Clues on the dynamics of DNA replication in Giardia lamblia.

Genomic replication is a critical, regulated process that ensures accurate genetic information duplication. In eukaryotic cells, strategies have evolved to prevent conflicts between replication and transcription. Giardia lamblia, a binucleated protozoan, alternates between tetraploid and octaploid genomes during its cell cycle. Using single-molecule techniques like DNA combing and nanopore-based sequencing, we investigated the spatio-temporal organization of DNA replication, replication fork progression and potential head-on replication-transcription collisions in Giardia trophozoites. Our findings indicate that Giardia chromosomes are replicated from only a few active origins, which are widely spaced and exhibit faster replication rates compared to those in other protozoan parasites. Immunofluorescence assays revealed that ∼20% of trophozoites show asynchronous replication between nuclei. Forksense and gene ontology analyses disclosed that genes in regions with potential head-on collisions are linked to chromatin dynamics, cell cycle regulation and DNA replication/repair pathways, possibly explaining the observed asynchronous replication in part of the population. This study offers the first comprehensive view of replication dynamics in Giardia, which is the pathogen that causes giardiasis, a diarrheal disease impacting millions worldwide.

Pleonotoquinones, Cytotoxic Oxepinenaphthoquinones from Pleonotoma jasminifolia.

Two unusual naphthoquinones, named here as pleonotoquinones A (1) and B (2), were isolated along with two known anthraquinones (3 and 4) via chromatographic separations of an ethyl acetate extract of the roots of Pleonotoma jasminifolia. Compounds 1 and 2 are the first examples of quinones bearing a 2-methyloxepine moiety. The compounds were isolated with the aid of mass spectrometry and molecular networking, and their structures were resolved using 1D and 2D NMR and HRESIMS data. The isolated compounds were evaluated for their antiproliferative activity against human cancer cell lines, and compounds 1 and 2 displayed cytotoxicity against human colon cancer HCT116 cells (IC50 = 2.6 µM for compound 1 and IC50 = 4.3 µM for compound 2) and human liver cancer HepG2 cells (IC50 = 1.9 µM for compound 1 and IC50 = 6.4 µM for compound 2).

Targeted Isolation of Anti-inflammatory Lignans from Justicia aequilabris by Molecular Networking Approach.

Herein, the isolation of secondary metabolites from the aerial parts of Justicia aequilabris guided by HPLC-MSn and molecular networking analyses is reported. Twenty-two known compounds were dereplicated. Three new lignans (aequilabrines A-C (1-3)) and three known compounds (lariciresinol-4'-O-ß-glucose (4), roseoside (5), and allantoin (6)) were obtained. The anti-inflammatory activity of compounds 1-3 was evaluated in vitro by inhibiting the nitric oxide production (NO) and pro-inflammatory activity on the cytokine IL-1ß. Compounds 2 and 3 showed significant inhibitory activity against NO production, with IC50 values of 9.1 and 7.3 µM, respectively. The maximum inhibition of IL-1ß production was 23.5% (1), 27.3% (2), and 32.5% (3).

New Pregnane Glycosides from Mandevilla dardanoi and Their Anti-Inflammatory Activity.

Mandevilla Lindl. is an important genus of the Apocynaceae family, not only as ornamental plants but also for its medicinal uses. In Brazil, Mandevilla species are indicated to treat asthma and skin infections, their anti-inflammatory potential and wound healing properties are also reported in the literature. Concerning their chemical composition, this group of plants is a conspicuous producer of pregnane glycosides. Mandevilla dardanoi is an endemic species from the Brazilian semiarid region not studied by any phytochemical methods. In view of the medicinal potential of Mandevilla species, this study aimed to isolate new pregnane glycosides from M. dardanoi. To achieve this main goal, modern chromatography techniques were employed. Five new pregnane glycosides, dardanols A-E, were isolated from the roots of M. dardanoi by HPLC. Their structures were determined using extensive 1D and 2D-NMR and mass spectrometry (MSn and HRESIMS) data. The cytotoxicity and the anti-inflammatory potential of these compounds were evaluated. The first was evaluated by measuring proinflammatory cytokines and nitric oxide production by stimulated macrophages. Dardanols were able to inhibit the production of nitric oxide and reduce IL-1ß and TNF-α. The current work demonstrates the chemodiversity of Brazilian semiarid species and contributes to amplifying knowledge about the biological potential of the Mandevilla genus.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

BACKGROUND: Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. METHODS AND RESULTS: Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. CONCLUSIONS: We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. GENERAL SIGNIFICANCE: LCalA is the first reported calmodulin that binds in vivo telomeric DNA.

Ribifolin, an orbitide from Jatropha ribifolia, and its potential antimalarial activity.

A new orbitide named ribifolin was isolated and characterized from Jatropha ribifolia using mass spectrometry, NMR spectroscopy, quantitative amino acid analysis, molecular dynamics/simulated annealing, and Raman optical activity measurements and calculations. Ribifolin (1) and its linear form (1a) were synthesized by solid-phase peptide synthesis, followed by evaluation of its antiplasmodial and cytotoxicity activities. Compound 1 was moderately effective (IC50 = 42 µM) against the Plasmodium falciparum strain 3D7.

Fermenting Acerola (Malpighia emarginata D.C.) and Guava (Psidium guayaba L.) Fruit Processing Co-Products with Probiotic Lactobacilli to Produce Novel Potentially Synbiotic Circular Ingredients.

This study evaluated the effects of acerola and guava fruit processing co-products fermented with probiotic Lactobacillus acidophilus LA-05 and Lacticaseibacillus paracasei L-10 on the abundance of different intestinal bacterial groups and microbial metabolic activity during 48 h of in vitro fecal fermentation. Digested fermented fruit co-products increased the relative abundance of beneficial bacterial groups while overall decreasing or maintaining the relative abundance of non-beneficial bacterial groups, suggesting selective stimulatory effects on beneficial bacterial intestinal populations. The fermented co-products stimulated microbial metabolic activity due to decreased pH, sugar consumption, short-chain fatty acid production, phenolic compound and metabolic profile alteration, and high antioxidant capacity during fecal fermentation. Acerola and guava co-products have high nutritional value and bioactive compounds whose fermentation with probiotics improves their potential functionalities. The results show that fermented fruit co-products could induce beneficial changes in the relative abundance of several bacterial groups as well as in the metabolic activity of the human intestinal microbiota. These results highlight their potential as novel and circular candidates for use as synbiotic ingredients.

Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.

Mass Spectrometry-Based Investigation of Sugarcane Exposed to Five Different Pesticides.

The use of agrochemicals has become a standard practice worldwide to ensure the productivity and quality of sugarcane crops. This study aimed to analyze the metabolic changes in sugarcane culms treated with five different nematicides. The experimental design was randomized in blocks, and agro-industrial and biometric variables were evaluated. The samples were extracted and then analyzed using LC-MS, LC-MS/MS, and LC-HRMS. The data obtained were submitted to statistical methods (PCA and PLS). Fragmentation patterns, retention time, and UV absorptions of the main features were analyzed. The plantations treated with carbosulfan (T4) obtained higher agricultural productivity and total recoverable sugar (TRS), while the use of benfuracarb (T3) was associated with lower growth and lower TRS. Statistical analysis revealed the contribution of the features at m/z 353 and m/z 515, assigned as chlorogenic acids, which discriminated the groups. The MS profile also supported the occurrence of flavonoids (C-glycosides and O-glycosides) in the samples.

Self-organizing maps of molecular descriptors for sesquiterpene lactones and their application to the chemotaxonomy of the Asteraceae family.

The Asteraceae, one of the largest families among angiosperms, is chemically characterised by the production of sesquiterpene lactones (SLs). A total of 1,111 SLs, which were extracted from 658 species, 161 genera, 63 subtribes and 15 tribes of Asteraceae, were represented and registered in two dimensions in the SISTEMATX, an in-house software system, and were associated with their botanical sources. The respective 11 block of descriptors: Constitutional, Functional groups, BCUT, Atom-centred, 2D autocorrelations, Topological, Geometrical, RDF, 3D-MoRSE, GETAWAY and WHIM were used as input data to separate the botanical occurrences through self-organising maps. Maps that were generated with each descriptor divided the Asteraceae tribes, with total index values between 66.7% and 83.6%. The analysis of the results shows evident similarities among the Heliantheae, Helenieae and Eupatorieae tribes as well as between the Anthemideae and Inuleae tribes. Those observations are in agreement with systematic classifications that were proposed by Bremer, which use mainly morphological and molecular data, therefore chemical markers partially corroborate with these classifications. The results demonstrate that the atom-centred and RDF descriptors can be used as a tool for taxonomic classification in low hierarchical levels, such as tribes. Descriptors obtained through fragments or by the two-dimensional representation of the SL structures were sufficient to obtain significant results, and better results were not achieved by using descriptors derived from three-dimensional representations of SLs. Such models based on physico-chemical properties can project new design SLs, similar structures from literature or even unreported structures in two-dimensional chemical space. Therefore, the generated SOMs can predict the most probable tribe where a biologically active molecule can be found according Bremer classification.

Single-cell transcriptomics reveals hidden information in trypanosomatids.

Dixenic parasites often encounter environmental extremes during the transition from vector to host. Preadapted transmission stages overcome these challenges to promote parasites' survival and ensure life cycle progression. Recently, Vigneron et al. and Briggs et al. used single-cell transcriptomics to investigate developmental stage specific gene expression patterns during parasite differentiation.

The Trypanosomatids Cell Cycle: A Brief Report.

Trypanosomatids are protozoan parasites among which are the etiologic agents of various infectious diseases in humans, such as Trypanosoma cruzi (causative agent of Chagas disease), Trypanosoma brucei (causative agent of sleeping sickness), and species of the genus Leishmania (causative agents of leishmaniases). The cell cycle in these organisms presents a sequence of events conserved throughout evolution. However, these parasites also have unique characteristics that confer some peculiarities related to the cell cycle phases. This review compares general and peculiar aspects of the cell cycle in the replicative forms of trypanosomatids. Moreover, a brief discussion about the possible cross-talk between telomeres and the cell cycle is presented. Finally, we intend to open a discussion on how a profound understanding of the cell cycle would facilitate the search for potential targets for developing antiparasitic therapies that could help millions of people worldwide.

Synchronization of Leishmania amazonensis Cell Cycle Using Hydroxyurea.

Leishmania spp. comprises a group of protozoan parasites that affect millions of people around the world. Understanding the main cell cycle-dependent events could provide an important route for developing specific therapies since some factors involved in cell cycle control may have low similarity relative to their homologs in mammals. Furthermore, accurate cell cycle-dependent analyses often require many cells, which can be achieved through cell cycle synchronization. Here, we described a useful method to synchronize procyclic promastigote forms of Leishmania amazonensis using hydroxyurea (HU) and the analysis of its DNA content profile. This approach can be extended to other trypanosomatids, such as Trypanosoma cruzi or Trypanosoma brucei, and provides an effective method for arresting more than 80% of cells at the G1/S phase transition.

Antitumor and antiangiogenic effects of Tonantzitlolone B, an uncommon diterpene from Stillingia loranthacea.

Natural products have played a pivotal role for the discovery of anticancer drugs. Tonantzitlolones are flexibilan-type diterpenes rare in nature; therefore, few reports have shown antiviral and cytotoxic activities. This study aimed to investigate the in vivo antitumor action of Tonantzitlolone B (TNZ-B) and its toxicity. Toxicity was evaluated in mice (acute and micronucleus assays). Antitumor activity of TNZ-B (1.5 or 3 mg/kg intraperitoneally - i.p.) was assessed in Ehrlich ascites carcinoma model. Angiogenesis and reactive oxygen species (ROS) and nitric oxide (NO) production were also investigated, in addition to toxicological effects after 7-day treatment. The LD50 (lethal dose 50%) was estimated at around 25 mg/kg (i.p.), and no genotoxicity was recorded. TNZ-B reduced the Ehrlich tumor's volume and total viable cancer cell count (p < 0.001 for both). Additionally, TNZ-B reduced peritumoral microvessel density (p < 0.01), suggesting antiangiogenic action. Moreover, a decrease was observed on ROS (p < 0.05) and nitric oxide (p < 0.001) levels. No significant clinical findings were observed in the analysis of biochemical, hematological, and histological (liver and kidney) parameters. In conclusion, TNZ-B exerts antitumor and antiangiogenic effects by reducing ROS and NO levels and has weak in vivo dose-repeated toxicity. These data contribute to elucidate the antitumor action of TNZ-B and point the way for further studies with this natural compound as an anticancer drug.

Tropane alkaloids from Erythroxylum caatingae Plowman.

Three tropane alkaloids, 1-3, were isolated from Erythroxylum caatingae, i.e., 6ß-benzoyloxy-3α-[(4-hydroxy-3,5-dimethoxybenzoyl)oxy]tropane (1), a new tropane alkaloid, along with the known alkaloids 3α,6ß-dibenzoyloxytropane (2) and 6ß-benzoyloxy-3α-[(3,4,5-trimethoxybenzoyl)oxy]tropane (catuabine B; 3). Their structures were determined by 2D- ((1) H and (13) C) NMR. By LC/ESI-MS/MS analysis of the fractions of alkaloids 1-3, it was possible to detect five more alkaloids, 4-8, two of these, 4 and 8, possibly being new natural products. X-Ray crystallography of the chloride derivate of 1, i.e., 6ß-benzoyloxy-3α-(4-hydroxy-3,5-dimethoxybenzoyloxy)tropane hydrochloride (1a) confirmed the structure of 1. Cytotoxicity was tested against the cell lines HEp-2, NCI-H292, and KB for the MeOH extract and alkaloid 3, and antitumor activity was tested against Sarcoma 180 only for the MeOH extract.

Autophagy buffers Ras-induced genotoxic stress enabling malignant transformation in keratinocytes primed by human papillomavirus.

Malignant transformation involves an orchestrated rearrangement of cell cycle regulation mechanisms that must balance autonomic mitogenic impulses and deleterious oncogenic stress. Human papillomavirus (HPV) infection is highly prevalent in populations around the globe, whereas the incidence of cervical cancer is 0.15%. Since HPV infection primes cervical keratinocytes to undergo malignant transformation, we can assume that the balance between transforming mitogenic signals and oncogenic stress is rarely attained. We showed that highly transforming mitogenic signals triggered by HRasG12V activity in E6E7-HPV-keratinocytes generate strong replication and oxidative stresses. These stresses are counteracted by autophagy induction that buffers the rapid increase of ROS that is the main cause of genotoxic stress promoted by the oncoprotein. As a result, autophagy creates a narrow window of opportunity for malignant keratinocytes to emerge. This work shows that autophagy is crucial to allow the transition of E6E7 keratinocytes from an immortalized to a malignant state caused by HRasG12V.

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

Leishmaniases belong to the inglorious group of neglected tropical diseases, presenting different degrees of manifestations severity. It is caused by the transmission of more than 20 species of parasites of the Leishmania genus. Nevertheless, the disease remains on the priority list for developing new treatments, since it affects millions in a vast geographical area, especially low-income people. Molecular biology studies are pioneers in parasitic research with the aim of discovering potential targets for drug development. Among them are the telomeres, DNA-protein structures that play an important role in the long term in cell cycle/survival. Telomeres are the physical ends of eukaryotic chromosomes. Due to their multiple interactions with different proteins that confer a likewise complex dynamic, they have emerged as objects of interest in many medical studies, including studies on leishmaniases. This review aims to gather information and elucidate what we know about the phenomena behind Leishmania spp. telomere maintenance and how it impacts the parasite's cell cycle.

In vitro and in silico studies of 8(17),12E,14-labdatrien-18-oic acid in airways smooth muscle relaxation: new molecular insights about its mechanism of action.

In the field of experimental pharmacology, researchers continuously investigate new relaxant agents of the airway smooth muscle cells (ASMCs), since the pathophysiology of respiratory illnesses, such as asthma, involves hyperresponsiveness and changes in ASMC homeostasis. In this scenario, labdane-type diterpenes, like forskolin (FSK), are a class of compounds known for their relaxing action on smooth muscle cells (SMCs), being this phenomenon related to the direct activation of AC-cAMP-PKA pathway. Considering the continuous effort of our group to study the mechanism of action and prospecting for compounds isolated from natural sources, in this paper, we presented how the diterpene 8(17),12E,14-labdatrien-18-oic acid (LBD) promotes relaxant effect on ASMC, performing in vitro experiments using isolated guinea pig trachea and in silico molecular docking/dynamics simulations. In vitro experiments showed that in the presence of aminophylline, FSK and LBD had their relaxant effect potentiated (EC50 from 1.4 ± 0.2 × 10-5 M to 1.5 ± 0.3 × 10-6 M for LBD and from 2.0 ± 0.2 × 10-7 M to 6.4 ± 0.4 × 10-8 M for FSK) while in the presence of Rp-cAMPS this effect was attenuated (EC50 from 1.4 ± 0.2 × 10-5 M to 3 × 10-4 M for LBD and from 2.0 ± 0.2 × 10-7 to 3.1 ± 1.0 × 10-6 M for FSK). Additionally, in silico simulations evidenced that the lipophilic character of LBD is probably responsible for its stability on AC binding site. LBD presented two preferential orientations, where the double bonds of the isoprene moiety as well as the unique polar group (carboxylic acid) in this compound form important anchoring points. In this sense, we consider that the LBD can interact stabilizing the catalytic dimmer of AC as the FSK, although less efficiently.

The Leishmania amazonensis TRF (TTAGGG repeat-binding factor) homologue binds and co-localizes with telomeres.

BACKGROUND: Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2. RESULTS: LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo. CONCLUSION: The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.

Tropane alkaloids from Erythroxylum genus: distribution and compilation of 13C-NMR spectral data.

Erythroxylum, the most representative genus of the Erythroxylaceae family, presents tropane alkaloids as main constituents. This class of compounds greatly contributes to the chemotaxonomic characterization of plants of this genus, and it has important medical uses and shows toxic effects. This review describes 186 tropane alkaloids in the 35 species of Erythroxylum distributed worldwide. In addition, a compilation of their (13)C-NMR spectral data is presented.