Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.     

Molecular characterization of the chalcone isomerase gene family in Deschampsia antarctica

Deschampsia antarctica (Poaceae) is one of the two vascular plants known to have colonized the Antarctic region. Studies examining the biosynthesis of flavonoids, compounds which plants use, for example, for protection against overexposure to UV light or as antioxidants that scavenge free radicals and other oxidative species, in D. antarctica may provide clues to its success in that extreme environment. We characterized the family of genes encoding chalcone isomerase (CHI EC 5.5.1.6), an important enzyme involved in flavonoid biosynthesis, in D. antarctica. Sequence analysis of the three family members revealed differences in numbers of introns and lengths of coding regions among the three and suggest that DaCHI3 is likely a pseudogene (ψDaChi2). Salinity stress resulted in differential mRNA expression of the DaCHI genes with ψDaCHI2 exhibiting the earliest response (3-h post-treatment), induced by as much as sevenfold, while DaCHI1 and DaCHI2 mRNAs accumulated later (3d and 5d post-treatment, respectively) and, in the case of DaCHI2, with a response of nearly sixfold. We discuss how differences in the proposed gene structures, deduced protein characteristics, and mRNA expression patterns suggest that the members of this gene family may have unique functions in the phenylpropanoid pathway in D. antarctica.     

Endophytic fungi from Peruvian highland and lowland habitats form distinctive and host plant-specific assemblages

Biodiversity and biogeography of leaf-inhabiting endophytic fungi have not been resolved yet. This is because host specificity, life cycles and species concepts, in this heterogeneous ecological guild of plant-associated microfungi, are far from being understood. Even though it is known that culture-based collection techniques are often biased, this has been the method of choice for studying fungal endophytes. Isolation of fungal endophytes only through culture-based methods could potentially mask slow growing species as well as species with low prevalence, preventing the capture of the communities’ real diversity and composition. This bias can be partially resolved by the use of cultivation-independent approaches such as direct sequencing of plant tissue by next generation techniques. Irrespective of the chosen sampling method, an efficient analysis of community ecology is urgently needed in order to evaluate the driving forces acting on fungal endophytic communities. In the present study, endophytic ascomyceteous fungi from three different plant genera (Vasconcellea microcarpa, Tillandsia spp., and Hevea brasiliensis) distributed in Peru, were isolated through culture-based sampling techniques and sequenced for their ITS rDNA region. These data sets were used to assess host preferences and biogeographic patterns of endophytic assemblages. This study showed that the effect of the host’s genetic background (identity) has a significant effect on the composition of the fungal endophytic community. In other words, the composition of the fungal endophytic community was significantly related to their host’s taxonomic identity. However, this was not true for all endophytic groups, since we found some endophytic groups (e.g. Xylariales and Pleosporales) occurring in more than one host genus. Findings from this study promote the formulation of hypotheses related to the effect of altitudinal changes on the endophytic communities along the Eastern Andean slopes. These hypotheses and perspectives for fungal biodiversity research and conservation in Peru are addressed and discussed.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Rhythms of high-grade block in an ionic model of a strand of regionally ischemic ventricular muscle

Electrical alternans, a beat-to-beat alternation in the electrocardiogram or electrogram, is frequently seen during the first few minutes of acute myocardial ischemia, and is often immediately followed by malignant cardiac arrhythmias such as ventricular tachycardia and ventricular fibrillation. As ischemia progresses, higher-order periodic rhythms (e.g., period-4) can replace the period-2 alternans rhythm. This is also seen in modelling work on a two-dimensional (2-D) sheet of regionally ischemic ventricular muscle. In addition, in the experimental work, ventricular arrhythmias are overwhelmingly seen only after the higher-order rhythms arise. We investigate an ionic model of a strand of ischemic ventricular muscle, constructed as a 3-cm-long 1-D cable with a centrally located 1-cm-long segment exposed to an elevated extracellular potassium concentration ([K(+)](o)). As [K(+)](o) is raised in this "ischemic segment" to represent one major effect of ongoing ischemia, the sequence of rhythms {1:1-->2:2 (alternans)-->2:1} is seen. With further increase in [K(+)](o), one sees higher-order periodic 2N:M rhythms {2:1-->4:2-->4:1-->6:2-->6:1-->8:2-->8:1}. In a 2N:M cycle, only M of the 2N action potentials generated at the proximal end of the cable successfully traverse the ischemic segment, with the remaining ones being blocked within the ischemic segment. Finally, there is a transition to complete block {8:1-->2:0-->1:0} (in an n:0 rhythm, all action potentials die out within the ischemic segment). Changing the length of the ischemic segment results in different rhythms and transitions being seen: e.g., when the ischemic segment is 2 cm long, the period-6 rhythms are not seen; when it is 0.5 cm long, there is a 3:1 rhythm interposed between the 2:1 and 1:0 rhythms. We discuss the relevance of our results to the experimental observations on the higher-order rhythms that presage reentrant ischemic ventricular arrhythmias.     

Regulation of Na,K-ATPase during acute lung injury

A hallmark of acute lung injury is the accumulation of a protein rich edema which impairs gas exchange and leads to hypoxemia. The resolution of lung edema is effected by active sodium transport, mostly contributed by apical Na⁺ channels and the basolateral located Na,K-ATPase. It has been reported that the decrease of Na,K-ATPase function seen during lung injury is due to its endocytosis from the cell plasma membrane into intracellular pools. In alveolar epithelial cells exposed to severe hypoxia, we have reported that increased production of mitochondrial reactive oxygen species leads to Na,K-ATPase endocytosis and degradation. We found that this regulated process follows what is referred as the Phosphorylation–Ubiquitination–Recognition–Endocytosis–Degradation (PURED) pathway. Cells exposed to hypoxia generate reactive oxygen species which activate PKCζ which in turn phosphorylates the Na,K-ATPase at the Ser18 residue in the N-terminus of the α1-subunit leading the ubiquitination of any of the four lysines (K16, K17, K19, K20) adjacent to the Ser18 residue. This process promotes the α1-subunit recognition by the μ2 subunit of the adaptor protein-2 and its endocytosis trough a clathrin dependent mechanism. Finally, the ubiquitinated Na,K-ATPase undergoes degradation via a lysosome/proteasome dependent mechanism.     

Assessing the need for adaptive radiotherapy in head and neck cancer patients using an automatic planning tool

BackgroundUnbiased analysis of the impact of adaptive radiotherapy (ART) is necessary to evaluate dosimetric benefit and optimize clinics’ workflows. The aim of the study was to assess the need for adaptive radiotherapy (ART) in head and neck (H&N) cancer patients using an automatic planning tool in a retrospective planning study.Materials and methodsThirty H&N patients treated with adaptive radiotherapy were analysed. Patients had a CT scan for treatment planning and a verification CT during treatment according to the clinic’s protocol. Considering these images, three plans were retrospectively generated using the iCycle tool to simulate the scenarios with and without adaptation: 1) the optimized plan based on the planning CT; 2) the optimized plan based on the verification CT (ART-plan); 3) the plan obtained by considering treatment plan 1 re-calculated in the verification CT (non-ART plan). The dosimetric endpoints for both target volumes and OAR were compared between scenarios 2 and 3 and the SPIDERplan used to evaluate plan quality.ResultsThe most significant impact of ART was found for the PTVs, which demonstrated decreased D98% in the non-ART plan. A general increase in the dose was observed for the OAR but only the spinal cord showed a statistical significance. The SPIDERplan analysis indicated an overall loss of plan quality in the absence of ART.ConclusionThese results confirm the advantages of ART in H&N patients, especially for the coverage of target volumes. The usage of an automatic planning tool reduces planner-induced bias in the results, guaranteeing that the observed changes derive from the application of ART.     

Functional assessment of morphological homoplasy in stem-gnathostomes

Osteostraci and Galeaspida are stem-gnathostomes, occupying a key phylogenetic position for resolving the nature of the jawless ancestor from which jawed vertebrates evolved more than 400 million years ago. Both groups are characterized by the presence of rigid headshields that share a number of common morphological traits, in some cases hindering the resolution of their interrelationships and the exact nature of their affinities with jawed vertebrates. Here, we explore the morphological and functional diversity of osteostracan and galeaspid headshields using geometric morphometrics and computational fluid dynamics to constrain the factors that promoted the evolution of their similar morphologies and informing on the ecological scenario under which jawed vertebrates emerged. Phylomorphospace, Mantel analysis and Stayton metrics demonstrate a high degree of homoplasy. Computational fluid dynamics reveals similar hydrodynamic performance among morphologically convergent species, indicating the independent acquisition of the same morphofunctional traits and, potentially, equivalent lifestyles. These results confirm that a number of the characters typically used to infer the evolutionary relationships among galeaspids, osteostracans and jawed vertebrates are convergent in nature, potentially obscuring understanding of the assembly of the gnathostome bodyplan. Ultimately, our results reveal that while the jawless relatives of the earliest jawed vertebrates were ecologically diverse, widespread convergence on the same hydrodynamic adaptations suggests they had reached the limits of their potential ecological diversity—overcome by jawed vertebrates and their later innovations.