Search

Chromatography techniques facilitate separation, purification, and identification of secondary compounds (natural products) in lichens and their mycobiont cultures. In particular, high-performance liquid chromatography (HPLC) plays a vital role in the identification of lichen substances because of its high sensitivity, speed, and reliability with the minimal sample. Therefore, we describe the extraction and HPLC protocol for the investigation of secondary compounds with a special focus on lichen mycobiont cultures.

Engraftment fever (EF) is a common complication of autologous HSCT (AHSCT). It is difficult to discern it from infectious fever (IF). We studied the significance of total blood leucocyte count (TLC) to C-reactive protein (CRP) ratio in differentiating EF from IF. 109 consecutive AHSCT patients were retrospectively analysed between March 2011 and August 2013. Breakthrough fever (BF) was defined as new-onset fever preceded by an afebrile period of at least 48 hours. The BF episodes were classified as IF or EF. Infectious fever was diagnosed in case of blood culture positivity, radiological signs of infection, or fever subsiding within 48 hours of changing the antibiotics. Engraftment fever was defined in cases associated with rising leucocyte counts without identifiable infective focus. EF responded well to steroid therapy. Daily TLC and CRP values were obtained from patients’ records. Optimal cut-off value of ratio on day of BF was obtained by plotting ROC curve. Sensitivity and specificity were calculated at this value. Among 109 cases, the breakthrough fever manifested in seventy patients. The median term for BF was day +9. Sixty-two patients had the EF. Median value of TLC/CRP ratio on the day of BF was significantly higher in patients with EF than with IF (0.139 vs 0.038, p=0.013). With ROC analysis, the AUC value was 0.78 (95%CI – 0.66-0.89, p0.056 for EF, the sensitivity and specificity were 63% (95%CI 50-75%) and 100% (95%CI 63-100%) respectively. TLC/CRP ratio >0.056 is highly specific for EF. Prospective studies are warranted to confirm these findings.

The choice of inoculum for successful isolation and establishment of axenic lichen mycobiont culture is a key step towards eliminating endolichenic and lichenicolous fungi and other microbial contamination. The nutritional requirements of each lichen species are unique. This work reports on the isolation, phenotypic plasticity, growth and secondary metabolites from mycobiont culture of the pantropical lichen Platygramme caesiopruinosa. Media composition [Malt yeast extract (MY), Modified Murashige and Skoog (MMS) and Lilly and Barnett (LB) media] was optimized to determine nutritional requirements for optimal growth of this species as assessed by dry biomass and the occurrence of secondary metabolite. Furthermore, the role of different carbon sources in affecting growth, growth stages, phenotypic plasticity, biomass and spectrum of secondary metabolites produced of this mycobiont was examined. The molecular identity of the mycobiont culture was confirmed by amplifying mitochondrial small subunit (mtSSU) sequences. Cultures showed optimum biomass production in MY medium with 10% sucrose. The secondary metabolite profiles for each culture treatment were characterized using High-performance Thin-Layer Chromatography (HPTLC) and Gas Chromatography with Mass Spectrometric (GC-MS) analysis. The HPTLC spectral comparison, phenolic and iodine confirmatory analysis revealed the absence of phenolic metabolites and the presence of non-phenolic metabolites in mycobiont extracts, while GC-MS analysis revealed the biosynthesis of side chain fatty acids, hydrocarbons and sugar alcohol in mycobiont cultures treated with 10% supplemented sucrose as a carbon source.

Algunos microbios beneficiosos del suelo ayudan al establecimiento y crecimiento de plantas medicinales exóticas. Evaluamos la presencia y el estado de la asociación de endófitos de raíces [hongos micorrízicos arbusculares (AM) y hongos endofíticos septados oscuros (DSE)] en diez especies de plantas medicinales exóticas cultivadas en Nilgiris (Ghats Occidentales). Los hongos AM colonizaron todas las especies examinadas y ocho plantas tuvieron la co-ocurrencia de hongos DSE. El alcance de las variables de los hongos endofíticos y las características del pelo radicular difirieron significativamente entre las plantas. Se identificaron seis morfotipos de esporas de hongos AM. Por lo tanto, este estudio indicó la asociación de plantas medicinales exóticas con hongos nativos AM y DSE que podrían explotarse para promover el crecimiento y aumentar la producción de metabolitos secundarios en estas especies de plantas. Some soil beneficial microbes help in the establishment and growth of exotic medicinal plants. We evaluated the presence and status of root endophyte [arbuscular mycorrhizal (AM) fungi and dark septate endophytic (DSE) fungi] association in ten exotic medicinal plant species cultivated in the Nilgiris (Western Ghats). The AM fungi colonized all the examined plant species, and eight plants had the co-occurrence of DSE fungi. The extent of fungal endophyte variables and root hair characteristics significantly differed among the plants. Six AM fungal spore morphotypes were identified. Thus, this study indicated the association of exotic medicinal plants with native AM and DSE fungi which could be exploited to promote growth and increase secondary metabolite production in these plant species.

The metabolites of the mycobiont culture of the lichen

The metabolites of the mycobiont culture of the lichen Trypethelium eluteriae were isolated by column chromatography and preparative TLC. Nine compounds (1–9) including two new trypethelones, 8-methoxytrypethelone (6) and 5′-hydroxy-8-ethoxytrypethelone (9), together with four known trypethelones (3–4, 7–8), and two known phenalenones (1–2) were characterized. It is the first report of 8-methoxytrypethelone methyl ether (5) purification as a racemic mixture in T. eluteriae. Earlier, 7-hydroxyl-8-methoxyltrypethelone (10) was reported as new compound with erroneous spectroscopic data. This compound was identified later as 8-hydroxytrypethelone methyl ether (4). X-ray crystallographic structures of compounds 5–7 were elucidated for the first time. Phenalenones (1–2) and trypethelones (5–6 and 9) were the additional compounds discovered in the cultured mycobiont of T. eluteriae. Six compounds (1–2, 5–8) were screened against Mycobacterium tuberculosis H37Rv and two compounds (7–8) against non-tuberculosis mycobacteria and other human pathogenic bacteria. Compound (7) inhibited M. tuberculosis H37Rv strain with an MIC of 12.5 µg/mL.

Lichen species have unique culture media preferences, and established cultures are known for the synthesis of secondary metabolites. This paper reports observations on the developmental stages and secondary compound biosynthesis by the mycobiont and whole thallus cultures of Buellia subsororioides. It also investigates the suitable media compositions for the culture growth, the role (nutrient or stressor) of sucrose concentrations on the growth stages, biomass, secondary compound profiles, and the quantity of biosynthesized known compounds/g of culture biomass for each treatment using mycobiont cultures. The ascospore-derived mycobiont cultures and thallus macerate-derived whole thallus cultures of B. subsororioides were established and grown using malt yeast extract (MY) medium. Mycobiont cultures were subcultured in MY medium supplemented with sucrose and its concentrations ranging from 0 to 30 % (with 2 % increment between treatments) for 120 days. The molecular identity of cultures was confirmed using nuclear ribosomal Internal transcribed spacer (ITS) DNA sequences obtained from the cultured mycobiont and from the natural thallus. The ITS DNA sequences of the mycobiont showed 99 % similarity with the sequences of the natural thallus. The mycobiont cultures under varying sucrose concentrations initiated as white cottony stages and transformed to brown compact mycelia, with optimum biomass and biosynthesis of nine secondary compounds in MY 10 %. The number of compounds (1–9) varies according to treatments. The whole thallus cultures (MY 0 %) showed a profile of secondary compounds similar to that of the natural thalli along with a trace of one unknown compound. The obtained results are encouraging for the synthesis of the desired quantities of lichen secondary compounds through cultures for relevant applications.

Ehler Danlos syndrome (EDS), is a group of genetically heterogenous connective tissue disorder. A very rare type of this syndrome is the Progeroid type which is included in the NIH group of rare diseases list. The prevalence is < 1 / 1000000. Along with the usual clinical features, patients with Ehler Danlos syndrome-Progeroid type have old age appearance. Here we report one such rare case of this syndrome diagnosed in a 8 yr old child with characteristic clinical features and supportive genetic confirmation.