Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.     

Living with a crucial decision: a qualitative study of parental narratives three years after the loss of their newborn in the NICU

Background

The importance of involving parents in the end-of-life decision-making-process (EOL DMP) for their child in the neonatal intensive care unit (NICU) is recognised by ethical guidelines in numerous countries. However, studies exploring parents'' opinions on the type of involvement report conflicting results. This study sought to explore parents'' experience of the EOL DMP for their child in the NICU.

Methods

The study used a retrospective longitudinal design with a qualitative analysis of parental experience 3 years after the death of their child in four NICUs in France. 53 face-to-face interviews and 80 telephone interviews were conducted with 164 individuals. Semi-structured interviews were conducted to explore how parents perceived their role in the decision process, what they valued about physicians'' attitudes in this situation and whether their long-term emotional well being varied according to their perceived role in the EOL DMP.

Findings

Qualitative analysis identified four types of perceived role in the DMP: shared, medical, informed parental decision, and no decision. Shared DM was the most appreciated by parents. Medical DM was experienced as positive only when it was associated with communication. Informed parental DM was associated with feelings of anxiousness and abandonment. The physicians'' attitudes that were perceived as helpful in the long term were explicit sharing of responsibility, clear expression of staff preferences, and respectful care and language toward the child.

Interpretation

Parents find it valuable to express their opinion in the EOL DMP of their child. Nonetheless, they do need continuous emotional support and an explicit share of the responsibility for the decision. As involvement preferences and associated feelings can vary, parents should be able to decide what role they want to play. However, our study suggests that fully autonomous decisions should be misadvised in these types of tragic choices.     

Surface microbial consortia from Livarot, a French smear-ripened cheese

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.     

Description of a new small-sized Synodontis species (Siluriformes: Mochokidae) that is important for local subsistence fisheries in the middle Lufira (upper Congo River,DR Congo)

Synodontis denticulatus sp. nov. is an endemic from the middle Lufira Basin and its associated tributaries and lakes. The species shows close morphological resemblance to Synodontis greshoffi and Synodontis unicolor, which are widespread Congo Basin and Bangweulu-Mweru endemic species, respectively. However, it differs from both S. greshoffi and S. unicolor by its non-villous skin (v. villous skin), strong and numerous serrations on the posterior margin of the dorsal spine (v. weak and fewer serrations), weak and few serrations on the posterior margin of the pectoral spine (v. strong and numerous serrations), relatively short maxillary barbels (v. long) and its small maximum standard length (89.1 mm L_S v. 148.0 and 190.7 mm L_S respectively). A DNA barcoding study (coI, mtDNA) revealed that S. denticulatus forms a distinct genetic clade with a genetic distance of 2.18% with S. greshoffi and 0.84% with S. unicolor. Synodontis denticulatus is caught regularly and abundantly as a by-catch in the gillnet fisheries in the middle Lufira lakes. Owing to its small overall size and large bony head, the species has usually no real commercial value but is an important food fish for the fishermen's families.     

Egg‐size plasticity in Apis mellifera: Honey bee queens alter egg size in response to both genetic and environmental factors

Social evolution has led to distinct life‐history patterns in social insects, but many colony‐level and individual traits, such as egg size, are not sufficiently understood. Thus, a series of experiments was performed to study the effects of genotypes, colony size and colony nutrition on variation in egg size produced by honey bee (Apis mellifera) queens. Queens from different genetic stocks produced significantly different egg sizes under similar environmental conditions, indicating standing genetic variation for egg size that allows for adaptive evolutionary change. Further investigations revealed that eggs produced by queens in large colonies were consistently smaller than eggs produced in small colonies, and queens dynamically adjusted egg size in relation to colony size. Similarly, queens increased egg size in response to food deprivation. These results could not be solely explained by different numbers of eggs produced in the different circumstances but instead seem to reflect an active adjustment of resource allocation by the queen in response to colony conditions. As a result, larger eggs experienced higher subsequent survival than smaller eggs, suggesting that honey bee queens might increase egg size under unfavourable conditions to enhance brood survival and to minimize costly brood care of eggs that fail to successfully develop, and thus conserve energy at the colony level. The extensive plasticity and genetic variation of egg size in honey bees has important implications for understanding life‐history evolution in a social context and implies this neglected life‐history stage in honey bees may have trans‐generational effects.     

Role of cleavage and shedding in human thyrotropin receptor function and trafficking.

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.     

Mutational analysis of the <Emphasis Type="Italic">TRE2</Emphasis> oncogene encoding an inactive RabGAP

The TRE2 oncoprotein is structurally related to the RabGAP (GTPase-activating protein) family. However, TRE2 seems enzymatically inactive. Two regions are important for its lack of GAP activity. First, the TBC domain, forming the catalytically active domain of RabGAPs, is non-functional in the oncoprotein. Also involved in TRE2 inactivity is the 93-aa region flanking the TBC domain on the C-terminal side. In order to identify the residues responsible for non-functionality, we performed hydrophobic cluster analysis of the oncoprotein sequence, combined with secondary structure prediction, receptor-binding domain analysis, and a tilted peptide calculation. These analyses were complemented with site-directed and random mutagenesis experiments. On the basis of our data, we hypothesize that the lack of secondary structure of the region flanking the TBC domain in TRE2 may explain why this region plays a role in the lack of GAP activity, even when a potentially functional TBC domain is present.     

Spatial differences in genetic diversity and northward migration suggest genetic erosion along the boreal caribou southern range limit and continued range retraction

With increasing human activities and associated landscape changes, distributions of terrestrial mammals become fragmented. These changes in distribution are often associated with reduced population sizes and loss of genetic connectivity and diversity (i.e., genetic erosion) which may further diminish a species' ability to respond to changing environmental conditions and lead to local population extinctions. We studied threatened boreal caribou (Rangifer tarandus caribou) populations across their distribution in Ontario/Manitoba (Canada) to assess changes in genetic diversity and connectivity in areas of high and low anthropogenic activity. Using data from >1,000 caribou and nine microsatellite loci, we assessed population genetic structure, genetic diversity, and recent migration rates using a combination of network and population genetic analyses. We used Bayesian clustering analyses to identify population genetic structure and explored spatial and temporal variation in those patterns by assembling networks based on R_ST and F_ST as historical and contemporary genetic edge distances, respectively. The Bayesian clustering analyses identified broad‐scale patterns of genetic structure and closely aligned with the R_ST network. The F_ST network revealed substantial contemporary genetic differentiation, particularly in areas presenting contemporary anthropogenic disturbances and habitat fragmentation. In general, relatively lower genetic diversity and greater genetic differentiation were detected along the southern range limit, differing from areas in the northern parts of the distribution. Moreover, estimation of migration rates suggested a northward movement of animals away from the southern range limit. The patterns of genetic erosion revealed in our study suggest ongoing range retraction of boreal caribou in central Canada.     

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) – Isoleucine (I) – Lysine (K) – Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite’s ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.