Molecular cytogenetic characterization of a constitutional complex intrachromosomal 4q rearrangement in a patient with multiple congenital anomalies

Constitutional Complex Chromosomal Rearrangements (CCRs) are very rare. While the vast majority of CCRs involve more than one chromosome, only seven cases describe CCRs with four or more breakpoints within a single chromosome. Here, we present a patient with multiple congenital anomalies and mental retardation. Array Comparative Genomic Hybridisation (array CGH), FISH and Multicolour Banding FISH revealed a de novo complex rearrangement with two deletions, a duplication and an inversion of 4q. This CCR involving at least seven breakpoints is one of the most complex rearrangements of a single chromosome reported thus far. Potential mechanisms generating such complex rearrangements are discussed.     

Use of multiple markers demonstrates a cryptic western refugium and postglacial colonisation routes of Atlantic salmon (Salmo salar L.) in northwest Europe

Glacial and postglacial processes are known to be important determinants of contemporary population structuring for many species. In Europe, refugia in the Italian, Balkan and Iberian peninsulas are believed to be the main sources of species colonising northern Europe after the glacial retreat; however, there is increasing evidence of small, cryptic refugia existing north of these for many cold-tolerant species. This study examined the glacial history of Atlantic salmon in western Europe using two independent classes of molecular markers, microsatellites (nuclear) and mitochondrial DNA variation. Alongside the well-documented refuge in the Iberian Peninsula, evidence for a cryptic refuge in northwest France is also presented. Critically, methods utilised to estimate divergence times between the refugia indicated that salmon in these two regions had diverged a long time before the last glacial maximum; coalescence analysis (as implemented in the program IMa2) estimated divergence times at around 60 000 years before present. Through the examination of haplotype frequencies, previously glaciated areas of northwest Europe, that is, Britain and Ireland, appear to have been colonised from salmon expanding out of both refugia, with the southwest of England being the primary contact zone and exhibiting the highest genetic diversity.     

Chicken domestication: an updated perspective based on mitochondrial genomes

Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A–I and W–Z. Common haplogroups A–G were shared by domestic chickens and red junglefowl. Rare haplogroups H–I and W–Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.     

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.     

Adjustments to McConville et al. and Young et al. body segment inertial parameters

Body segment inertial parameters (BSIPs) are important data in biomechanics. They are usually estimated from predictive equations reported in the literature. However, most of the predictive equations are ambiguously applicable in the conventional 3D segment coordinate systems (SCSs). Also, the predictive equations reported in the literature all include two assumptions: the centre of mass and the proximal and distal endpoints are assumed to be aligned, and the inertia tensor is assumed to be principal in the segment axes. These predictive equations, restraining both position of the centre of mass and orientation of the principal axes of inertia, become restrictive when computing 3D inverse dynamics, when analyzing the influence of BSIP estimations on joint forces and moments and when evaluating personalized 3D BSIPs obtained from medical imaging. In the current study, the extensive data from McConville et al. (1980. Anthropometric relationships of body and body segment moments of inertia. AFAMRL-TR-80-119, Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Dayton, Ohio) and from Young et al. (1983. Anthropometric and mass distribution characteristics of the adults female. Technical Report AFAMRL-TR-80-119, FAA Civil Aeromedical Institute, Oklaoma City, Oklaoma) are adjusted in order to correspond to joint centres and to conventional segment axes. In this way, scaling equations are obtained for both males and females that provide BSIPs which are directly applicable in the conventional SCSs and do not restrain the position of the centre of mass and the orientation of the principal axes. These adjusted scaling equations may be useful for researchers who wish to use appropriate 3D BSIPs for posture and movement analysis.     

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.     

Hypoxia tolerance and air-breathing ability correlate with habitat preference in coral-dwelling fishes

Hypoxia tolerance and air-breathing occur in a range of freshwater, estuarine and intertidal fishes. Here it is shown for the first time that coral reef fishes from the genera Gobiodon, Paragobiodon and Caracanthus, which all have an obligate association with living coral, also exhibit hypoxia tolerance and a well-developed air-breathing capacity. All nine species maintained adequate respiration in water at oxygen concentrations down to 15–25% air saturation. This hypoxia tolerance is probably needed when the oxygen levels in the coral habitat drops sharply at night. Air-breathing abilities of the species correlated with habitat association, being greatest (equaling oxygen uptake in water) in species that occupy corals extending into shallow water, where they may become air exposed during extreme low tides. Air-breathing was less well-developed or absent in species inhabiting corals from deeper waters. Loss of scales and a network of subcutaneous capillaries appear to be key adaptations allowing cutaneous respiration in air. While hypoxia tolerance may be an ancestral trait in these fishes, air-breathing is likely to be a more recent adaptation exemplifying convergent evolution in the unrelated genera Gobiodon and Caracanthus in response to coral-dwelling lifestyles.     

FAK competes for Src to promote migration against invasion in melanoma cells

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK^Y397F reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.Patients with spreading melanoma diseases have a very poor prognosis with a 5-year survival rate <5%. The metastatic spread of melanoma is a complex process involving several genetic alterations. In melanoma,¹ as in many highly invasive cancer cell types like head and neck squamous cell carcinoma² or breast carcinoma,³ specialized matrix-degrading organelles termed invadopodia have been identified. Invadopodia consist of dynamic actin-based protrusions of 0, 1 to 2 μm in diameter emanating from the ventral edge of tumor cells.⁴ Besides their actin scaffold, these structures are enriched in proteolytic enzymes such as matrix metalloproteinases (MMPs), which mediate extracellular matrix (ECM) degradation. Indeed, MMP are upregulated in invasive melanoma and there is extensive evidence that they have a role in promoting the dissemination of melanoma.^{5, 6, 7} Several proteins like integrins, Src and paxillin, found at sites of cell adhesion to the matrix, are also present in invadopodia.^{8, 9} On the other hand, other proteins like the Src substrate proteins cortactin¹⁰ and the tyrosine kinase substrate with five SH3 domain (Tks-5)¹¹ are specifically localized at invadopodia and not found at focal adhesion (FA). In addition, reactive oxygen species (ROS)¹² have been localized at invadopodia and are supposed to have a prominent role in inducing invadopodia function.^{13, 14} Although significant efforts have been made to characterize components of invadopodia, the precise mechanisms of their regulation, especially in a melanoma context, remain poorly understood.Tumor invasion is a multistep process that requires cell adhesion to the environing substratum, migration and invasion. In many cell types, migration requires fine control of FA turn-over. FAs are formed by the cluster of up to 200 proteins¹⁵ ensuring cell anchorage to the ECM. The cyclic process of FA formation and disruption is crucial for cell migration. Because both anchorage and migration involve cellular interactions with ECM components, FAs are endowed with transmembrane ECM receptor proteins such as integrins that interact with ECM molecules and intra-cellular proteins composed of scaffold proteins, as well as signal-transducing molecules. Among those, focal adhesion kinase (FAK) is a crucial signaling protein that integrates signals from integrins to the actin filaments during cell migration.¹⁶ Structurally, FAK is a 125-kDa protein that contains an N-terminal 4.1-ezrin–radixin–moesin domain, a central kinase domain and a C-terminal domain that contains the focal adhesion targeting site.¹⁷ The phosphorylation of FAK at Y397 creates a binding site for Src, which can phosphorylate other tyrosines on the FAK sequence, thus creating new binding sites for SH2 domain-containing proteins.FAK is involved in many aspects of the metastatic process and thus, overexpression, hyperphosphorylation and/or elevated activity of FAK have been reported in a variety of human cancers, including sarcomas and carcinomas of the breast, colon, thyroid, prostate, oral cavity, liver, stomach and ovary.¹⁸ In human melanoma cell lines, early studies reported high FAK expression and requirement of FAK for cell substrate adhesion.¹⁹ Later, it was reported that FAK promotes the aggressive melanoma phenotype.²⁰ Indeed, immunohistochemical analyses revealed high levels of FAK phosphorylation at Tyr397 and Tyr576, a marker of FAK kinase activity, in late-stage cutaneous and uveal melanoma, which correlated with their increased invasion and migration properties.²¹ Furthermore, melanoma differentiation-associated gene-9 (mda-9)/syntenin was also reported to mediate adhesion-dependant activation of protein kinase Cα (PKCα) and FAK in melanoma cells. Thus, inhibiting either mda-9/syntenin or PKCα suppressed fibronectin-induced formation of integrin-β1/FAK/c-Src signaling complexes and reduced migration and invasion toward fibronectin.²² Therefore, FAK appears to be a major player of melanoma invasion, but how this kinase controls the formation and proteolytic activity of invadopodia in melanoma cells was never investigated.In this study, we uncovered a surprising negative regulation of invadopodia activity in B16F10 cells by FAK. The depletion of FAK was associated with increased ROS production and Tks-5 phosphorylation. Using mutation of FAK at Tyr397, a binding site for Src, we found that these sites are implicated in FAK-mediated inhibition of invadopodia activity. In addition, we report that this mutation induced decreased migration speed but increased invasive properties. Taken together, our data suggest a competition between FA and invadopodia substrates for Src phosphorylation that might depend on environmental cues, thus leading to the engagement of either migration or degradation pathways.