Deletion of the lipid droplet protein kinase gene affects lipid droplets biogenesis, parasite infectivity, and resistance to trivalent antimony in Leishmania infantum.

The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized CRISPR/Cas9 technology to generate LDK-knockout lines of Leishmania infantum to investigate its role in this parasite. Our findings demonstrate that LDK is not an essential gene for L. infantum, as its deletion did not impede parasite survival. Furthermore, removing LDK did not impact the growth of promastigote forms of L. infantum lacking LDK. However, a noticeable reduction in LDs occurred during the stationary phase of parasite growth following LDK deletion. In the presence of myriocin, a LD inducer, LDK-knockout parasites displayed reduced LD abundance during both logarithmic and stationary growth phases compared to control parasites. Moreover, an infection analysis involving THP-1 cells revealed that 72 h post-infection, LDK-knockout L. infantum lines exhibited fewer infected macrophages and intracellular amastigotes than control parasites. LDK-knockout L. infantum lines also displayed 1.7 to 1.8 -fold greater resistance to trivalent antimony than control parasites. There were no observed alterations in susceptibility to amphotericin B, miltefosine, or menadione in LDK-knockout L. infantum lines. Our results suggest that LDK plays a crucial role in the biogenesis and/or maintenance of LDs in L. infantum, as well as in parasite infectivity and resistance to trivalent antimony.

Secondary metabolites related to the resistance of Psidium spp. against the nematode Meloidogyneenterolobii.

The guava tree (Psidium guajava) is a tropical species native to South America and is recognized as the 11th most economically important fruit tree in Brazil. However, the presence of the nematode Meloidogyne enterolobii and the fungus Fusarium solani in the roots of guava plants leads to the development of root galls, causing significant damage. In contrast, the species P. guineense and P. cattleianum have been identified as resistant and immune to the nematode, respectively. In this study, the researchers aimed to compare the metabolomic profiles of infected and uninfected roots of P. guajava, P. cattleianum, and P. guineense using mass spectrometry coupled with liquid chromatography (LC-MS). The goal was to identify secondary metabolites that could potentially be utilized as biochemical resources for nematode control. The findings of the study demonstrated that the plant metabolism of all three species undergoes alterations in response to the phytopathogen inoculation. By employing molecular networks, the researchers identified that the secondary metabolites affected by the infection, whether produced or suppressed, are primarily of a polar chemical nature. Further analysis of the database confirmed the polar nature of the regulated substances after infection, specifically hydrolysable tannins and lignans in P. guineense and P. cattleianum. Interestingly, a group of non-polar substances belonging to the terpene class was also identified in the resistant and immune species. This suggests that these terpenes may act as inhibitors of M. enterolobii, working as repellents or as molecules that can reduce oxidative stress during the infection process, thus enhancing the guava resistance to the nematode. Overall, this study provides valuable insights into the metabolic alterations occurring in different Psidium spp. in response to M. enterolobii infection. The identification of specific secondary metabolites, particularly terpenes, opens up new possibilities for developing effective strategies to control the nematode and enhance guava resistance.

Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the chronic phase of the disease. The existence of drug-resistant T. cruzi strains and the occurrence of cross-resistance between BZ and NFX have also been described. In this context, it is urgent to study the metabolism of these drugs in T. cruzi, to better understand the mechanisms of resistance. Prostaglandin F2α synthase (PGFS) is an enzyme that has been correlated with parasite resistance to BZ, but the mechanism by which resistance occurs is still unclear. Our results show that the genome of the CL Brener clone of T. cruzi, contains five PGFS sequences and three potential pseudogenes. Using CRISPR/Cas9 we generated knockout cell lines in which all PGFS sequences were disrupted, as shown by PCR and western blotting analyses. The PGFS deletion did not alter the growth of the parasites or their susceptibility to BZ and NFX when compared to wild-type (WT) parasites. Interestingly, NTR-1 transcripts were shown to be upregulated in ΔPGFS mutants. Furthermore, the ΔPGFS parasites were 1.6 to 1.7-fold less tolerant to oxidative stress generated by menadione, presented lower levels of lipid bodies than the control parasites during the stationary phase, and were less infective than control parasites.

Development of the prickly pear cactus Opuntia stricta (Haw.) Haw. (Cactaceae) in vitro in response to the replacement of potassium nitrate for a commercial kno 3 fertilizer / Desenvolvimento da palma Opuntia stricta (Haw.) Haw. (Cactaceae) in vitro em resposta à substituição de nitrato de potássio (P.A.) por fertilizante comercial KNO 3

ABSTRACT: In micropropagation, potassium nitrate (KNO3), an ACS reagent grade chemical, used in the preparation of growing mediums is expensive and its procurement depends on bureaucratic procedures, as it is controlled by the Brazilian Army. This research to assessed the effect of replacing the ACS KNO3 for a commercially available fertilizer (KNO3- based) on the micropropagation of the prickly pear cactus (Opuntia stricta (Haw.) Haw. cv. Elephant Ear. Treatments used six different fertilizer concentrations (0, 0.5, 1, 1.5, 2 and 2.5 g L-1) and a control consisting of 1.9 g L-1 KNO3, as shown in the MS salts. The survival, size and number of sprouts and the value of fresh biomass were evaluated. After seedling acclimation, we assessed the survival, number of sprouts, length, and number of roots, racket formation, average fresh biomass mass, macronutrient absorption and morphological changes of the seedlings. Explants inoculated with fertilizers at concentrations of 0.0; 2.0 and 2.5 g L-¹ did not grow. The response of explants at concentrations of 0.5 and 1.5 g L-1 of the fertilizer were the same as those developed in a KNO3 medium, and at a concentration of 1.0 g L-1, in all variables, the means were higher than those of the control medium. Therefore, it showed the feasibility of using fertilizers in the in vitro cultivation of the prickly pear cactus, which may remove bureaucratic barriers and reduce product costs by 99.12%.

RESUMO: Na micropropagação, o nitrato de potássio (KNO3), reagente puro para análise (P.A.), utilizado no preparo dos meios de cultura, possui custo elevado e a sua aquisição depende de trâmites burocráticos, por se tratar de substância controlada pelo Exército Brasileiro. O objetivo deste trabalho foi avaliar o efeito da substituição do KNO3 P.A. por fertilizante comercial (com fonte de KNO3), encontrado livremente no comércio, na micropropagação de palma (Opuntia stricta (Haw.) Haw. cv Orelha de Elefante. Os tratamentos foram de seis concentrações do fertilizante (0; 0,5; 1; 1,5; 2 e 2,5 g L-1) e um controle constituído de 1,9 g L-1 de reagente KNO3, conforme mostrado nos sais MS. Avaliou-se a sobrevivência, tamanho e número de brotações do explante, e o valor da biomassa fresca. Após a aclimatização das mudas avaliou-se a sobrevivência, número de brotações, comprimento da parte aérea, número de raízes, formação da raquete, massa média da biomassa fresca, absorção de macronutrientes e alterações morfológicas das mudas. Os explantes inoculados em meio com fertilizantes nas concentrações de 0,0; 2,0 e 2,5 g L-¹ não se desenvolveram. A resposta dos explantes nas concentrações de 0,5 e 1,5 g L-1 do fertilizante foram iguais aos desenvolvidos em meio contendo KNO3, e na concentração de 1,0 g L-1, em todas as variáveis, as médias foram superiores em relação as do controle. Dessa forma, constatou-se a viabilidade do uso do fertilizante no cultivo in vitro da palma, o que propiciou a eliminação dos entraves burocráticos e redução no custo de 99,12% na compra do produto.

Evaluation of cobalt(III) complexes as potential hypoxia-responsive carriers of esculetin.

Differentiation between hypoxic and normoxic tissues have been exploited for the development of selective chemotherapeutic agents. In this context, cobalt(III)-based coordination compounds have been designed and investigated as prospective hypoxia-responsive drug delivery systems. Three cobalt(III) complexes, namely [CoIII(esc)(py2en)]ClO4·(CH3OH)2 (1) [CoIII(esc)(TPA)]ClO4·3H2O (2) and [CoIII(bipy)2(esc)]ClO4·2.5H2O (3) (py2en = N,N'-bis(pyridin-2-ylmethyl)ethylenediamine, TPA = tris(2-pyridylmethyl)amine, bipy = 2,2'-bipyridine and esc = 6,7-dihydroxycoumarin or esculetin), were prepared and investigated as potential carriers of esculetin. The spectroscopic and electrochemical properties of 1-3 were investigated and compared. Reactions of the complexes with biologically relevant reducing agents, viz. ascorbic acid, cysteine and glutathione, were monitored spectroscopically for 24 h, in pH 6.2 and 7.4 PBS phosphate buffer saline (PBS) solutions at 37 °C, under air, argon and dioxygen atmospheres. Dissociation of esculetin was observed upon Co3+/Co2+ reduction preferably under hypoxic conditions, with more effective conversion rates for 3 > 2 > 1. These results illustrate the importance to modulate the Co3+/Co2+ redox potential through the donor-acceptor properties of the ancillary ligands. Complex 3 is cytotoxic against HCT-116 but not against HT-29 and HEK-293 cells. In addition, DNA-binding studies indicate that interactions of 1 and 3 with the biomolecule are electrostatic.

Lapachol acetylglycosylation enhances its cytotoxic and pro-apoptotic activities in HL60 cells.

Lapachol is a plant-derived naphthoquinone that kills several types of cancer cells. Derivatives of this molecule may therefore prove to be useful chemotherapeutic agents. In this study, we explored whether glycosylation increases the cytotoxic potency of lapachol towards HL-60 human leukemia cells. Two beta-glycosides were synthesized and characterized: LA4A (lapachol-ß-glucoside) and LA4C (lapachol-N-acetylglucosamine-ß-glucoside). The sugar moieties of both novel molecules were per-acetylated to facilitate cellular uptake. The IC50 values (in µM) for LA4A (5.7) and LA4C (5.3) were lower than those for lapachol (25). LA4A and LA4C triggered typical signs of apoptosis, such as the exposure of phosphatidylserine on the outside of cells, chromatin condensation, DNA fragmentation and a decrease of the mitochondrial transmembrane potential (ΔΨm) prior to cell lysis. Moreover, DNA fragmentation triggered by the lapachol-glycosides was reduced by pre-treatment with the caspase inhibitor, z-VAD-fmk. While LA4A and LA4C activated caspases-3, -8 and -9, lapachol failed to activate these apoptotic proteases, even when used at high concentrations. Finally, the toxicity of lapachol and its derivatives was also tested on non-tumor cells. We used human peripheral neurons (PeriTox test) to evaluate the side effect potential of these compounds. LA4C was clearly less toxic than LA4A. We conclude that LA4C had the most favorable profile as drug candidate (high tumor cell toxicity, reduced neurotoxicity). In general, this study shows that the cytotoxicity of lapachol towards HL-60 can be enhanced by glycosylation, and that the therapeutic ratio may be modified by the type of sugar added.

Synthetic Mn(III) porphyrins as biomimetic catalysts of CYP450: Degradation of antibiotic norfloxacin in aqueous medium.

Norfloxacin (NOR) is a synthetic broad-spectrum fluoroquinolone antibiotic classified as an emerging contaminant. Here, we investigate Mn(III) porphyrin-catalyzed NOR degradation using peroxides or peracids (H2O2, t-BuOOH, or Oxone®) as oxidants. We evaluate three Mn(III) porphyrins: the 1st-generation tetraphenylporphyrin and 2nd -generation porphyrins bearing halogen atoms at the ortho-positions of the porphyrin macrocycle meso-aryl groups. Experiments were carried out in aqueous medium under mild conditions. NOR degradation was 67%. Products were proposed by mass spectrometry (MS) analysis. Oxone® was the best oxidant for NOR degradation despite its possible decomposition in the reaction medium. The second-generation Mn(III) porphyrins were more resistant than the first-generation Mn(III) porphyrin, indicating that the bulky groups introduced into the porphyrin macrocycle meso-aryl groups led to more robust catalysts. The degradation products did not present cytotoxic behavior under the employed conditions. In conclusion, Mn(III) porphyrin-catalyzed NOR degradation is a promising strategy to degrade fluoroquinolones and other pollutants.

Efficient atrazine degradation catalyzed by manganese porphyrins: Determination of atrazine degradation products and their toxicity evaluation by human blood cells test models.

Atrazine (ATZ) is an herbicide that has been considered an environmental pollutant worldwide. ATZ contaminates groundwaters and can persist in soils for up to a year causing several environmental and health problems. This study aimed to investigate ATZ degradation catalyzed by manganese porphyrins as biomimetic cytochrome P450 models. We used PhIO, PhI(OAc)2, H2O2, t-BuOOH, m-CPBA, or Oxone® as oxidant under mild conditions and evaluated a range of manganese porphyrins as catalyst. Concerning oxidant, iodosylbenzene provided the best result-ATZ degradation catalyzed by one of the studied manganese porphyrins in acetonitrile was as high as 47%. We studied the same catalyst/oxidant systems in natural water from a Brazilian river as solvent and obtained up to 100% ATZ degradation when iodobenzene diacetate was the oxidant, regardless of the manganese porphyrin. Besides the already known ATZ degradation products, we also identified unexpected degradation compounds (ring-opening products). Toxicity tests showed that the latter products were capable of proliferate blood cells because they did not show toxicity under the evaluated conditions.

Caracterização e estoques de carbono de sistemas agroflorestaisno Cerrado de Minas Gerais / Characterization and carbon storage of agroforestry systems in brazilian savannas of Minas Gerais, Brazil

Os sistemas agroflorestais (SAFs) além de geração de renda e conservação da biodiversidade pode ser uma alternativa para a estocagem de carbono. O trabalho teve como objetivo caracterizar três sistemas agroflorestais (SAFs) e estimar os estoques de carbono na fitomassa aérea, na serapilheira e no solo. Os SAFs e suas respectivas vegetações nativas adjacentes foram caracterizados quanto ao número, classificação botânica,distribuição diamétrica e estratificação vertical dos indivíduos arbóreose. Em cada um dos sistemas, foram estimados os estoques de carbono na fitomassa aérea, na serapilheira e no solo. Os SAFs,implantados a partir do manejo da vegetação nativa, apresentaram uma biodiversidade vegetal e estoque de carbono na fitomassa, na serapilheira e no solo semelhantes à área de vegetação nativa adjacente. Por outro lado, os estoques de carbono no SAF implantado em área anteriormente cultivada com culturas anuais apresentou menor diversidade de espécies arbóreas e estoques de carbono na fitomassa, serapilheira e no solo, em relação à vegetação nativa adjacente. Sistemas agroflorestais bem manejados se assemelham à vegetação nativa em estoque de carbono e conservação da biodiversidade dos recursos naturais.

Agroforestry systems (SAFs) as well as income generation and biodiversity conservation can be an alternative to carbon stocks. The study aimed to characterize and estimate the soil and plant carbon stocks of SAFs in Brazilian savannas of Minas Gerais State, Brazil. Three agroforestry systems and their adjacent native vegetation were evaluated. In addition, the forest inventory, the diameter distribution and vertical stratification of tree species were measured. The SAFs deployed from the management of native vegetation had a plant biodiversity and carbon stock in biomass, litter and soil similar to that of the adjacent area of native vegetation. On the other hand, carbon stocks in SAF deployed in the area cultivated with annual crops had lower species diversity and carbon stocks in biomass, litter and soil in relation to native vegetation adjacent. Agroforestry systems well managed have carbon stocks and biodiversity of natural resources like native vegetation.

Teor de óleo essencial de alecrim-pimenta em função do horário de colheita / Essential oil content of pepper-rosmarin as a function of harvest time

O alecrim-pimenta é uma importante planta medicinal, nativa do nordeste brasileiro, que, devido ao seu óleo essencial, apresenta grande potencial antifúngico e antibacteriano. Um dos fatores que afetam o teor de metabólitos secundários das plantas é o horário de colheita, assim, o conhecimento desse fator subsidia as decisões agronômicas da espécie, favorecendo o aumento do teor de óleo essencial no momento da colheita. Dessa forma, o objetivo do presente trabalho foi avaliar o efeito do horário de colheita na produção de óleo essencial de alecrim-pimenta (Lippia sidoides Cham.). O experimento foi desenvolvido no Instituto de Ciências Agrárias da Universidade Federal de Minas Gerais (ICA/UFMG), Montes Claros, Minas Gerais, Brasil. Utilizou-se o delineamento experimental em blocos casualizados com cinco tratamentos, definidos pelos horários de coleta das folhas (6:00, 9:00, 12:00, 15:00 e 18:00) e seis repetições, sendo esse o número de coletas em cada tratamento. As folhas foram obtidas de plantas matrizes do Horto Medicinal do ICA/UFMG. O óleo essencial foi obtido pelo método de hidrodestilação usando o aparelho de Clevenger. A análise de regressão apresentou um efeito cúbico para o teor de óleo essencial, sendo que o maior valor, 5,78 por cento em relação à matéria seca, foi obtido às 9h59min. Portanto, recomenda-se a colheita das folhas de alecrim-pimenta próximo das 10h.

The pepper-rosmarin is an important medicinal plant, native of Northeast of Brazil, that, because of its essential oil, presents great antifungal and antibacterial potential. One of the factors that affect the secondary metabolites content of plants is the harvest time, thus, the knowledge of this factor subsidizes the decisions agronomic species, favoring the increase of essential oil content at the harvest moment. Thus, the aim of this research was to evaluate the effect of harvest time on the essential oil production of pepper-rosmarin (Lippia sidoides Cham.). The experiment was carried out in the Instituto of Ciências Agrárias of Universidade Federal of Minas Gerais (ICA/UFMG), Montes Claros, Minas Gerais, Brazil. It was installed in a randomized block design, with five treatments consisted by harvest times (6:00, 9:00, 12:00, 15:00 and 18:00) with six replications (number of collections in each treatment). The leaves were obtained from stock plants of medicinal vegetable garden of ICA/ UFMG. The essential oil was obtained by hydrodistillation in a Clevenger apparatus. The regression analysis presented a cubic effect to the essential oil content, being that the highest value, 5,78 percent in relation to dry matter, was obtained at 9h59min. Therefore, it was recommended the harvest time of pepper-rosmarin leaves next to 10h.

Substituição de nitrato de potássio (PA) por salitre potássico no preparo de meio de cultura de tecidos vegetais esterilizado com hipoclorito de sódio / Potassium nitrate substitution for potassic saltpetre on preparation of plant tissue culture nutritive media esterilized with sodium hypochlorite

A aquisição de nitrato de potássio PA, para qualquer finalidade, depende de autorização do Ministério da Defesa, o que resulta em dificuldades na sua aquisição. Com a finalidade de estudar a possibilidade da substituição desse reagente por outro produto encontrado livremente no comércio, foi conduzido um experimento onde se testaram várias concentrações de salitre potássico em comparação com o nitrato de potássio PA, como componente dos sais de MS. Utilizou-se o produto granulado MURER® nas seguintes concentrações (g L-1): a) 7,0; b) 7,4; c) 7,8; d) 8,2. Foi adicionado um tratamento-controle, com o KNO3 PA, conforme aparece nos sais de MS (1,9 g L-1), sendo a espécie testada à fáfia, a partir de segmentos nodais. O meio de cultura constituiu-se dos sais de MS, vitaminas de White, 30 g L-1 sacarose, 2,0 mg L-1 ácido indol butírico, 2,0 mg L-1 ácido naftalenoacético, 2,0 mg L-1 benzilaminopurina, pH 6,0 + 0,1 e 1,5 g L-1 Phytagel® como gelificante. O número médio de ramos formados para todos os tratamentos foi inferior ao do tratamento-controle; mas, em compensação, o comprimento médio deles foi superior, também em todos os tratamentos, sendo que o comprimento total de ramos na menor concentração de salitre, atingiu 17,0 cm, enquanto no tratamento-controle foi de 12,6 cm. A média da biomassa seca foi de 68,3 mg para a menor concentração de salitre e de 50,0 mg para o tratamento-controle. A análise dos resultados indicou ser vantajosa a substituição do nitrato de potássio PA pelo salitre potássico.

The acquisition of potassium nitrate for any purpose, including preparation of nutritive media for micropropagation, depends on authorization from the Ministry of Defense, what turns out in difficulties in its acquisition. With the purpose of studying the possibility of replacing this component of the culture for another product found freely in the market, an experiment was carried out, where several concentrations of potassic saltpetre were tried, in comparison to the chemically pure potassium nitrate, as a component of the MS salts formulation. The commercial granulated product MURER® was used in the following (g L-1): a) 7,0; b) 7,4; c) 7,8; d) 8,2; and e) chemically pure potassium nitrate (control-treatment) as it appears in the MS salts, or 1,9 g L-1Fáfia was tested, starting from nodal segments. The basal nutrient medium comprised the MS salts, White vitamins, 30 g L-1 sucrose, 2.0 mg L-1 indol butyric acid, 2.0 mg L-1 naftaleneacetic acid, 2.0 mg L-1 benzilaminopurine, pH 6.0 + 0.1 and 1.5 g L-1 Phytagel® as the gelling agent. The number of branches formed in all concentrations of potassic saltpetre was lower than those ones in the control treatment; however, their lengths were higher. The mean length of branches in the treatment with the smallest potassic saltpetre concentration, reached 17.0 cm in length, while in the control-treatment it was 12,6 cm. The dry biomass weight reached 68.3 mg for the smallest potassic saltpetre concentration, but only 50,0 mg for the control treatment. The results indicated to be advantageous the substitution of the chemically pure potassium nitrate for the potassic saltpeter, when either shoot elongation or biomass production is desired, being the nutrient medium sterilized with sodium hipoclorite. However, when a faster multiplication rate is the main objective, the nutrient medium should be prepared with chemically pure potassium nitrate.