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.

A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.

BACKGROUND: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. METHODS AND RESULTS: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3'-5' Exonuclease I digestion, and unfold telomeric G-quadruplex. CONCLUSIONS: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.

Landscape of heart proteome changes in a diet-induced obesity model.

Obesity is a pandemic associated with a high incidence of cardiovascular disease; however, the mechanisms are not fully elucidated. Proteomics may provide a more in-depth understanding of the pathophysiological mechanisms and contribute to the identification of potential therapeutic targets. Thus, our study evaluated myocardial protein expression in healthy and obese rats, employing two proteomic approaches. Male Wistar rats were established in two groups (n = 13/group): control diet and Western diet fed for 41 weeks. Obesity was determined by the adipose index, and cardiac function was evaluated in vivo by echocardiogram and in vitro by isolated papillary muscle analysis. Proteomics was based on two-dimensional gel electrophoresis (2-DE) along with mass spectrometry identification, and shotgun proteomics with label-free quantification. The Western diet was efficient in triggering obesity and impaired contractile function in vitro; however, no cardiac dysfunction was observed in vivo. The combination of two proteomic approaches was able to increase the cardiac proteomic map and to identify 82 differentially expressed proteins involved in different biological processes, mainly metabolism. Furthermore, the data also indicated a cardiac alteration in fatty acids transport, antioxidant defence, cytoskeleton, and proteasome complex, which have not previously been associated with obesity. Thus, we define a robust alteration in the myocardial proteome of diet-induced obese rats, even before functional impairment could be detected in vivo by echocardiogram.

Moderate exercise training does not prevent the reduction in myocardial L-type Ca2+ channels protein expression at obese rats.

Authors have showed that obesity implicates cardiac dysfunction associated with myocardial L-type calcium channels (LTCCs) activity impairments, as well as moderate exercise training (MET) seems to be an important therapeutic tool. We tested the hypothesis that MET promotes improvements on LTCCS activity and protein expression at obesity induced by unsaturated high-fat diets, which could represent a protective effects against development of cardiovascular damage. Male Wistar rats were randomized in control (C, n = 40), which received a standard diet and obese (Ob; n = 40), which received high-fat diet. After 20 weeks, the animals were assigned at four groups: control (C; n = 12); control submitted to exercise training (ET; n = 14); obese (Ob; n = 10); and obese submitted to exercise training (ObET; n = 11). ET (5 days/week during 12 weeks) began in the 21th week and consisted of treadmill running that was progressively increased to reach 60 min. Final body weight (FBW), body fat (BF), adiposity index (AI), comorbidities, and hormones were evaluated. Cardiac remodeling was assessed by morphological and isolated papillary muscles function. LTCCs activity was determined using specific blocker, while protein expression of LTCCs was evaluated by Western blot. Unsaturated high-fat diet promoted obesity during all experimental protocol. MET controlled obesity process by decreasing of FBW, BF, and AI. Obesity implicated to LTCCs protein expression reduction and MET was not effective to prevent this condition. ET was efficient to promote several improvements to body composition and metabolic parameters; however, it was not able to prevent or reverse the downregulation of LTCCs protein expression at obese rats.