Cell morphology impact on the set-up of electroporation protocols for in-suspension and adhered breast cancer cells.

In order to establish cancer-type-specific electroporation protocols for breast cancer, electroporation was performed in vitro in two modalities: in-suspension and adhered cells. Electroporation of cell suspensions was carried out through commercial electroporation cuvettes whereas a novel electrode for electroporation of adhered cells was designed and manufactured aimed to preserve cell structure, to provide a closer model to an in vivo scenario, and as a means to visualize the mechanical effects of electroporation on the cell membrane by using scanning electron microscopy. Electroporation protocols and electric field thresholds were predicted in silico and experimentally tuned through propidium iodide uptake and cell viability. Three breast-cancer cell lines (BT-20, MCF-7 and HCC1419) and a non-cancerous cell line (BEAS-2B) were used. Cancerous cells responded differently to electroporation depending on the electric parameters, cell histology, the cell culture modality, and the cell morphology (membrane thickness mainly), which was evaluated trough confocal and transmission electron microscopy. Particularly, it was found that electrochemotherapy may represent a promising alternative as an adjuvant treatment of metastatic breast tumours, and as a neoadjuvant therapy for Her2/neu tumours. Oppositely, triple negative breast tumours may show a high sensitivity to electroporation and therefore, they could be efficiently treated with irreversible electroporation. On the other hand, noncancerous cells demanded the highest voltage in both cell culture modalities in order to be electroporated. Hence, these cells in suspension may provide a reliable, easy-to-perform, low-cost model for the development of electroporation protocols for eradication of healthy tissue around a tumour in a safety margin.

Myo1g is an active player in maintaining cell stiffness in B-lymphocytes.

B-lymphocytes are migrating cells that specialize in antigen presentation, antibody secretion, and endocytosis; these processes implicate the modulation of plasma membrane elasticity. Cell stiffness is a force generated by the interaction between the actin-cytoskeleton and the plasma membrane, which requires the participation of several proteins. These proteins include class I myosins, which are now considered to play a role in controlling membrane-cytoskeleton interactions. In this study, we identified the motor protein Myosin 1g (Myo1g) as a mediator of this phenomenon. The absence of Myo1g decreased the cell stiffness, affecting cell adhesion, cell spreading, phagocytosis, and endocytosis in B-lymphocytes. The results described here reveal a novel molecular mechanism by which Myo1g mediates and regulates cell stiffness in B-lymphocytes. © 2016 Wiley Periodicals, Inc.

Morphological and proteomic characterization of midgut of the malaria vector Anopheles albimanus at early time after a blood feeding.

The midgut of anopheline mosquito is the entry of Plasmodium, the causative agent of malaria.When the mosquito feeds on parasite infected host, Plasmodium parasites reach the midgut and must confront digestive enzymes, the innate immune response and go across the peritrophic matrix (PM), a thick extracellular sheath secreted by the mosquito midgut epithelial cells. Then, to continue its development, the parasite must reach the salivary glands to achieve transmission to a vertebrate host. We report here the morphological and biochemical descriptions of the midgut changes after a blood meal in Anopheles albimanus. Before blood feeding, midgut epithelial cells contained numerous electrondense vesicles distributed in the central to apical side. These vesicles were secreted to the luminal side of the midgut after a blood meal. At early times after blood ingest, the PM is formed near microvilli as a granulous amorphous material and after it consolidates forming a highly organized fibrillar structure, constituted by layers of electrondense and electronlucent regions. Proteomic comparative analysis of sugar and blood fed midguts showed several molecules that modify their abundance after blood intake; these include innate immunity, cytoskeletal, stress response, signaling, and digestive, detoxifying and metabolism enzymes. Biological significance In the midgut of mosquitoes during bloodfeeding, many simultaneous processes occur, including digestion, innate immune activities, cytoskeleton modifications, construction of a peritrophic matrix and hormone production, between others. Mechanical forces are very intense during bloodfeeding and epithelial and muscular cells must resist the stress, modifying the actin cytoskeleton and coordinating intracellular responses by signaling. Microorganisms present in midgut contents reproduce and interact with epithelial cells triggering innate immune response. When infectious agents are present in the blood meal they must traverse the peritrophic matrix, an envelope formed from secretion products of epithelial cells, and evade the immune system in order to reach the epithelium and continue their journey towards salivary glands, in preparation for the transmission to the new hosts. During all these processes, proteins of mosquitoes are modified in order to deal with mechanical and biological challenges, and the aim of this work is to study these changes.

Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

Src and PI3 K inhibitors affect the virulence factors of Entamoeba histolytica.

Protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. A large number of protein kinases within the protein kinome of Entamoeba histolytica strongly suggest that protein phosphorylation is a key component of pathogenesis regulation by this parasite. PI3 K and Src are kinases previously described in this parasite, but their role is poorly understood. Here, the effect of Src-1-inhibitor and PI3 K inhibitor (Wortmannin) on the virulence factors of E. histolytica was evaluated. Results show that both inhibitors affect the actin cytoskeleton and the amoebic movement. Also, the proteolytic activity is diminished by Wortmannin, but not by Src-inhibitor-1; however, the phagocytic capacity is diminished by Wortmannin and Src-1-inhibitor. Finally, we found that the virulence in vivo of E. histolytica is affected by Wortmannin but not by Src-1-inhibitor. This study opens the way for the design of anti-amoebic drugs based on kinase inhibition.

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis.

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.

Evaluation of the in vitro effect of albendazole, metronidazole and nitazoxanide on viability and structure of Giardia lamblia cysts.

Effect of albendazole, metronidazole and nitazoxanide on the viability and structure of Giardia lamblia cysts isolates from infected children. The viability was evaluated by inducing excystation in a low-pH solution followed by an incubation in TYI-S culture medium. Nitazoxanide exhibited potent inhibitory effect (100%), metronidazole (79%) and albendazole (31%). The analysis among groups indicated a rs = 0.75 and p < 0.05. By TEM the cysts incubated with albendazole did not show morphological changes; with metronidazole, the formation of residual bodies in the nucleus border was observed. Incubated with nitazoxanide the damage to the cyst wall was evident, with the formation of areas with a granular content and the presence of cytoplasmic components in the peritrophic space. Our results propose that nitazoxanide showed a high effect on the viability and structure of G. lamblia cysts.

Ultrastructural study of Entamoeba invadens encystation and excystation.

In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.

Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus).

Gerbils (Meriones unguiculatus) were intragastrically inoculated with axenic Giardia lamblia cultures from symptomatic and asymptomatic children. All isolates were able to colonize the duodenum. However, the colonization capacity of the symptomatic isolates was significantly higher compared to that of the asymptomatic ones. Despite the different colonization capacity of the isolates, the growth curves of infected animals were significantly lower than those of controls. The study demonstrates that acute giardia infections are capable of altering the corporal development of the host. These results may suggest that not only symptomatic, but also asymptomatic giardiasis in children, often unnoticed by parents and clinicians, could be causing a silent detriment in their nutritional status.

Trichinella spiralis: ultrastructure of the body wall of newborn and muscle larvae.

The body wall structure of muscle and newborn larvae of Trichinella spiralis was studied using transmission and scanning electron microscopy. Differences were found in the structure of the cuticle of the two developmental stages. In the case of the cuticle of the muscle larvae only transverse striae were present whereas the newborn larvae cuticle showed both transverse and longitudinal striations. In the two parasite stages the outer surface of the cuticle appears as a three-layered structure. The hypodermis presents well defined cellular components similar in fine structure in both stages. The plasma membrane of the hypodermal cells in the muscle larvae shows abundant short finger-like projections, that are not present in newborn larvae.