[A Uncommon Case: Kasabach-Merritt syndrome with VACTERL Association]. / Ein seltener Fall: Kasabach-Merrit-Syndrom bei VACTERL-Assoziation.

The Kasabach-Merrit syndrome is characterized as the association of a vascular tumor, typically a caposiform hemangioendothelioma and rarely a tufted hemangioma, and a severe consumptive coagulopathy with potentially life-threatening thrombocytopenia. The severe coagulopathy with increased bleeding tendency must be considered before invasive procedures and often requires repeated platelet concentrate substitutions. We present a case of a mature male neonate with Kasabach-Merritt- Syndrome as well as VACTERL association. The VACTERL association describes a group of malformations. Our patient presented with anal atresia combined with tethered cord, and left renal agenesis. The VACTERL association as well as Kasabach-Merritt syndrome were found to be independent pathologies within this patient. A common occurrence or an association with each other has not been described in the literature so far. The challenging coagulation setting due to severe thrombocytopenia complicated the surgical management so far. Finally, mTOR-inhibitor sirolimus was successful in terms of tumor reduction and especially reduction of platelet consumption.

Off-loading strategies in diabetic foot syndrome-evaluation of different devices.

INTRODUCTION: Diabetic foot syndrome is one of the most dreaded complications in diabetes mellitus. The purpose of this study was to assess the value of different offloading devices compared to walking in barefoot condition and in normal shoes both in healthy subjects and in patients with diabetes and neuropathy. METHODS: Twenty patients with diabetes and polyneuropathy and ten healthy probands were included. Pedobarographic examination was performed in barefoot condition, with sneakers, postoperative shoes, Aircast® Diabetic Pneumatic Walker™ and VACO®diaped. In the diabetic group, a total contact cast was additionally tested. RESULTS: The most effective reduction of force was achieved by TCC (75%) and VACOdiaped (64.3%) with the VACO®diaped resulting in the most homogeneous distribution of forces all over the foot. DISCUSSION/CONCLUSION: A customized device like the TCC is still the most proven offloading device. However, a removable cast walker being based on vacuum pads and a cushioning sole, provides better results concerning force distribution.

Cytosolic free calcium dynamics as related to hyphal and colony growth in the filamentous fungal pathogen Colletotrichum graminicola.

Tip growth of pollen tubes and root hairs of plants is oscillatory and orchestrated by tip-focussed variations of cytosolic free calcium ([Ca(2+)]cyt). Hyphae of filamentous fungi are also tubular tip-growing cells, and components of the Ca(2+) signalling machinery, such as Ca(2+) channels and Ca(2+) sensors, are known to be important for fungal growth. In this study, we addressed the questions if tip-focussed [Ca(2+)]cyt transients govern hyphal and whole-colony growth in the maize pathogen Colletotrichum graminicola, and whether colony-wide [Ca(2+)]cyt dynamics rely on external Ca(2+) or internal Ca(2+) stores. Ratiometric fluorescence microscopy of individual hyphae expressing the Ca(2+) reporter Yellow Cameleon 3.6 revealed that Ca(2+) spikes in hyphal tips precede the re-initiation of growth after wounding. Tip-focussed [Ca(2+)]cyt spikes were also observed in undisturbed growing hyphae. They occurred not regularly and at a higher rate in hyphae growing at a medium-glass interface than in those growing on an agar surface. Hyphal tip growth was non-pulsatile, and growth speed was not correlated with the rate of spike occurrence. A possible relationship of [Ca(2+)]cyt spike generation and growth of whole colonies was assessed by using a codon-optimized version of the luminescent Ca(2+) reporter Aequorin. Depletion of extracellular free Ca(2+) abolished [Ca(2+)]cyt spikes nearly completely, but had only a modest effect on colony growth. In a pharmacological survey, some inhibitors targeting Ca(2+) influx or release from internal stores repressed growth strongly. However, although some of those inhibitors also affected [Ca(2+)]cyt spike generation, the effects on both parameters were not correlated. Collectively, the results indicate that tip growth of C. graminicola is non-pulsatile and not mechanistically linked to tip-focused or global [Ca(2+)]cyt spikes, which are likely a response to micro-environmental parameters, such as the physical properties of the growth surface.

A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi.

To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses.

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.

An inexpensive technique for dot blotting in a 96-well format.

Dot blotting for the quantification of proteins usually requires expensive devices. Here we present an inexpensive way to facilitate dot blotting in a 96-well format. An agarose gel was used as sample reservoir, and proteins were electrophoretically transferred from the gel to a membrane. Dots produced by this technique were applied to reproducible immunoquantification of proteins such as beta-actin and doublecortin.

Calcium Transport Proteins in Fungi: The Phylogenetic Diversity of Their Relevance for Growth, Virulence, and Stress Resistance.

The key players of calcium (Ca2+) homeostasis and Ca2+ signal generation, which are Ca2+ channels, Ca2+/H+ antiporters, and Ca2+-ATPases, are present in all fungi. Their coordinated action maintains a low Ca2+ baseline, allows a fast increase in free Ca2+ concentration upon a stimulus, and terminates this Ca2+ elevation by an exponential decrease - hence forming a Ca2+ signal. In this respect, the Ca2+ signaling machinery is conserved in different fungi. However, does the similarity of the genetic inventory that shapes the Ca2+ peak imply that if "you've seen one, you've seen them all" in terms of physiological relevance? Individual studies have focused mostly on a single species, and mechanisms elucidated in few model organisms are usually extrapolated to other species. This mini-review focuses on the physiological relevance of the machinery that maintains Ca2+ homeostasis for growth, virulence, and stress responses. It reveals common and divergent functions of homologous proteins in different fungal species. In conclusion, for the physiological role of these Ca2+ transport proteins, "seen one," in many cases, does not mean: "seen them all."

Radial glial elements in the cerebral cortex of the lesser hedgehog tenrec.

We investigated astroglial cells in several areas of the telencephalic cortex of the lesser hedgehog tenrec (Echinops telfairi). Compared to other mammals, the cortex of the tenrec has a relatively large paleocortex and a low encephalization index. We stained sections from tenrec forebrains with structural and functional glia markers focusing on selected cortical areas, the paleocortex, rhinal cortex, neocortex and the dentate gyrus of the hippocampal formation. We found that in all parts of the tenrec forebrain cortex, radial processes exist which are positive for glial fibrillary acidic protein (GFAP) although with differential localization: in the rhinal cortex and neocortical region radial glial fibers are located in the subventricular regions, whereas in the dentate gyrus and paleocortex they appear to arise from the cells in the respective granular layers. The relatively high abundance of the radial fibers in layer III of the paleocortex was very conspicuous. Only few of these radial processes were also co-labeled with doublecortin (DCX), yet most of the DCX-positive cells were negative for GFAP. The GFAP-positive radial fibers were in turn neither positive for glutamine synthetase, nor did they show immunoreactivity for the astroglia-specific water channel aquaporin-4 (AQP4). Star-shaped astrocytes, however, displayed the typical perivascular and subpial expression patterns for AQP4. We conclude that the radial glia in the adult tenrec represents an immature form of astroglia that persists in these animals throughout life.

The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction.