minus, 1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL&minus, 1 2,4-D and 0.5 mgL&minus, 1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury&mdash, an emerging model in developmental biology.

Centaurium erythraea Rafn. (common centaury, Gentianaceae) is a medicinal plant with great regeneration potential and developmental plasticity in vitro. Centaury can be regenerated from leaf explants by both somatic embryogenesis (SE) and shoot development (SD). We believe that its regeneration potential and developmental plasticity rest on high activity of certain genes, which may not be active or present in species recalcitrant to in vitro regeneration. However, there are no sequenced Gentianaceae genomes to support investigation of the molecular events during SE or SD. To this end, we have sequenced six centaury transcriptomes (embryogenic calli, globular somatic embryos, cotyledonary somatic embryos, adventitious buds, leaves and roots of in vitro grown plants) and de novo assembled centaury referent transcriptome comprising 105.726 genes. The high quality and completeness transcriptome was functionally annotated against NCBI nt, Swissprot and PFAM databases with KOG and GO enrichment. In addition, 11 housekeeping and functional genes were validated for expression stability in 27 tissue samples representing the processes of SE and SD, plants from nature and wounded tissues using GeNorm and NormFinder. The most stable genes that can be used for expression studies during SE, SD and in vitro manipulations are Ribosomal protein L2 (RPL2) and TATA binding protein 1 (TBP1) in combination with RAS (Rat Sarcoma)-related Nuclear protein (RAN) or Adenosine kinase (AK). These results comprise a complete framework for the search for genes involved in SE and SD, but may also be useful in identifying genes involved in biosynthesis of C. erythraea secondary metabolites. De novo assembled and fully annotated Centaurim erythraea transcriptome is publically available along with validated housekeeping genes with stable expression during in vitro development and mechanical injury.

Medicinal plant Gentiana lutea is considered as an official drug for gastrointestinal disorders in many pharmacopoeias. Its uncontrolled overexploitation led to the protection regime and encouraged development of in vitro culture. The aim of this work was to chemically characterize root and leaf methanol extracts of plant grown in plantation and in vitro conditions, and to determine their antigenotoxicity against heterocyclic aromatic amines IQ and PhIP, the food-borne mutagens that are formed during high temperature cooking of protein-reach foods. High yields and growth ratios were determined for in vitro plants. Higher content of polyphenols and flavonoids were determined in aerial parts, while UPLC-MS/MS analysis pointed at the richness of in vitro grown shoot extract (GLvS) in active constituents, and high amounts of gentiopicroside in all tested extracts. Notable amounts of sweroside were detected in plantation root extract (GLR) and in GLvS, and of loganic acid in plantation leaf extract (GLL) and GLvS. Two experimental models for genotoxicity/antigenotoxicity study, i.e. SOS/umuC test with metabolic activation (addition of rat liver S9 fraction) and alkaline comet assay on hepatocellur carcinoma HepG2 cells indicated dual effects: genotoxic of high and antigenotoxic of lower non-genotoxic doses. While in vitro extracts were genotoxic in both models, plantation extracts were active only in SOS/umuC with S9. In contrast, inhibition of genotoxicity was observed in both models for all extracts (up to 83 %). Results of DPPH assay indicated that antioxidativity could be responsible for antigenotoxic properties. In conclusion, genoprotective effect encourages further investigation of antigenotoxicity, while high growth ratio and richness of GLvS in active compounds justify the use of in vitro cultivation method.

Objectives – Although congenital toxoplasmosis (CTox) is asymptomatic in most cases, some infants/neonates present with variable clinical manifestations, including anaemia, jaundice, hepatosplenomegaly, seizures, hydrocephalus, chorioretinitis and sensorineural deafness. Involvement of the hypothalamic-pituitary axis is rarely reported with CTox. Case report – We present a full term baby girl who was admitted to the hospital on the 15th day of her life, due to an intense tremor and irritability. Brain ultrasound and CT exam revealed calcifications of the brain parenchyma and severe ventriculomegaly. Serological and molecular findings (positive specific immunoglobulin M and immunoglobulin G antibodies, polymerase chain reaction) confirmed CTox. After diagnosis was established, specific therapy with pyrimethamine, sulfodiazine and folic acid was started. On the fifth day of hospitalisation, laboratory findings revealed high serum sodium and chloride, high serum osmolality, low urine osmolality and low urine specific gravity. Since these findings strongly suggested the diagnosis of diabetes insipidus (DI) in this new born, further endocrinological workup was performed to assess the pituitary function. An ACTH (synacthen) stimulation test showed a low basal cortisol level with a positive response after administration of synacthen. The free thyroxin level was also low and repeated prolactin levels were high. On the basis of these findings, it was concluded that this new born has panhypopituitarism and diabetes insipidus. Hormonal substitution therapy with hydrocortisone, L-thyroxine and desmopressin was started and gradual normalization of electrolytes and hormonal status was noted. Conclusion – A continuous endocrinological monitoring of a child with CTox is important and necessary for early detection, timely therapy and prevention of severe sequelae.

Nepeta rtanjensis Diklic & Milojevic (fam. Lamiaceae) is an endemic, critically endangered plant, protected by the law in Serbia. Various biological activities have been ascribed to its major constituents, nepetalactones. In this study we describe for the first time cytotoxic activity of N. rtanjensis essential oil (EO), obtained from field cultivated plants, which was found to be especially rich in trans,cis-nepetalactone. MTT assays indicated that after 72 h of treatment the EO exhibited cytotoxic activity on investigated cancer cell lines: HeLa, K562, A549, LS-174 and MDA-MB-231. Normal cell line (MRC-5) was the least sensitive to the treatment and IC50 value for this cell line was not reached within the tested range of EO concentrations (up to 0.1 μL/mL). Analysis of morphological changes of treated cells confirmed the higher sensitivity of tumor cells than normal cells to the tested EO. Application of N. rtanjensis EO resulted in the appearance of morphological changes in tested cancer cell lines characteristic for apoptotic cell death, and induced perturbations of the cell cycle of HeLa cells. In addition, upregulation of Bax and p53, and downregulation of Bcl-2, and Skp2 genes, involved in apoptotic signalling cascades, confirmed an apoptosis-inducing effect of N. rtanjensis EO on HeLa cells. Presented results highlighted the potential of N. rtanjensis EO in anticancer therapy.

Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin – a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms – GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.

Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin – a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms – GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.

International audience; Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis of leaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology.

Glandular trichomes are secretory organs that vary greatly in size, shape, location, type, and composition of secondary metabolites that are synthesized in them. They represent a protective chemical barrier and metabolic factories for the synthesis of bioactive compounds. We evaluated the effect of sucrose on the growth and biomass production, as well as on the distribution, density, and chemical composition of leaf glandular trichomes developed in vitro cultured Inula britannica, an important medical and aromatic species. Nodal segments (5 mm) were cultivated on the basal Murashige and Skoog medium with different concentrations (0 M, 0.01 M, 0.06 M, 0.1 M and 0.3 M) of sucrose. The lack of sugar does not retard growth, while a high sugar concentration inhibits biomass accumulation in vitro. The density and number of leaf glandular trichomes are changed with the medium composition variation. Morphoanatomical and histochemical analyses of the trichomes of the cultured I. britannica applying SEM, conventional light, fluorescent and Raman microscopy revealed the presence of non- glandular and biseriate glandular trichomes on the leaf surface. The histochemical analysis proved that glandular trichomes synthesized a complex mixture of biomolecules. The Raman microscopy analysis of glandular trichomes confirmed the presence of terpenes, most probably the bicyclic monoterpenes and sesquiterpenes. The multivariate analysis, based on PCA, was applied to distinguish among the glandular trichomes of three sugar concentrations: sugar-free medium (0 M), optimal (0.1 M) and the highest sucrose concentration (0.3 M). The main differences between the chemical profile of the leaf glandular trichomes of the plants grown on the 0 M and the chemical profile of the trichomes developed on the 0.1 and 0.3 M medium come mainly from phenolic compounds and to a smaller degree from cyclic terpenes, while the chemical profile of the glandular trichomes grown on the 0.1 M and 0.3 M differs considerably from the trichomes grown on the sugar-free medium in the polysaccharide cell wall ingredients. Most differences between the chemical profile of the glandular trichomes developed on 0 M and 0.3 M and the chemical profile of the trichomes grown on the 0.1 M of sucrose, come from phenolic compounds.

Background/Aim. An ideal insulin regimen for children and adolescents with type 1 diabetes mellitus (T1DM) should be physiological, flexibile and predictable, protecting against hypoglycaemia. The aim of this study was to evaluate the influence of insulin analogues on glycaemic control and the occurrence of hypoglycaemic episodes in children and adolescents with T1DM. Methods. The study group consisted of 151 children and adolescents (90 boys, 61 girls) treated with human insulins for at least 12 months before introducing insulin analogues. All the patients were divided into two groups: the group I consisted of 72 (47.7%) patients treated with three injections of regular human insulin before meals and long-acting analogue (RHI/LA), and the group II of 79 (52.3%) patients treated with a combination of rapid-acting and long-acting analogue (RA/LA). The levels of glycated hemoglobin (HbA1c) and the number of hypoglycaemic episodes were assessed at the beginning of therapy with insulin analogues, and after 6 and 12 months. Results. The mean HbA1c was significantly lower in the group I (RHI/LA) after 6 months (9.15% vs 8.20%, p < 0.001) and after 12 months (9.15% vs 8.13%, p < 0.001) as well as in the group II (RA/LA) after 6 months (9.40% vs 8.24%, p < 0.001) and after 12 months of insulin analogues treatment (9.40% vs 8.38%, p < 0.001). The frequency of severe hypoglycaemia was significantly lower in both groups after 6 months (in the group I from 61.1% to 4.2% and in the group II from 54.4% to 1.3%, p < 0.001), and after 12 months (in the group I from 61.1% to 1.4% and in the group II from 54.4% to 1.3%, p < 0.001). Conclusion. Significantly better HbA1c values and lower risk of severe hypoglycaemia were established in children and adolescents with T1DM treated with insulin analogues.

Chicory ( Cichorium intybus L.) is traditionally recognized for its anti-inflammatory, antimicrobial, anticancer and nutritive properties. Among active secondary metabolites detected in chicory, the most important are sesquiterpene lactones and phenolics, including chlorogenic acid (CA). Hereby we have analyzed the content of CA in previously obtained Agrobacterium rhizogenes – transformed chicory hairy root cultures and transformed regenerants. Among three analyzed hairy root clones, clone 13 had exceptionally high biomass production, so the amount of CA in this culture was the highest, e.g. 400 times higher in comparison to the untransformed root culture. Since the roots spontaneously regenerated, the system was upgraded to allow the comparison of CA content not only among the clones, but also between different developmental phases of the regenerants (vegetative vs. flowering plants) and their organs (roots vs. leaves). It was shown that the CA production varies from clone to clone, and also depends on the phenophase of the clone. In the rosette stage of clones 13 and 36, the highest amount of CA was detected in roots, while the same clones in the flowering stage had significantly lower CA content. The clone 35 in the vegetative phase produced the lowest amounts of CA. However, in the flowering stage the roots of clone 35 produced the highest CA amount. Among the regenerants, the clone 13 had the fastest growth, and hence the best CA production. The current paper presents for the first time the CA content in transformed chicory regenerants. The obtained results suggest that the culture of transformed chicory plants can be equally good source of CA as liquid hairy root culture.

Five new sesquiterpene lactones (1-5) were isolated from Inula britannica collected in the wild from Serbia along with five known compounds (6-10). Sesquiterpene lactones were isolated using centrifugal partition chromatography followed by combination of flash chromatography and semi-preparative HPLC. Isolated compounds were screened for cytotoxic activity on four different human cancer cell lines and their multi-drug resistant counterparts, as well as on normal human keratinocytes. Sesquiterpene lactones showed similar cytotoxic activity toward drug sensitive and drug resistant cancer cell lines. (C) 2013 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved. European Union [227448]; Center for Biosystems Genomics part of the Netherlands Genomics Initiative; Netherlands Metabolomics Center part of the Netherlands Genomics Initiative

Cistus creticus subsp. creticus is a native plant of the Mediterranean region and it has been used since ancient times for its medicinal properties. The leaves secrete labdane type diterpenes that exhibit considerable antibacterial and cytotoxic activities. However there are no data about implementation of in vitro culture of C. creticus in order to obtain applicable production of labdane diterpenes. We successfully established in vitro culture of shoots and roots on solid or liquid MS (Murashige and Skoog) medium without addition of plant growth regulators. GC/FID and GC/MS detections were used to carry out characterization of ethanol extracts of shoots and roots from in vitro culture. Labdane diterpenes were the most abundant compounds in shoot extract, but absent from the root extract. Total phenolic and flavonoid contents of C. creticus shoot and root ethanol extracts were also determined. In order to evaluate possible cytotoxic activity of extracts, sulforhodamine B (SRB) assay was performed on five human cancer cell lines. Only shoot extract had cytotoxic activity on HeLa (cervix), MDA-MB-453 (breast) and FemX (melanoma) cancer cells with IC50 reaching 80.83 μg/ml (HeLa), 76.18 μg/ml (MDA-MB-453), and 87.52 μg/ml (FemX) respectively. This is the first report of cytotoxic effect of labdane diterpenes from Cistus on HeLa cells.

Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.

The Empress tree (Paulownia tomentosa Steud.) is a fast growing deciduous tree originating from East Asia. It is grown as an ornamental plant, but can also be used as industrial wood. However, in some parts of North America, this species is considered invasive, with high impact on autochthonous populations. We have investigated whether post-fire expansion of this species can be explained at the germination level by analyzing the interaction between physical (light and temperature) and chemical (smoke and nitrogenous compounds) fire-related cues in the regulation of P. tomentosa germination. Light is the key environmental signal in the induction of germination of positively photoblastic P. tomentosa seeds. Smoke, applied in the form of extract (liquid smoke [LS]), stimulates light-induced germination if applied during imbibition, or shortly after the inductive irradiation. This stimulatory effect of LS can be effectively prevented by far-red light, suggesting that the mechanism of smoke action is phytochrome related. The LS had no effect on the germination percentage when applied during the phase of radicle protrusion. However, LS reduced radicle elongation and slowed its protrusion. The LS had no effect on the optimal temperature range of germination (23–27°C). The combined action of LS and KNO3 in light-induced germination was more effective then when each chemical was applied separately. It can be concluded that all tested components of fire, except elevated temperature, operate to stimulate germination of P. tomentosa seeds, suggesting that the post-fire invasive potential of this species can, at least in part, be explained at the germination level.

To evaluate sodium salicylate (NaSA) as an elicitor of rosmarinic acid (RA) and phenolic diterpenes, carnosol (C) and carnosic acid (CA) production, in a culture of Salvia officinalis shoots. In sage shoots grown in vitro, 28 polyphenolic compounds (phenolic acids, flavonoids, and phenolic diterpenes) were identified. In shoots treated for 1 week with increasing NaSA concentrations, the content of C increased from 2.3 in control to 5.7 mg g(-1) DW in shoots treated with 500 A mu M NaSA. In shoots that were recovered on basal medium for 3 weeks, the maximal amount of C (14 mg/g(-1) DW) was with 150 A mu M NaSA treatment. In treated and recovered shoots, the increase in C was accompanied with a decrease in CA, resulting in 1.9-fold increase in the C/CA ratio. Accumulation of RA was not affected by the NaSA treatment. However, elicitation by NaSA was accompanied with growth retardation. NaSA can improve C production in sage shoot culture, probably by stimulating the conversion of CA to C. European Union Seventh Framework Program {[}227448]; Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173024]

The germination of Empress tree (Paulownia tomentosa Steud.) seeds is phytochrome-controlled. Liquid smoke could not induce germination in darkness but red light irradiation of liquid smoke imbibed seeds induced a high percentage of germination. Maximum germination was achieved at liquid smoke concentration of 0.1% (v/v) when present during the imbibition phase or during the phase of phytochrome activity. The light requirement of these seeds could be completely substituted by exogenously applied gibberellins. In the presence of liquid smoke, optimal concentrations of GA3, GA4, and GA9 necessary for inducing germination were several times lower than in the controls, while that of GA7 was equally active when applied at a concentration one order of magnitude lower. The inhibitory effect of the applied growth retardants was strongly reduced and liquid smoke, in the presence of retardants, allowed light-induced germination, if applied simultaneously or after retardants treatment.

Chicory (Cichorium intybus L) is rich in bitter sesquiterpene lactones, mainly guaianolides: lactucin, 8-deoxylactucin, lactupicrin and their 11(S),13-dihydroderivatives-compounds recognized for their antimicrobial and anti-cancer effects. In vitro plant tissue culture, and particularly Agro bacterium rhizogeries-generated hairy root (HR) cultures, have many advantages as systems for production of valuable secondary metabolites. Although chicory HRs grow better than control culture, having nearly 60 times greater fresh weight gain, they do not contain a higher content of guaianolides than wild type (wt) roots. Thus we have established in vitro system comprised of wt root and HR cultures, and wt and transformed regenerated plants of the same age, in rosette and flowering stage, in order to study the effects of transformation, organogenesis and flowering on guaianolides production. Both regeneration and flowering in vitro were spontaneous, so the results were not influenced by exogenous growth regulators. Some of the transformed clones grew better, but all flowered earlier in comparison to wt plants. Floral transition increased guaianolides content in both roots and leaves of transformed, but not of wt plants. Expression of RolC oncogene correlated with floral transition and with guaianolides accumulation. We propose A. rhizogenes transformed plants at the flowering stage as an alternative source of free guaianolides, where, in contrast to HRs, entire plants can be used for the extraction. (C) 2014 Elsevier B.V. All rights reserved. Serbian Ministry of Education, Science and Technological Development {[}OI 173024]; EU {[}227448]

Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1; 5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1 :1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers. (C) 2014 Elsevier Masson SAS. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}ON173024]

Ornamental plants Impatiens hawkerii and Impatiens walleriana are susceptible to Tomato spotted wilt virus infection that can be eradicated by meristem-tip propagation. Comparison of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) profiles of healthy, infected and virus-eliminated plants showed that long-term infection did not cause major changes in I. hawkerii, but caused induction of several APX and GPX isoforms, increase in Mn-SOD and APX and a decrease in CAT activity in I. walleriana. This is the first report on the effects of virus elimination by meristem-tip propagation on the antioxidative metabolism in plants. (C) 2012 Elsevier Ltd. All rights reserved. Serbian Ministry of Education and Science [TR-31019]

Tanacetum parthenium produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 SL from T. parthenium with centrifugal partition chromatography and semipreparative HPLC. Compounds were screened in vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All SL containing the alpha-methylene-gamma-lactone moiety were able to activate the ARE and cause cellular toxicity. The structure-activity relationship among the SL isolated indicates that the guaianolides were more active and when lacking the endoperoxide functionality less toxic then the germacranolides. European Union [227448]; National Institute of Environmental Health Sciences [R01ES08089, R01ES10042]

The effect of high Mn2+ content on Centaurium pulchellum seed germination has been investigated. Seeds containing extremely high Mn2+ content were produced by culturing single-node flowering explants for 2 months in the MS-media, supplemented with Mn in concentrations ranging from 1 to 10,000 mu M. Although the seeds displayed the capacity to accumulate high amount of Mn, their germination was undisturbed. EPR spectroscopy was used to measure the ratio of free (aqueous) Mn to bound Mn and it was found that over 97% of total Mn was in the bound form. With elevating the external Mn supply, seed Mn concentration also increased, but the proportion of free Mn2+ fraction decreased from 3% in the control (1 mu M Mn) to 0.35% and 0.15% in high Mn supply (1000 mu M and 10,000 mu M, respectively). These results suggest that an elevation of internal Mn concentration in seeds is associated with increased Mn binding pools, hence Mn remains bound during germination. Consequently, the action of potentially harmful Mn2+ ions, which may generate ROS and affect seed viability, is alleviated. (C) 2008 Elsevier B.V. All rights reserved. Serbian Ministry of Science and Environmental Protection [143031, 143016, 143020]

The effect of sucrose on fruiting, seed production, and seed germination of lesser centaury [Centaurium pulchellum (Sw.) Druce] was examined using explants of flowers and flower buds. Sucrose concentrations in the culture medium ranged from 0.003 to 0.3 M. It has been shown that the number of auxiliary buds, capsules dimension, number of viable seeds per capsule and seed dimensions increased with the increase of sucrose concentrations. The highest values were recorded at sucrose concentrations higher than 0.03 M, except for seeds size, which were larger at sucrose concentration ranging from 0.003 to 0.1 M. The germination of in vitro produced seeds was affected by previous culture history: a higher germination percentage was obtained in seeds that were raised from explants originally grown on medium with sucrose concentrations higher than 0.003 M. null

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data.

Rindera umbellata (Waldst. & Kit.) Bunge is a rare, critically endangered and horticulturally appealing plant with unexplored pharmaceutical potential. Its distribution is restricted to sandy habitats, whereas propagation in nature is limited by fungal infections of the seeds. To initiate its ex situ conservation and provide material for metabolomic studies, we have introduced R. umbellata into in vitro culture using immature embryos as primary explants. A 72% of the embryos germinated on growth regulator-free medium. The optimization of growth conditions was based on varying carbohydrates (sucrose, glucose, and fructose) in the medium. In vitro growth and development of R. umbellata plants were significantly affected by both the type and concentration of the applied sugars. For most recorded parameters, including leaf elongation, biomass production, rooting percentage, and the number and length of roots, 0.1 m sucrose was optimal. The highest percentage of explants with developed buds was achieved on 0.06 m sucrose (38.77%) or 0.3 m glucose (27.43%). The plantlets obtained on 0.1 m sucrose were successfully acclimatized to greenhouse and field conditions with survival rates of 71.43% and 42.86%, respectively. To our best knowledge, this is the first publication dedicated to this species.