Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT)

Congenital amegakaryocytic thrombocytopenia (CAMT) without physical anomalies is a rare disease, presenting isolated thrombocytopenia and megakaryocytopenia in infancy, which can evolve into aplastic anemia and leukemia. Recently, two heterozygous truncating mutations of the thrombopoietin (TPO) receptor MPL, coded by the c-mpl gene, were identified in a 10-year-old Japanese patient with CAMT transmitted in an autosomal recessive manner. Here, we report for the first time two different MPL amino-acid substitutions in a 2-year-old Italian boy with CAMT and compound heterozygosis for two (c-mpl point mutations. C-to-T transitions were detected on exons 5 and 12 at the 769 and 1904 cDNA nucleotide positions, respectively. The mutation in exon 5 substitutes an arginine with a cysteine (R257C) in the extracellular domain, 11 amino acids distant from the WSXWS motif conserved in the cytokine-receptor superfamily. The mutation in exon 12 substitutes a proline with a leucine (P635L) in the last amino acid of the C-terminal intracellular domain, responsible for signal transduction. As in the Japanese family, the mutations were both transmitted from the parents. TPO plasma levels were highly increased in the patient. The patient's 7-year-old brother, who was a candidate donor for allografting, turned out to be an asymptomatic heterozygous carrier of P635L and showed defective megakaryocyte colony formation from bone-marrow progenitor cells. The present study provides important confirmation that CAMT can be associated with (c-mpl) mutations.     

Identification of the Bovine Arachnomelia Mutation by Massively Parallel Sequencing Implicates Sulfite Oxidase (SUOX) in Bone Development

Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development.     

Microbiological quality of Pecorino Siciliano "primosale" cheese on retail sale in the street markets of Palermo, Italy

Pecorino Siciliano (PS) "primosale" is a traditional Sicilian fresh soft cheese made from sheep's milk. Short-ripening time and production from unpasteurized or raw milk can facilitate bacterial contamination of PS "primosale". The microbiological quality of "primosale" on retail sale in the street markets of Palermo, Italy was studied by detecting the common food pathogens Listeria monocytogenes and Staphylococcus aureus and indicator microorganisms, such as Escherichia coli, Enterobacteriaceae and Staphylococcaceae. In our study, 4% and 44% of the samples, respectively, did not comply with the acceptability levels fixed by European regulations for S. aureus and E. coli. A high contamination of bacteria belonging to Enterobacteriaceae and Staphylococcaceae was found in 42% and 50% of the cheeses analyzed, respectively. Such results indicate poor husbandry and poor hygiene practices during milk collection or preservation or during cheese production processes and handling. In addition, the retail sale conditions may have played a role in cheese contamination since a correlation was found between poor microbiological quality and some selling parameters. This study emphasizes the need to improve production hygiene throughout the PS food chain in line with the traditional cheese-making procedures. Labelling of PS with clear information on whether the cheese was prepared from raw milk also requires improvement.     

Changes of Plasma Membrane Properties in a Human Pre-T Cell Line Undergoing Apoptosis

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca²⁺ concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.     

KDM2B Is Implicated in Bovine Lethal Multi-Organic Developmental Dysplasia

In the last decade breeders of Romagnola cattle observed an outbreak of a new congenital anomaly. This lethal multi-organ developmental dysplasia is mainly characterized by facial deformities, ascites and hepatic fibrosis. Affected stillborn calves were inbred to a single founder sire suggesting autosomal monogenic recessive inheritance. We localized the causative mutation to a 1.2 Mb interval on BTA 17 by genome-wide association and identical by descent mapping. A solution-based method for targeted DNA capture combined with massively parallel sequencing was used to analyze the entire critical region containing 24 genes. Homozygosity for two non-synonymous coding sequence variants affecting the RNF34 and KDM2B genes was detected by evaluating one affected calf. Here we show that the disease phenotype is associated with a KDM2B missense mutation (c.2503G>A) leading to an amino acid exchange (p.D835N) in an evolutionary strongly conserved domain. In addition, the genetic makeup of three inbred cattle strongly supports the causality of the KDM2B mutation. This report of a naturally-occurring spontaneous mutation of a JmjC domain containing histone demethylase gene provides evidence for their important role in the endo- and mesodermal organ development.     

Electronic and vibrational polarizabilities of the twenty naturally occurring amino acids

The geometries, relative energies, gas-phase static and dynamic dipole polarizabilities of the two most stable neutral forms and of the zwitterionic form of the twenty naturally occurring amino acids have been obtained by Density Functional and conventional ab initio Hatree-Fock theories using correlation consistent basis sets. Mean electronic polarizabilities (s) are encompassed in the 40-160 a.u. range and are little dependent on the amino acid framework conformation and structure. The relation between and the number of electrons in the molecule makes to classify the amino acids as one of the most polarizable family of compounds. Calculated values of the neutral forms linearly relate to the molecular volume and molecular hardness as well as, rather unexpectedly, with the experimental values in water solution, where amino acids are known to be in a zwitterionic form. Vibrational polarizabilities amount to 15-45 a.u.. They come essentially from the low-frequency angular deformation modes of the -OH and -NH(2) groups.     

Febrile temperature reprograms by redox-mediated signaling the mitochondrial metabolic phenotype in monocyte-derived dendritic cells

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3 h exposure at 39 °C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca²⁺ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.     

The body beyond the body: expectation of a sensory event is enough to induce ownership over a fake hand

More than 100 papers have been published on the rubber hand illusion since its discovery 14 years ago. The illusion has been proposed as a demonstration that the body is distinguished from other objects by its participation in specific forms of intermodal perceptual correlation. Here, we radically challenge this view by claiming that perceptual correlation is not necessary to produce the experience of this body as mine. Each of 15 participants was seated with his/her right arm resting upon a table just below another smaller table. Thus, the real hand was hidden from the participant''s view and a life-sized rubber model of a right hand was placed on the small table in front of the participant. The participant observed the experimenter''s hand while approaching—without touching—the rubber hand. Phenomenology of the illusion was measured by means of skin conductance response and questionnaire. Both measures indicated that participants experienced the illusion that the experimenter''s hand was about to touch their hidden hand rather than the rubber hand, as if the latter replaced their own hand. This did not occur when the rubber hand was rotated by 180° or replaced by a piece of wood. This illusion indicates that our brain does not build a sense of self in a merely reactive way, via perceptual correlations; rather it generates predictions on what may or may not belong to itself.     

Arachnomelia in Brown Swiss cattle maps to chromosome 5

Arachnomelia in Brown Swiss cattle is a monogenic autosomal recessive inherited congenital disorder of the skeletal system giving affected calves a spidery look (OMIA ID 000059). Over a period of 20 years 15 cases were sampled in the Swiss and Italian Brown cattle population. Pedigree data revealed that all affected individuals trace back to a single acknowledged carrier founder sire. A genome scan using 240 microsatellites spanning the 29 bovine autosomes showed homozygosity at three adjacent microsatellite markers on bovine Chr 5 in all cases. Linkage analysis confirmed the localization of the arachnomelia mutation in the region of the marker ETH10. Fine-mapping and haplotype analysis using a total of 34 markers in this region refined the critical region of the arachnomelia locus to a 7.19-Mb interval on bovine Chr 5. The disease-associated IBD haplotype was shared by 36 proven carrier animals and allows marker-assisted selection. As the corresponding human and mouse chromosome segments do not contain any clear functional candidate genes for this disorder, the mutation causing arachnomelia in the Brown Swiss cattle might help to identify an unknown gene in bone development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The molecular anatomy of human Hsp60 and its similarity with that of bacterial orthologs and acetylcholine receptor reveal a potential pathogenetic role of anti-chaperonin immunity in myasthenia gravis

Heat-shock protein 60 (Hsp60) is ubiquitous and highly conserved being present in eukaryotes and prokaryotes, including pathogens. This chaperonin, although typically a mitochondrial protein, can also be found in other intracellular sites, extracellularly, and in circulation. Thus, it can signal the immune system and participate in the development of inflammation and immune reactions. Both phenomena can be elicited by human and foreign Hsp60 (e.g., bacterial GroEL), when released into the blood by infectious agents. Consequently, all these Hsp60 proteins become part of a complex autoimmune response characterized by multiple cross reactions because of their structural similarities. In this study, we demonstrate that Hsp60 proteins from humans and two common pathogens, Chlamydia trachomatis and Chlamydia pneumoniae, share various sequence segments of potentially highly immunogenic epitopes with acetylcholine receptor α1 subunit (AChRα1). The structural data indicate that AChRα1 antibodies, implicated in the pathogenesis of myasthenia gravis, could very well be elicited and/or maintained by self- and/or bacterial Hsp60.