Important Morphological Alterations Research Articles

Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of M. abscessus infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of M. abscessus in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat M. abscessus skin infections requires further evaluations.IMPORTANCEMycobacterium abscessus causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against M. abscessus in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against M. abscessus skin infections.

Treatment with Candida albicans biotherapic influences in vitro fungal adhesion to Ma-104 cells

Background: Oral candidiasis is an opportunist fungal infection in humans, mainly caused by Candida albicans. It occurs when the host presents an imbalance in the immune system and Candida spp., normally found in human flora, become able to develop the infection [1]. This disease is very common in HIV patients, and in all individuals that present immunossupression, such as patients treated with chemotherapy. Considering this scenario, the development of new medicines to treat oral candidiasis is mandatory.
 
 Aims: The aim of this study was to evaluate citotoxicity, morphology and quantify the adhesion rates of C. albicans to biotherapic-treated Ma104 cells.
 
 Methodology: The biotherapic was prepared following the Roberto Costa technique and Brazilian Homeopathic Pharmacopeia protocol [2]. Briefly, biotherapic 1X was prepared with 1 mL of aqueous solution containing 108 yeasts of living Candida albicans plus 9 ml of sterile distilled water. This solution was submmited to 100 mechanical succussions. Biotherapic 2X was obtained after addition of 1 ml of 1X solution in 9 ml of sterile distilled water and it was also submitted to 100 mechanical succussions. This procedure was repeated until biotherapic 30X was obtained. As a control, sterile dynamized water (30X) was used. The inhibition of fungal growth induced by biotherapic was evaluated by MTT method after 24 hours of treatment. The morphological aspects of Ma104-biotherapic-treated cells were analyzed by Giemsa staining after 5, 10 and 60 days, and compared with control groups (water 30X and untreated cells). Additionally, Ma104 cells were treated during 5 and 30 days with biotherapic in parallel with respective controls, and the index adhesion of yeast cells was quantified. 
 
 Results: The biotherapic was not able to reduce the viability of treated C. albicans when compared with controls. On the other hand, Ma104 treated cells presented important morphological alterations after 60 days, such as: cytoplasmic vacuoles, halos around the nucleolus and elongation of the plasmatic membrane. These changes were not observed in ,untreated cells nor in ones treated with water 30X. The adhesion index to Ma104 cells was reduced around 27% after 5 and 30 days of treatment when compared to controls. 
 
 Conclusion: These results showed that the biotherapic did not present any citotoxicity, but was able to modify the morphological aspects of Ma-104 cells. Additionally, the interaction between host cells and ethilogic agent is directly influenced by biotherapic treatment, suggesting a promising antifungal potential of this medicine.

Safety analysis of percutaneous needle electrolysis: a study of needle composition, morphology, and electrical resistance

Percutaneous needle electrolysis (PNE) consists of a galvanic current combined with the insertion of a solid needle into the tissues of the musculoskeletal system. The application of a galvanic current through a needle can alter the morphology and composition during treatment application. This procedure may also provoke a localized temperature increase. The aim was to evaluate the safety of the PNE procedure by analyzing possible alterations of the needles employed. Physio Invasiva® and AguPunt EPI® brand needles, commonly used for the application of this technique, were analyzed in response to three different treatment protocols. Temperature changes were evaluated with the needles immersed in a test tube containing Ringer's solution, and electrical resistance was evaluated with a multimeter. The morphology of the needles, pre- and post-treatment, was examined with a scanning electron microscope (FEI Quanta 600), and the composition of the needles was evaluated using RX diffusion with Oxford Instruments software. Ringer's solution contained in the test tubes examined did not present temperature changes. No changes were observed in the needles under investigation with respect to electrical resistance, morphology, or composition with a protocol applying 3-mA intensity for 3 s and three applications. However, important morphological alterations were observed that affected needle composition after 50 applications (at 3 mA for 3 s). PNE, applied according to conventional protocols, appeared to be safe and athermal, and did not provoke a loss of metal particles or modify the morphology of the needles used when studied in vitro.

Induction of micronuclei, nuclear anomalies, and dimensional changes in erythrocytes of the rare minnow (Gobiocypris rarus) by lanthanum.

The genotoxicity and cytotoxicity of lanthanum (La(III)) were studied in fish micronucleus analysis in erythrocytes of the rare minnow (Gobiocypris rarus). Fish were exposed to 0.04, 08, 0.16, 0.32, and 0.80 mg L-1 of La concentration for 21 days. Several important morphological alterations of the nucleus were noted, such as the ratio of micronucleated erythrocyte, and total ratio of erythrocytes with nuclear anomalies, blebs, notches, and so on. The total ratio of nuclear anomalies was significantly higher (P < 0.05) in G. rarus exposed to La(III) (except for 0.04 mg L-1) compared with the control. Hypsometric analysis indicated significant dose-dependent changes in erythrocyte and nucleus dimensions (P < 0.05). Various abnormal morphological forms of erythrocytes were also observed. These results showed that La(III) was cytotoxic to erythrocytes of the rare minnow.

Characterization of the adenosinergic system in a zebrafish embryo radiotherapy model

Adenosine is a nucleoside that acts as a signaling molecule by activating P1 purinergic receptors (A1, A2A, A2B and A3). This activation is involved in immune responses, inflammation, and tissue remodeling and tumor progression. Gamma rays are a type of ionizing radiation widely adopted in radiotherapy of tumors. Although it brings benefits to the success of the therapeutic scheme, it can trigger cellular damages, inducing a perpetual inflammatory response that culminates in adverse effects and severe toxicity. Our study aims to characterize the adenosinergic system in a zebrafish embryo radiotherapy model, relating the adenosine signaling to the changes elicited by radiation exposure. To standardize the radiotherapy procedure, we established a toxicological profile after exposure. Zebrafish were irradiated with different doses of gamma rays (2, 5, 10, 15 and 20 Gy) at 24 hpf. Survival, hatching rate, heartbeats, locomotor activity and morphological changes were determined during embryos development. Although without significant difference in survival, gamma-irradiated embryos had their heartbeats increased and presented decreased hatching time, changes in locomotor activity and important morphological alterations. The exposure to 10 Gy disrupted the ecto-5′-nucleotidase/CD73 and adenosine deaminase/ADA enzymatic activity, impairing adenosine metabolism. We also demonstrated that radiation decreased A2B receptor gene expression, suggesting the involvement of extracellular adenosine in the changes prompted by radiotherapy. Our results indicate that the components of the adenosinergic system may be potential targets to improve radiotherapy and manage the tissue damage and toxicity of ionizing radiation.

Evaluating the effect of roasting on coffee lipids using a hybrid targeted-untargeted NMR approach in combination with MRI

An integrated targeted-untargeted 1H and 13C Nuclear Magnetic Resonance (NMR) analysis was applied to determine the impact of roasting on coffee lipids. For targeted analysis, both an internal standard (IS) method, as well as the ERETIC2 tool based on PULCON (Pulse Length-based Concentration determination), were used for quantitation. PULCON allows for quantitative analysis without sample contamination with an IS and was found to be in very good agreement with the traditional IS approach as indicated by a systematic Bland-Altman comparison study. For the untargeted analysis, NMR was coupled with multivariate statistical analysis (MVSA), namely Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), and Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS-DA). 13C NMR spectra were acquired using a z-stored spin-echo sequence to achieve higher spectral quality, which is important for both targeted and untargeted analysis. Results showed that roasting has a clear effect on coffee lipids, with diterpenes, oxidation/hydrolysis products and unsaturated fatty acid chains being the most significant markers. In addition, the application of MRI indicated important morphological alterations in bean structure and lipid migration from the endosperm to the surface of the coffee bean.

Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170.

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.

The phenotype alterations showed by the res tomato mutant disappear when the plants are grown under semi-arid conditions: Is the res mutant tolerant to multiple stresses?

ABSTRACTThe res (restored cell structure by salinity) mutant, recently identified as the first tomato mutant accumulating jasmonate (JA) without stress, exhibited important morphological alterations when plants were grown under control conditions but these disappeared under salt stress. Since the defense responses against stresses are activated in the res mutant as a consequence of the increased expression of genes from the JA biosynthetic and signaling pathways, the mutant may display a tolerance response not only to salt stress but also to multiple stresses. Here, we show that when res mutant plants are grown under the summer natural conditions of the Mediterranean area, with high temperatures and low relative humidity, the characteristic leaf chlorosis exhibited by the mutant disappears and leaves become dark green over time, with a similar aspect to WT leaves. Moreover, the mutant plants are able to achieve chlorophyll and fluorescence levels similar to those of WT. These results hint that research on res tomato mutant may allow very significant advances in the knowledge of defense responses activated by JA against multiple stresses.

Water availability preceding long-term drought defines the tolerance of Eucalyptus to water restriction

Plantations of Eucalyptus species are expanding across South America into regions where drought conditions can reduce growth rate and result in substantial commercial loss. Understanding the mechanisms of drought tolerance in Eucalyptus is essential for the successful production in drought-regions. The main objectives of this study were to evaluate how water availability preceding a long-term drought period affects morphological, physiological and molecular traits of four Eucalyptus clones grown under field conditions. The study areas are located in north-eastern Brazil with an average rainfall of 800 and 1500 mm per year. At each rainfall regime, the following clones were evaluated: 1404 (Eucalyptus urophylla), 1407 (E. urophylla × E. camaldulensis), 1296 and 6500 (E. grandis × E. urophylla). Our results indicate that trees growing in the area with higher annual precipitation were more stressed after long-term drought, compared to those stands previously exposed to mild water-restriction period. The genetic materials showed distinct responses to drought, which allowed their separation in two groups: drought-tolerant (1404 and 1407) and drought sensitive (6500 and 1296). The former group shows some important adaptations to drought, such as decreased leaf area (avoiding excessive transpiration rates), higher antioxidant activity and carotenoid concentration (leading to lower lipid peroxidation). In conclusion, previous exposure to water deficit may provide the benefit of increased defense protection during future water deficit. From all measured variables, the leaf area, antioxidant compounds and changes in 13C and 18O isotope abundance reflect some of the most important morphological and physiological alterations in order to mitigate the water stress damage in drought-tolerant genotypes.

Phospho-Tau Accumulation and Structural Alterations of the Golgi Apparatus of Cortical Pyramidal Neurons in the P301S Tauopathy Mouse Model.

The Golgi apparatus (GA) is a highly dynamic organelle involved in the processing and sorting of cellular proteins. In Alzheimer’s disease (AD), it has been shown to decrease in size and become fragmented in neocortical and hippocampal neuronal subpopulations. This fragmentation and decrease in size of the GA in AD has been related to the accumulation of hyperphosphorylated tau. However, the involvement of other pathological factors associated with the course of the disease, such as the extracellular accumulation of amyloid-β (Aβ) aggregates, cannot be ruled out, since both pathologies are present in AD patients. Here we use the P301S tauopathy mouse model to examine possible alterations of the GA in neurons that overexpress human tau (P301S mutated gene) in neocortical and hippocampal neurons, using double immunofluorescence techniques and confocal microscopy. Quantitative analysis revealed that neurofibrillary tangle (NFT)-bearing neurons had important morphological alterations and reductions in the surface area and volume of the GA compared with NFT-free neurons. Since in this mouse model there are no Aβ aggregates typical of AD, the present findings support the idea that the progressive accumulation of phospho-tau is associated with structural alterations of the GA, and that these changes may occur in the absence of Aβ pathology.

Lipases and lipid droplet-associated protein expression in subcutaneous white adipose tissue of cachectic patients with cancer

BackgroundCancer cachexia is a multifactorial metabolic syndrome characterized by marked loss of adipose tissue and skeletal muscle. Fat loss from adipose tissue in cancer cachexia is partly the result of increased lipolysis. Despite the growing amount of studies focused on elucidating the mechanisms through which lipolysis-related proteins regulate the lipolytic process, there are scarce data concerning that profile in the adipose tissue of cancer cachectic patients. Considering its fundamental importance, it was our main purpose to characterize the expression of the lipolysis-related proteins in the white adipose tissue of cachectic cancer patients.MethodsPatients from the University Hospital were divided into three groups: control, cancer cachexia (CC), and weight-stable cancer patients (WSC). To gain greater insight into adipose tissue wasting during cancer cachexia progression, we have also analyzed an experimental model of cachexia (Walker 256 carcinosarcoma). Animals were divided into: control, intermediate cachexia (IC) and terminal cachexia (TC). Subcutaneous white adipose tissue of patients and epidydimal white adipose tissue of animals were investigated regarding molecular aspects by determining the protein content and gene expression of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58), perilipin 1, leptin, adiponectin, visfatin, and tumour necrosis factor alpha (TNF-alpha).ResultsWe found augmented lipolysis in CC associated with increased HSL expression, as well as upregulation of ATGL expression and reduction in perilipin 1 content. In IC, there was an imbalance in the secretion of pro- and anti-inflammatory factors. The alterations at the end-stage of cachexia were even more profound, and there was a reduction in the expression of almost all proteins analyzed in the animals.ConclusionsOur findings show that cachexia induces important morphological, molecular, and humoral alterations in the white adipose tissue, which are specific to the stage of the syndrome.

In vitro and In vivo Antifungal Activity of Culture Filtrates and Organic Extracts of Penicillium sp. and Gliocladium spp. against Botrytis cinerea

Eight isolates of Penicillium sp. and two isolates of Gliocladium spp. were tested in vitro and in vivo for their inhibitory effects against Botrytis cinerea, causal agent of tomato fruit grey mold. The biocontrol essays conducted in vitro revealed that the culture filtrates of the isolates tested have significantly reduced the mycelial growth of the pathogen. The filtrates of the isolate CH6 of Penicillium sp. applied at different concentrations (10, 15 and 20% v/v), was most effective in reducing B. cinerea colony diameter. The ethyl acetate and chloroform extracts of the isolates CH6 of Penicillium sp., Gc1 of G. catenulatum and Gv1 of G. virens have shown an inhibitory effect of the pathogen radial growth, at the concentrations used (1, 2.5 and 5% v/v). In addition to the reduction of the mycelial growth of B. cinerea, these antagonistic agents have induced important morphological alterations to the mycelium of the pathogen. These antagonists were applied to tomato fruits 2 hours before their inoculation with the pathogen. Tested as culture filtrates, the most effective isolates CH11 and MC1 of Penicillium sp. and Gv1 of G. virens had significantly reduced the severity of the disease compared to the inoculated and untreated control fruits. Similar effects were recorded using the ethyl acetate and chloroform extracts of the tested antagonists; those of CH6 and CH5 of Penicillium sp. and Gc1 of G. catenulatum were found to be the most effective in reducing severity grey mold. Thus, this study showed the presence of bioactive molecules in the culture filtrates of the antagonistic agents used and also allowed the selection of effective isolates for grey mold disease control.

Influence of oral administration of HMB to pregnant dams on calbindin expression in the dentate gyrus of the hippocampus during postnatal development in spiny mice offspring

The aim of the study was to investigate the morphology, density and immunostaining intensity of calbindin (CB)-positive neurons of dentate gyrus (DG) in new-born (P0) and 21-day-old (P21) male Acomys cahirinus mice from dams receiving β-hydroxy-β-methylbutyrate (HMB) during pregnancy. Different substances administrated to pregnant dams may affect the calcium ion homeostasis which is crucial for the proper brain development of their offspring. DG with hilus (H) plays an important role in memory and learning processes. Calcium levels in DG are regulated by buffering proteins like calbindin D28k (CB). Experimental dams were orally treated with HMB at a dose of 0.2g/kg b.w. Half of new-born animals were euthanised after birth and the rest after the 21st day of life. The brains were dissected and embedded in paraffin blocks using a routine histological technique. In order to demonstrate CB protein expression an immunohistochemical peroxidase-antiperoxidase reaction was conducted. The results of the study did not reveal important morphological alterations. There were no statistically significant changes in the density of the studied cells either in P0 and P21 animals. However, the authors have demonstrated a statistically significant increase of the average CB-immunostaining intensity in nuclei and cytoplasm in both age groups. It may be a result of a compensation effect to alterations that occurred under the influence of HMB. On the basis of the conducted research, it may be assumed that HMB activity in DG may provide long-term consequences.

In vitro exposure of Ulva lactuca Linnaeus (Chlorophyta) to gasoline – Biochemical and morphological alterations

Refined fuels have considerable share of pollution of marine ecosystems. Gasoline is one of the most consumed fuel worldwide, but its effects on marine benthic primary producers are poorly investigated. In this study, Ulva lactuca was chosen as a biological model due to its cosmopolitan nature and tolerance to high levels and wide range of xenobiotics and our goal was to evaluate the effects of gasoline on ultrastructure and metabolism of that seaweed. The experimental design consisted of in vitro exposure of U. lactuca to four concentrations of gasoline (0.001%, 0.01%, 0.1%, and 1.0%, v/v) over 30 min, 1 h, 12 h, and 24 h, followed by cytochemical, SEM, and biochemical analysis. Increase in the number of cytoplasmic granules, loss of cell turgor, cytoplasmic shrinkage, and alterations in the mucilage were some of the ultrastructural alterations observed in thalli exposed to gasoline. Decrease in carotenoid and polyphenol contents, as well as increase of soluble sugars and starch contents were associated with the time of exposure to the xenobiotic. In combination, the results revealed important morphological and biochemical alterations in the phenotype of U. lactuca upon acute exposure to gasoline. This seaweed contain certain metabolites assigned as candidates to biomarkers of the environmental stress investigated and it is thought to be a promise species for usage in coastal ecosystems perturbation monitoring system. In addition, the findings suggest that U. lactuca is able to metabolize gasoline hydrocarbons and use them as energy source, acting as bioremediator of marine waters contaminated by petroleum derivatives.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BackgroundThe human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents.Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections.ResultsAntifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25–64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4–16) for both planktonic and sessile cells.Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms.An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles.ConclusionsThe direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

In vitro biological action of aqueous extract from roots of Physalis angulata against Leishmania (Leishmania) amazonensis.

BackgroundLeishmaniasis is an infectious disease caused by various species of the protozoan parasites of the Leishmania genus and transmitted by phlebotomine sandflies. The protozoa multiply in phagocytic cells, mainly macrophages, which play an important role defending the organism from pathogens. The most effective treatment for leishmaniasis is the chemotherapy and besides the high cost, these drugs are toxic and require a long period of treatment. Currently, some herbal products are considered an important alternative source of a new leishmanicidal agent, which includes the plant Physalis angulata, . We evaluated effects of an aqueous extract from roots of Physalis angulata (AEPa) on Leishmania proliferation, morphology and also determined whether physalins were present in the extract contributing to the knowledge of its pharmacological efficacy.MethodsMorphological alterations were determined by light microscopy, transmission and scanning electron microscopy. Host cell viability was evaluated by MTT, and propidium iodide. AEPa were submitted in full HRESITOF analysis.ResultsAEPa promoted a dose-dependent reduction on promastigotes (IC50 = 39.5 μg/mL ± 5.1) and amastigotes (IC50 = 43.4 μg/mL ± 10.1) growth. This growth inhibition was associated with several morphological alterations observed in promastigote forms. No cytotoxic effect in mammalian cells was detected (IC50 > 4000 μg/mL). Furthemore, the presence of physalins A, B, D, E, F, G and H were described, for the first time, in the P. angulata root.ConclusionsResults demonstrate that AEPa effectively promotes antileishmanial activity with several important morphological alterations and has no cytotoxic effects on host cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0717-1) contains supplementary material, which is available to authorized users.

The tomato res mutant which accumulates JA in roots in non-stressed conditions restores cell structure alterations under salinity.

Jasmonic acid (JA) regulates a wide spectrum of plant biological processes, from plant development to stress defense responses. The role of JA in plant response to salt stress is scarcely known, and even less known is the specific response in root, the main plant organ responsible for ionic uptake and transport to the shoot. Here we report the characterization of the first tomato (Solanum lycopersicum) mutant, named res (restored cell structure by salinity), that accumulates JA in roots prior to exposure to stress. The res tomato mutant presented remarkable growth inhibition and displayed important morphological alterations and cellular disorganization in roots and leaves under control conditions, while these alterations disappeared when the res mutant plants were grown under salt stress. Reciprocal grafting between res and wild type (WT) (tomato cv. Moneymaker) indicated that the main organ responsible for the development of alterations was the root. The JA-signaling pathway is activated in res roots prior to stress, with transcripts levels being even higher in control condition than in salinity. Future studies on this mutant will provide significant advances in the knowledge of JA role in root in salt-stress tolerance response, as well as in the energy trade-off between plant growth and response to stress.

Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease

It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10−5M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10−6M ShPI-I, in the same periods of time, reduced parasite viability about 91–95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.

Biliary Obstruction After Short-Time Choledochal Clamping in Rats

IntroductionBiliary complications after liver procedures can lead to morbidity and poor survival. AimThe aim of this study was to evaluate the hepatic function after quick clamping of the common bile duct (BD) in Wistar rats. MethodsTwelve male Wistar rats with a mean weight of 323.14 g were anesthetized with sodium thiopental intravenous (IV). The common BD Clamping Group (BDCG; n = 6) was submitted to an abdominal incision (2 cm); the BD was isolated, dissected, and underwent clamping for 10 minutes with a microvascular clamp. After this time, the clamp was removed and the incision closed. The Sham Operation Group (SOG; n = 6 rats), under normal conditions, were subjected only to anesthesia and laparotomy and later control tests. On the 28th day liver and choledoch biopsy and biochemical tests were performed on all animals: total bilirubin (TB), alkaline phosphatase (ALK-P), aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), and gamma-glutamyl transferase (GGT). After the tests all the rats were humanely killed while still under anesthesia. ResultsIn this study 83% of the animals in the BDCG had large dilatation of the common BD with ductular proliferation, formation of septae, as well as multiple foci of parenchymal necrosis including micro-abscess formation. We also observed alterations in biochemical tests (P < .05). ConclusionOur study demonstrated that BD clamping even for a short time was sufficient to generate important morphological alterations in the liver and BD, as confirmed by enzymatic and histological analysis. Therefore, this technique can be used as a model of biliary obstruction for future studies.

Cisgenesis: a modern way of domesticating traits of the breeders' gene pool.

Abstract Plant breeding is a multidisciplinary scientific activity with tool development as driving force. It is clear from history that availability of genetic variation and selection methods are bottom lines for variety development. The genetic source of traditional plant breeding is restricted to domestication of traits from the so-called 'breeders' gene pool', consisting of crossable sources. Gene cloning and genetic transformation broaden the available genetic variation to genes from all living organisms. The so-called new genes in genetically modified organism (GMO) plants, consist of transgenes, with (chimaeric) genes from outside the 'breeder's gene pool'. Transgenic plants needed additional biosafety rules such as Directive 2001/18EC. However, these are not needed after transformation of the four rol -genes from wild-type Agrobacterium rhizogenes . In the meantime, cloned cisgenes, natural dominant genes from 'breeders' gene pool', are available, enabling cisgenic crops after marker-free transformation, which extends plant breeding with traditional traits. From long-term experience, it is clear that traditional breeding with the 'breeders' gene pool' has a history of safe use. Different scientific committees concluded that cisgenic crops are as safe as traditionally bred varieties. So, cisgenesis is a powerful new tool for plant breeding with traditional traits as indicated in the potential examples on: (1) breeding for durable resistance to potato late blight and apple scab by R -gene stacking; (2) the new possibility to come to stacking of monogenic resistance alleles in wheat; (3) engineering of restoration of cytoplasmic male sterility by cloned restorer genes and of altering gametophytic incompatibility by introducing additional S -alleles; (4) increasing phytase activity by gene dosage effect in barley; and (5) the possibility of changing hormone metabolism in (fruit) trees leading to important morphological alterations. In near future, because of availability of many more cisgenes, it is expected that the possibilities of cisgenesis will increase rapidly as the next step in plant breeding with traditional traits, if treatment as non-GMO is approved.