In vitro growth and content of vincristine and vinblastine of Catharanthus roseus L. hairy roots in response to precursors and elicitors

Catharanthus roseus L. is a medicinal plant that produces numerous indole terpenoid alkaloids, including vincristine and vinblastine, which are used for cancer treatment. The effect of specified precursors (L-phenylalanine, L-tyrosine) and elicitors (chitosan, methyl jasmonate) on C. roseus hairy roots (CHR) growth has been examined in order to increase the content of vincristine and vinblastine. Our results showed that CHR generated by an Agrobacterium rhizogenes strain isolated in Vietnam was capable of producing both vincristine and vinblastine when subjected to precursors, but only vinblastine when exposed to elicitors. However, both precursors and elicitors were evaluated to have an effect on increasing the accumulation of TIAs in CHR. In particular, the use of elicitors required more time to find the appropriate induction conditions, while the use of precursors gave outstanding efficiency in the treatment with 1 µM phenylalanine. The greatest yields of vincristine (51.99 µg g-1 DW) and vinblastine (699.92 µg g-1 DW) were obtained in the 7th week (with 0.306 g DW biomass). This result is the first time we might boost the levels of vincristine and vinblastine in our CHR clone generated by the Vietnam strain of A. rhizogenes.

In vitro growth of uropathogenic Escherichia coli isolated from a snow leopard treated with homeopathic and isopathic remedies: a pilot study

This paper reports the results of incubation of a strain of uropathogenic Escherichia coli (UPEC) isolated from a snow leopard - which had died of septicemia secondary to necro-hemorrhagic cystitis - with homeopathic and isopathic remedies. Methods: UPEC was isolated from heart blood and previously typified for virulence factors; it was incubated with homeopathic remedies Cantharis vesicatoria (urinary tract infection affinity), Mercurius solubilis (from symptoms analysis) and nosode prepared from the actual strain, all in dilution 12cH. Results: 2 patterns of bacterial growth were observed, associated to the quality of nutrients in the culture medium; in rich-nutrient medium, nosode of E. coli 12cH had a significant inhibitory effect; in poor-nutrient medium, Merc 12cH exerted significant inhibitory effect. Conclusion: results suggest that the previous conditions of prokaryote systems may influence the in vitro response to homeopathic and isopathic remedies. Keywords: urinary tract infection; Felines; Uropathogenic Escherichia coli; Homeopathy; Isopathy.  Estudo do crescimento (in vitro) de Escherichia coli uropatogénica isolada a partir de um leopardo das neves e tratada com medicamentos homeopáticos e isopáticos: estudo piloto. ResumoEste artigo relata os resultados da incubação de uma linhagem de Escherichia coli uropatogénica (UPEC) isolada a partir de um leopardo das neves, que morreu de septicemia secundária a cistite necrótica-hemorrágica. A UPEC foi tratada com preparados homeopáticos e isopáticos. Métodos: UPEC foi isolada de sangue cardíaco e previamente tipificada para fatores de viruléncia; foi incubada com o medicamento homeopático Cantharis vesicatoria (afinidade com infecção do trato urinário), Mercurius solubilis (a partir da análise de sintomas) e nosódio preparado a partir da mesma linhagem de bactérias, todas em 12 cH. Resultados: 2 padrões de crescimento bacteriano foram observados, associados àqualidade dos nutrientes do meio de cultura; em meios ricos em nutrientes, nosódio de E. coli 12 cH teve um significativo efeito inibitório; em meio pobre de nutrientes, Merc 12 cH exerceu efeito inibitório significativo. Conclusão: os resultados sugerem que as condições prévias do sistema procarioto estudado podem influenciar as respostas proliferativas in vitro para preparados homeopáticos e isopáticos. Palavras-chave: infecção do trato urinário, felinos, Escherichia coli uropatogénica; homeopatia, isopatia.  Crecimiento in vitro de Escherichia coli uropatogenica, aisladas de un leopardo de la nieve, tratadas con remedios homeopáticos y isopáticos: un estudio preliminar ResumenEste trabajo presenta los resultados de la incubación de una cepa de Escherichia coli uropatogenica (UPEC) aislada de un leopardo de la nieve, que había muerto de septicemia secundaria a la necro-hemorrágica cistitis, con remedios homeopáticos y isopáticos. Métodos: Se aisló UPEC de la sangre del corazón y caracterizado por factores de virulencia, y se incubó con los remedios homeopáticos Cantharis vesicatoria (afinidad con la infección del tracto urinario), Mercurius solubilis (a partir de la análisis de los síntomas) y nosódio preparado a partir de la cepa, todos em la dilución 12cH. Resultados: 2 patrones de crecimiento de las bacterias se han observado, asociado a la calidad de los nutrientes en el medio de cultivo. En medio rico en nutrientes, nosódios de E. coli 12cH tuvo un importante efecto inhibitorio; en médios pobres en nutrientes, Merc 12cH haz ejercido importante efecto inhibitorio. Conclusión: los resultados sugieren que las condiciones anteriores de los sistemas procariotos pueden influir en la respuesta in vitro a los remedios homeopáticos y isopáticos. Palabras-clave: Infección del tracto urinario; Felinos; Escherichia coli Uropatogenica ; Homeopatía; Isopatía  Correspondence author: Leoni Villano Bonamin, [email protected] How to cite this article: Kawakami AP; Osugui L; César AT; Priven SW; Carvalho VM; Bonamin LV. In vitro growth of uropathogenic Escherichia coli isolated from a snow leopard treated with homeopathic and isopathic remedies: a pilot study. Int J High Dilution Res [online]. 2009 [cited YYYY Month dd]; 8 (27): 41-44. Available from: http://journal.giri-society.org/index.php/ijhdr/article/view/341/394. ÂÂÂ

Cross-feeding between intestinal pathobionts promotes their overgrowth during undernutrition

Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition.

Compounds from the Medicines for Malaria Venture Box Inhibit In Vitro Growth of Babesia divergens, a Blood-Borne Parasite of Veterinary and Zoonotic Importance

Babesiosis is an infectious disease with an empty drug pipeline. A search inside chemical libraries for novel potent antibabesial candidates may help fill such an empty drug pipeline. A total of 400 compounds (200 drug-like and 200 probe-like) from the Malaria Box were evaluated in the current study against the in vitro growth of Babesia divergens (B. divergens), a parasite of veterinary and zoonotic importance. Novel and more effective anti-B. divergens drugs than the traditionally used ones were identified. Seven compounds (four drug-like and three probe-like) revealed a highly inhibitory effect against the in vitro growth of B. divergens, with IC50s ≤ 10 nanomolar. Among these hits, MMV006913 exhibited an IC50 value of 1 nM IC50 and the highest selectivity index of 32,000. The atom pair fingerprint (APfp) analysis revealed that MMV006913 and MMV019124 showed maximum structural similarity (MSS) with atovaquone and diminazene aceturate (DA), and with DA and imidocarb dipropionate (ID), respectively. MMV665807 and MMV665850 showed MMS with each other and with ID. Of note, a high concentration (0.75 IC50) of MMV006913 caused additive inhibition of B. divergens growth when combined with DA at 0.75 or 0.50 IC50. The Medicines for Malaria Venture box is a treasure trove of anti-B. divergens candidates according to the obtained results.

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

In vitro growth inhibitory activity of Medicines for Malaria Venture pathogen box compounds against Leishmania aethiopica

Introduction Leishmania aethiopica (L. aethiopica) is responsible for different forms of cutaneous leishmaniasis (CL) in Ethiopia. Treatment heavily depends on limited drugs, together with drawbacks like toxicity and microbial resistance. The current research aimed to investigate in vitro growth inhibitory activity of Medicines for Malaria Ventures - Pathogen Box (MMV - PB) compounds against L. aethiopica clinical isolate. Methodology Four hundred MMV – PB compounds were screened against L. aethiopica using resazurin based colourimetric assay. Compounds with > 70% inhibition were further tested using macrophage based intracellular amastigote assay. Cytotoxic and hemolytic activity of candidate hits were assessed on THP1- cells and sheep red blood cells (RBCs), respectively. In vitro drug interaction study was also conducted for the most potent hit using the combination index method. Results At the test concentration of 1 μM, twenty-three compounds showed > 50% inhibition of promastigotes parasite growth, of which 11 compounds showed > 70% inhibition. The 50% growth inhibition (IC50) of the 11 compounds was ranged from 0.024 to 0.483 μM in anti-promastigote assay and from 0.064 to 0.899 μM in intracellular amastigote assay. Candidate compounds demonstrated good safety on sheep RBCs and THP-1 cell lines. MMV688415 demonstrated a slight hemolytic activity on sheep RBC (5.3% at 25 μM) and THP-1 cell line (CC20 = 25 μM) while MMV690102 inhibited half of THP-1 cells at 36.5 μM (selectivity index = 478). No synergistic activity was observed from the combinations of MMV690102 and amphotericin B (CI > 1), and MMV690102 and Pentamidine (CI > 1) at lower and higher combination points. Conclusion The present study identified a panel of compounds that can be used as a novel starting point for lead optimization. MMV690102 appears to be the most potent inhibitor against L. aethiopica promastigotes and amastigotes. Future works should investigate the antileishmanial mechanism of action and in vivo antileishmanial activities of identified hits.

Insights from Leishmania (Viannia) guyanensis in vitro behavior and intercellular communication

Background Pentavalent antimonial-based chemotherapy is the first-line approach for leishmaniasis treatment and disease control. Nevertheless antimony-resistant parasites have been reported in some endemic regions. Treatment refractoriness is complex and is associated with patient- and parasite-related variables. Although amastigotes are the parasite stage in the vertebrate host and, thus, exposed to the drug, the stress caused by trivalent antimony in promastigotes has been shown to promote significant modification in expression of several genes involved in various biological processes, which will ultimately affect parasite behavior. Leishmania (Viannia) guyanensis is one of the main etiological agents in the Amazon Basin region, with a high relapse rate (approximately 25%). Methods Herein, we conducted several in vitro analyses with L. (V.) guyanensis strains derived from cured and refractory patients after treatment with standardized antimonial therapeutic schemes, in addition to a drug-resistant in vitro-selected strain. Drug sensitivity assessed through Sb(III) half-maximal inhibitory concentration (IC50) assays, growth patterns (with and without drug pressure) and metacyclic-like percentages were determined for all strains and compared to treatment outcomes. Finally, co-cultivation without intercellular contact was followed by parasitic density and Sb(III) IC50 measurements. Results Poor treatment response was correlated with increased Sb(III) IC50 values. The decrease in drug sensitivity was associated with a reduced cell replication rate, increased in vitro growth ability, and higher metacyclic-like proportion. Additionally, in vitro co-cultivation assays demonstrated that intercellular communication enabled lower drug sensitivity and enhanced in vitro growth ability, regardless of direct cell contact. Conclusions Data concerning drug sensitivity in the Viannia subgenus are emerging, and L. (V.) guyanensis plays a pivotal epidemiological role in Latin America. Therefore, investigating the parasitic features potentially related to relapses is urgent. Altogether, the data presented here indicate that all tested strains of L. (V.) guyanensis displayed an association between treatment outcome and in vitro parameters, especially the drug sensitivity. Remarkably, sharing enhanced growth ability and decreased drug sensitivity, without intercellular communication, were demonstrated. Graphical Abstract