Noma: experiences with a microvascular approach under West African conditions

Noma (cancrum oris) is a serious ulcerating disease that generally begins in the gingival sulcus of children. One of the main areas of prevalence today is West Africa. If noma is survived, it results in disfiguring midfacial defects and intense scarring. Oral incompetence is often combined with trismus resulting from scar formation or bony fusion between the maxilla and the mandible. Reconstructive approaches with pedicled flaps from the head or shoulder area for closure of the outer defects have been prone to donor-site complications or have not properly addressed the trismus, leading to high recurrence rates. During three West African Interplast missions, a single-stage procedure was developed for reconstruction of the inner and outer linings of the oral, nasal, and paranasal cavities, with restoration of jaw function. The procedure consists of radical scar excision, placement of an external distractor for mouth opening, and primary closure of the defect with a folded free parascapular flap for full-thickness coverage. Twenty-three patients with various noma-related defects were treated with this procedure; two cases are described in detail. This combined treatment can be a safe successful procedure for patients with noma, especially those with severe soft-tissue destruction and profound trismus, even under demanding surgical conditions.     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.     

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H₂, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (α-FeOOH) forms, and above pH 7.2 magnetite (Fe₃O₄) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ~10⁻⁵. To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B₁₂) biosynthesis.     

Phenotype and regulation of persistent intracerebral T cells in murine Toxoplasma encephalitis

Toxoplasma gondii is a parasite causing asymptomatic, persistent encephalitis. Protective CD4 and CD8 T cells are recruited to and accumulate in the brain in acute Toxoplasma encephalitis (TE), with slowly decreasing numbers in chronic TE. It is unclear how the size of the intracerebral T cell pool is regulated. Conceivably, permanent recruitment, proliferation, and apoptosis may be involved. We observed that in murine TE recruitment of T cells to the brain was terminated in chronic TE. In vivo 5-bromo-2'-deoxyuridine incorporation and in vitro T cell proliferation experiments revealed that intracerebral T cells did not proliferate, which was explained by the expression of the cell cycle inhibitors p21(Waf/cip1) and p27(Kip1) and the inhibitory activity of intracerebral F4/80(+) cells. TUNEL staining detected apoptotic T cells at low frequency corresponding to an increased expression of the anti-apoptotic molecules Bcl-2 and Bcl-x(L) and a reduced expression of the pro-apoptotic molecules Bad, Bax, and Fas ligand in CD4 and CD8 T cells. During progression from acute to chronic TE, both CD4 and CD8 T cells down-regulated CD45RB expression and expressed a differential pattern of cytokines. From these experiments it is concluded that the number of intracerebral T cells increases by recruitment of T cells during acute infection, whereas proliferation of intracerebral T cells does not play a role. In chronic TE, T cell recruitment is terminated, the phenotype of intracerebral T cells changes, and their number is gradually downsized by low level apoptosis, which, however, does not completely resolve the T cell infiltrates.     

BAFF,APRIL, TWEAK,BCMA, TACI and Fn14 Proteins Are Related to Human Glioma Tumor Grade: Immunohistochemistry and Public Microarray Data Meta-Analysis

Gliomas are common and lethal tumors of the central nervous system (CNS). Genetic alterations, inflammatory and angiogenic processes have been identified throughout tumor progression; however, treatment still remains palliative for most cases. Biological research on parameters influencing cell survival, invasion and tumor heterogeneity identified several cytokines interfering in CNS inflammation, oxidative stress and malignant transformation, including TNF-superfamily (TNFSF) members. In this report we performed a meta-analysis of public gene-array data on the expression of a group of TNFSF ligands (BAFF, APRIL, TWEAK) and their receptors (BAFF-R, TACI, BCMA, Fn14) in gliomas. In addition, we investigated by immunohistochemistry (IHC) the tumor cells'' expression of these ligands and receptors in a series of 56 gliomas of different grade. We show that in IHC, BAFF and APRIL as well as their cognate receptors (BCMA, TACI) and Fn14 expression correlate with tumor grade. This result was not evidenced in micro-arrays meta-analysis. Finally, we detected for the first time Fn14, BAFF, BCMA and TACI in glioma-related vascular endothelium. Our data, combined with our previous report in glioma cell lines, suggest a role for these receptors and ligands in glioma biology and advance these molecules as potential markers for the classification of these tumors to the proliferative, angiogenic or stem-like molecular subtype.     

Mitochondrial matrix calcium is an activating signal for hormone secretion

Mitochondrial Ca(2+) signals have been proposed to accelerate oxidative metabolism and ATP production to match Ca(2+)-activated energy-consuming processes. Efforts to understand the signaling role of mitochondrial Ca(2+) have been hampered by the inability to manipulate matrix Ca(2+) without directly altering cytosolic Ca(2+). We were able to selectively buffer mitochondrial Ca(2+) rises by targeting the Ca(2+)-binding protein S100G to the matrix. We find that matrix Ca(2+) controls signal-dependent NAD(P)H formation, respiration, and ATP changes in intact cells. Furthermore, we demonstrate that matrix Ca(2+) increases are necessary for the amplification of sustained glucose-dependent insulin secretion in β cells. Through the regulation of NAD(P)H in adrenal glomerulosa cells, matrix Ca(2+) also acts as a positive signal in reductive biosynthesis, which stimulates aldosterone secretion. Our dissection of cytosolic and mitochondrial Ca(2+) signals reveals the physiological importance of matrix Ca(2+) in energy metabolism required for signal-dependent hormone secretion.     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

Quantum dots – a versatile tool in plant science?

An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitve than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes.