Mutations in Multidomain Protein MEGF8 Identify a Carpenter Syndrome Subtype Associated with Defective Lateralization

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.     

Determination of isocyanate specific albumin-adducts in workers exposed to toluene diisocyanates

Toluene diisocyanates (2,4-TDI and 2,6-TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. It is therefore necessary to have sensitive and specific methods to monitor isocyanate exposure of workers. Urinary metabolites or protein adducts have been used as biomarkers in workers exposed to TDI. However, with these methods it was not possible to determine if the biomarkers result from exposure to TDI or to the corresponding toluene diamines (TDA). This work presents a new procedure for the determination of isocyanate-specific albumin adducts. Isotope dilution mass spectrometry was used to measure the adducts in albumin present in workers exposed to TDI. 2,4-TDI and 2,6-TDI formed adducts with lysine: N(?)-[({3-amino-4-methylphenyl}amino)carbonyl]-lysine, N(?)-[({5-amino-2-methylphenyl}amino)carbonyl]-lysine, and N(?)- [({3-amino-2-methylphenyl}amino)carbonyl]-lysine. In future studies, this new method can be applied to measure TDI-exposures in workers.     

An archaeal protein with homology to the eukaryotic translation initiation factor 5A shows ribonucleolytic activity

To identify proteins that are involved in RNA degradation and processing in Halobacterium sp. NRC-1, we purified proteins with RNA-degrading activity by classical biochemical techniques. One of these proteins showed strong homology to the eukaryotic initiation factor 5A (eIF-5A) and was accordingly named archaeal initiation factor 5A (aIF-5A). Eukaryotic IF-5A is known to be involved in mRNA turnover and to bind RNA. Hypusination of eIF-5A is required for sequence-specific binding of RNA. This unique post-translational modification is restricted to Eukarya and Archaea. The exact function of eIF-5A in RNA turnover remained obscure. Here we show for the first time that aIF-5A from Halobacterium sp. NRC-1 exhibits RNA cleavage activity, preferentially cleaving adjacent to A nucleotides. Detectable RNA binding could be shown for aIF-5A purified from Halobacterium sp. NRC-1 but not from Escherichia coli, while both proteins possess RNA cleavage activity, indicating that hypusination of aIF-5A is required for RNA binding but not for its RNA cleavage activity. Furthermore, we show that the hypusinated form of eIF-5A also shows RNase activity while the unmodified protein does not. Charged amino acids in the N-terminal domain of aIF-5A as well as in the C-terminal domain, which is highly similar to the cold shock protein A (CspA), an RNA chaperone of E. coli, are important for RNA cleavage activity. Moreover our results reveal that activity of aIF-5A depends on its oligomeric state.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

Modelling of the provisional side-branch stenting approach for the treatment of atherosclerotic coronary bifurcations: effects of stent positioning

The most common approach to treat atherosclerosis in coronary bifurcations is the provisional side-branch (PSB) stenting, which consists sequentially of the insertion of a stent in the main branch (MB) of the bifurcation and a dilatation of the side branch (SB) passing through the struts of the stent at the bifurcation. This approach can be followed by a redilatation of the MB only or by a Final Kissing Balloon (FKB) inflation, both strategies leading to a minor stent distortion in the MB. The positioning of the stent struts in the bifurcation and the stresses generated in the stent and vessel wall are worthy of investigation for a better understanding of the mechanobiology of the system. For this purpose, a computer model of an atherosclerotic coronary bifurcation based on the finite element method was developed; the effects of performing the final redilatation with the two strategies utilising one or two balloons and those created by a different stent strut positioning around the SB were investigated. Results correlate well with previous experimental tests regarding the deformation following balloon expansion. Furthermore, results confirm firstly that the re-establishment of an optimal spatial configuration of the stent after the PSB approach is achieved with both strategies; secondly, results show that case of stent positioning with one cell placed centrally (with regard to the SB) should be preferred, avoiding the presence of struts inside the vessel lumen, which may reduce hemodynamic disturbances. The central positioning also resulted in a better solution in terms of lower stresses in the stent struts and, more importantly, in the vascular tissues.     

A systematic investigation of multiheme c-type cytochromes in prokaryotes

Multiheme c-type cytochromes (MHCs) are metalloproteins that can play various biochemical roles, including enzymatic activity and electron transfer. As electron transfer proteins, the presence of multiple heme cofactors in the vicinity allows electrons to rapidly travel relatively long distances. MHCs are often characterized by relatively low structural complexity, with the heme cofactors being largely responsible for maintaining the structure in place, owing to the protein–heme covalent linkages. In this work, we analyzed an extensive ensemble of 594 complete prokaryotic proteomes, amounting to more than 1.9 million sequences, to characterize their content in MHCs. We identified 1,659 MHCs in 258 organisms. The presence of MHCs was found to correlate quite well with the capability of an organism to synthesize or take up heme. For two organisms, the presence of MHCs in the proteome could be taken as a hint to the presence of divergent heme uptake pathways. The most common numbers of heme-binding motifs in a sequence were four (25%) and two (23%), followed by five (13%) and ten (9.8%). The average protein-to-heme ratio was relatively similar for all MHCs, except diheme proteins, regardless of the number of motifs at around 60 ± 30. The latter ratio could in favorable cases be a useful indicator for functional assignments of novel MHCs. Finally, we showed that the amount of structural information currently available for MHCs is limited with respect to the diversity of this broad class of metalloproteins. Experimental efforts in the structural investigation of MHCs are thus warranted.     

Lunar-Rhythmic Molting in Laboratory Populations of the Noble Crayfish Astacus astacus (Crustacea,Astacidea): An Experimental Analysis

Juvenile noble crayfish, Astacusastacus (Crustacea, Astacidea) in the second year of age were kept in the laboratory for a twelve-month period under continuing “summer conditions” (LD 16:8, 19°C). Molting processes in this population could be synchronized by artificial moonlight cycles. Peaks of exuviations occurred at “new moons”. Males showed a slightly higher degree of synchronization than females. A phase-shift of the artificial lunar cycle in relation to the natural cycle resulted in a corresponding shift of the molting cycle. This clearly demonstrates that changes in the nocturnal light regime provide the primary external information for the lunar-monthly molting rhythm. There is a first indication that lunar photic stimuli do not act directly but as a zeitgeber which entrains an endogenous molting rhythm to the lunar cycle. Moreover, the results of the long-term experiments suggest that the hibernal resting period of A. astacus in the field (no molts between October and April) may also involve some endogenous programming. Continuing artificial summer conditions can delay but not completely suppress this resting period. The adaptive significance of the phenomena and how the findings may be applied to improve the management of crowded crayfish stocks are discussed.     

Female circumcision in multicultural Singapore: The hidden cut

In recent years discussion about female genital mutilation (FGM) has expanded and the UN has recently called for a universal ban of the practice. The practice in Southeast Asia is widespread among Malay Muslims and, although different styles and practices exist, procedures conducted in medical clinics are extremely minor and, according to gynaecological research, have no effect on sexuality due to the clitoris being left totally untouched. One of the states in which Malay Muslims maintain such a tradition is Singapore. Nonetheless, Singapore is rarely mentioned in academic studies or even in reports discussing the ritual. Even inside Singapore, only Malays tend to know of the tradition, while other ethnic groups remain oblivious to the fact that Singapore is among the states that allow such an operation. The present article does not discuss FGM per se and avoids contributing to the diatribe about labels and values, although these are, of course, extremely relevant. Instead it focuses on the reasons for the practice remaining hidden and undiscussed in Singapore, so much so that some respondents did not know that they had been circumcised.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.