Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI = 5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0-12.0), after heating to 55 °C for 30 min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H₂O₂ and other reactive components since it was not counteracted by H₂O₂ scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.     

Transcervical Ultrasonography Is Feasible to Visualize and Evaluate Base of Tongue Cancers

Background

Base of tongue (BOT) is a difficult subsite to examine clinically and radiographically. Yet, anatomic delineation of the primary tumor site, its extension to adjacent sites or across midline, and endophytic vs. exophytic extent are important characteristics for staging and treatment planning. We hypothesized that ultrasound could be used to visualize and describe BOT tumors.

Methods

Transcervical ultrasound was performed using a standardized protocol in cases and controls. Cases had suspected or confirmed BOT malignancy. Controls were healthy individuals without known malignancy.

Results

100% of BOT tumors were visualized. On ultrasound BOT tumors were hypoechoic (90.9%) with irregular margins (95.5%). Ultrasound could be used to characterize adjacent site involvement, midline extent, and endophytic extent, and visualize the lingual artery. No tumors were suspected for controls.

Conclusions

Ultrasonography can be used to transcervically visualize BOT tumors and provides clinically relevant characteristics that may not otherwise be appreciable.     

Endothelium Trans Differentiated from Wharton's Jelly Mesenchymal Cells Promote Tissue Regeneration: Potential Role of Soluble Pro-Angiogenic Factors

Background

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton''s jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton''s jelly (hWMSCs) can accelerate tissue repair in vivo.

Methods

Mesenchymal stem cells were isolated from human Wharton''s jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO).

Results

hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures.

Conclusion

These results demonstrate that mesenchymal stem cells isolated from Wharton''s jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.     

Genetic Diversity of Giardia duodenalis: Multilocus Genotyping Reveals Zoonotic Potential between Clinical and Environmental Sources in a Metropolitan Region of Brazil

Background

Giardia duodenalis is a flagellate protozoan that parasitizes humans and several other mammals. Protozoan contamination has been regularly documented at important environmental sites, although most of these studies were performed at the species level. There is a lack of studies that correlate environmental contamination and clinical infections in the same region. The aim of this study is to evaluate the genetic diversity of a set of clinical and environmental samples and to use the obtained data to characterize the genetic profile of the distribution of G. duodenalis and the potential for zoonotic transmission in a metropolitan region of Brazil.

Methodology/Principal Findings

The genetic assemblages and subtypes of G. duodenalis isolates obtained from hospitals, a veterinary clinic, a day-care center and important environmental sites were determined via multilocus sequence-based genotyping using three unlinked gene loci. Cysts of Giardia were detected at all of the environmental sites. Mixed assemblages were detected in 25% of the total samples, and an elevated number of haplotypes was identified. The main haplotypes were shared among the groups, and new subtypes were identified at all loci. Ten multilocus genotypes were identified: 7 for assemblage A and 3 for assemblage B.

Conclusions/Significance

There is persistent G. duodenalis contamination at important environmental sites in the city. The identified mixed assemblages likely represent mixed infections, suggesting high endemicity of Giardia in these hosts. Most Giardia isolates obtained in this study displayed zoonotic potential. The high degree of genetic diversity in the isolates obtained from both clinical and environmental samples suggests that multiple sources of infection are likely responsible for the detected contamination events. The finding that many multilocus genotypes (MLGs) and haplotypes are shared by different groups suggests that these sources of infection may be related and indicates that there is a notable risk of human infection caused by Giardia in this region.     

Host Genetic Background Influences the Response to the Opportunistic Pseudomonas aeruginosa Infection Altering Cell-Mediated Immunity and Bacterial Replication

Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P. aeruginosa infection. These results now provide a basis for mapping genomic regions underlying host susceptibility to P. aeruginosa infection.     

The ketogenic diet component decanoic acid increases mitochondrial citrate synthase and complex I activity in neuronal cells

The Ketogenic diet (KD) is an effective treatment with regards to treating pharmaco‐resistant epilepsy. However, there are difficulties around compliance and tolerability. Consequently, there is a need for refined/simpler formulations that could replicate the efficacy of the KD. One of the proposed hypotheses is that the KD increases cellular mitochondrial content which results in elevation of the seizure threshold. Here, we have focussed on the medium‐chain triglyceride form of the diet and the observation that plasma octanoic acid (C8) and decanoic acid (C10) levels are elevated in patients on the medium‐chain triglyceride KD. Using a neuronal cell line (SH‐SY5Y), we demonstrated that 250‐μM C10, but not C8, caused, over a 6‐day period, a marked increase in the mitochondrial enzyme, citrate synthase along with complex I activity and catalase activity. Increased mitochondrial number was also indicated by electron microscopy. C10 is a reported peroxisome proliferator activator receptor γ agonist, and the use of a peroxisome proliferator activator receptor γ antagonist was shown to prevent the C10‐mediated increase in mitochondrial content and catalase. C10 may mimic the mitochondrial proliferation associated with the KD and raises the possibility that formulations based on this fatty acid could replace a more complex diet.

     

Northern richness and cryptic refugia: phylogeography of the Italian smooth newt Lissotriton vulgaris meridionalis

Recent phylogeographical studies have re‐evaluated the role of refugia in central and northern Europe for glacial persistence and postglacial assembly of temperate biota. Yet, on a regional scale within Mediterranean peninsulas, putative ‘northern’ refugia's contribution to the current structure of biodiversity still needs to be fully appreciated. To this end, we investigated the phylogeographical structure and the evolutionary history of the Italian smooth newt, Lissotriton vulgaris meridionalis, through phylogeographical, molecular dating and historical demographic analyses. We found ten differentiated mitochondrial lineages with a clear geographical association, mainly distributed in northern Italy. The most ancient divergence among these lineages was estimated at the Early Pleistocene and was followed by a series of splits throughout the Middle Pleistocene. No haplogroup turned out to be derived from another one, each one occupying terminal positions within the phylogenetic network topologies. These results suggest an unprecedented scenario involving long‐term survival of distinct evolutionary lineages in multiple northern Mediterranean refugia. This scenario mirrors on a smaller geographical scale what has been previously observed in the literature concerning northern European environments; it also sheds more light on how northern Italy has contributed to temperate species' long‐term survival and to the assembly of regional biota. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 590–603.     

Asymmetric synthesis of the four diastereoisomers of a novel non-steroidal farnesoid X receptor (FXR) agonist: Role of the chirality on the biological activity

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.