SV2B/miR-34a/miR-128 axis as prognostic biomarker in glioblastoma multiforme.

Glioblastoma (GBM) is a heterogenous primary brain tumour that is characterised with unfavourable patient prognosis. The identification of biomarkers for managing brain malignancies is of utmost importance. MicroRNAs (miRNAs) are small, non-coding RNAs implicated in cancer development. This study aimed to assess the prognostic significance of miRNAs and their gene targets in GBM. An in silico approach was employed to investigate the differentially expressed miRNAs in GBM. The most dysregulated miRNAs were identified and analysed via Sfold in association with their gene target. The candidate gene was studied via multi-omics approaches, followed by in vitro and in vivo experiments. The in silico analyses revealed that miR-128a and miR-34a were significantly downregulated within GBM. Both miRNAs displayed high binding affinity to the synaptic vesicle glycoprotein 2B (SV2B) 3' untranslated region (3'UTR). SV2B exhibited upregulation within brain regions with high synaptic activity. Significantly higher SV2B levels were observed in high grade brain malignancies in comparison to their normal counterparts. SV2B expression was observed across the cytoplasm of GBM cells. Our findings underscored the downregulated expression patterns of miR-128a and miR-34a, alongside the upregulation of SV2B in GBM suggesting the importance of the SV2B/miR-34a/miR-128 axis as a potential prognostic approach in GBM management.

Genetic mosaics of Caenorhabditis elegans: a tissue-specific fluorescent mutant.

Genetic mosaics can be generated by x-irradiation in the simple nematode Caenorhabditis elegans. A mutation in the gene flu-3 alters the characteristic autofluorescence of intestinal cells under ultraviolet light and can be used as a cell- and tissue-specific marker. Embryos heterozygous for flu-3 give rise to adults with patches of these altered intestinal cells. The previously established intestinal cell lineage in Caenorhabditis elegans and the distribution and sizes of the fluorescent patches are consistent with a somatic segregation of the flu-3 allele.

Selective expression of the tba-1 alpha tubulin gene in a set of mechanosensory and motor neurons during the development of Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, a monoclonal antibody 3A5 raised against Drosophila alpha tubulins selectively stains the nervous system immuno-cytochemically. Direct screening of a C. elegans cDNA expression library with 3A5 has allowed cloning of the tba-1 (tubulin alpha-1) gene from C. elegans. The corresponding genomic DNA encodes a protein of 449 amino acid residues that has a high homology with the vertebrate alpha tubulins but a lower homology with yeast alpha tubulins. Interestingly, the carboxyl-terminus sequence EEEGEEY (Glu-Glu-Glu-Gly-Glu-Glu-Tyr) of the nematode tba-1 encoded isotype is identical to these residues in human, mouse, rat, pig and chicken alpha-1 tubulin isotypes that are expressed in the brain. Temporal and spatial expression studies of the tba-1 gene using Northern blot analysis and tba-1::lacZ fusion gene expression analysis during embryonic and the postembryonic development of C. elegans reveal that the tba-1 tubulin is preferentially expressed in the nematode nervous system, especially in a set of mechanosensory neurons and a set of ventral cord motor neurons (DA, DB, VA, and VB) during embryonic and postembryonic development. Our results indicate an inter-species conservation of the alpha tubulin carboxyl-terminal domain in functionally related brain specific isotypes from metazoans as divergent as mammals and nematodes. These results also suggest specificity of the individual alpha tubulin isotypes during neural development.

Molecular cloning and developmental expression of the alpha-2 tubulin gene of Caenorhabditis elegans.

Alpha tubulin isotypes are encoded by at least four genes designated alpha-1 to alpha-4 in the nematode Caenorhabditis elegans. We describe here, molecular cloning of the alpha-2 tubulin gene, located on chromosome I, that encodes a protein of 449 amino acids that has high homology to human, mouse and Drosophila alpha tubulins, but relatively lower homology to the yeast alpha tubulins. The alpha-2 tubulin gene is trans-spliced to the SL1 leader sequence. Northern analysis shows that the gene is increasingly transcribed during the early (L1-L3) larval stages but has a lower level of transcription in L4 L4 larvae, adults, and embryos. Using an alpha-2-lacZ fusion gene expression in transgenic animals, we show that the gene is expressed in a tissue-specific manner in the intestine, pharyngeal muscle cells, and a subset of neurons which include a class of DB and VB motor neurons in the ventral nerve cord, posterior touch receptor neurons, PLML, PLMR, in the lumbar ganglia; PVT in the pre-anal ganglion, and ALA in the dorsal ganglion in the head. Our results support the notion that tubulin structure may contribute to the functional specialization of microtubules.

Exclusive expression of C. elegans osm-3 kinesin gene in chemosensory neurons open to the external environment.

In Caenorhabditis elegans three genetic loci osm-3, unc-104 and unc-116 have been identified, which encode anterograde motor kinesin. Here we show that osm-3 encodes a 672 amino acid long kinesin-like protein (KLP) that contains all three functional domains similar to the kinesin heavy chain, including a globular motor region, an alpha-helical coiled-coil rod, and a globular tail region. OSM-3 shows homology in both the motor and rod domains with kinesins from divergent species such as mouse KIF3, and sea urchin KRP95, and also with the rod domains of several non-kinesin proteins, such as myosin, ezrin, outer membrane proteins alpha precursor OMPA, yeast intracellular protein transport USO1, and the rat neurofilament NF-H. Temporal and spatial expression of the osm-3::lacZ fusion gene during development is limited to an exclusive set of 26 chemosensory neurons whose dendritic endings are exposed to the external environment, including six IL2 neurons of the inner labial sensilla, eight pairs of amphid neurons (ADF, ADL, ASE, ASG, ASH, ASI, ASJ, ASK) in the head, and two pairs of phasmid neurons (PHA and PHB) in the tail. Our data are consistent with the known structural defects in the amphid and phasmid sensilla in osm-3 mutants and also show the expression of the gene in IL2 neurons. Temporally, the gene is differentially expressed in all three types of chemosensory sensilla. Further work on osm-3, unc-104 and unc-116 mutants should give insight into the in vivo functions of the kinesin family during C. elegans neurogenesis.

Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd.

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.

Central neuraxial blockade for splenectomy in myeloproliferative disease: A word of caution.

We describe management of portal vein thrombosis (PVT) in a patient with myeloproliferative disease after splenectomy. This case posed a unique therapeutic challenge in maintaining a fine balance between life-saving thrombolysis and the risk of neuraxial complications due to bleeding. The incidence of PVT after splenectomy in patients with myeloproliferative disorders is high (40%). Anaesthesiologists should be aware of this and avoid central neuraxial blockade in such cases. If post-operative emergency thrombolysis is required in a patient having an epidural catheter in situ, it should be done under close monitoring, weighing the risks and benefits. Fibrinogen levels should be monitored to evaluate the presence of residual thrombolytic effects and to time the catheter removal.

Molecular cloning and three-dimensional structure prediction of a novel alpha-tubulin in Caenorhabditis elegans.

This paper reports on the isolation of a cDNA clone (tba-6) encoded by a novel alpha-tubulin gene in the nematode C. elegans. The tba-6 gene is located on chromosome I, that encode a protein of 460 amino acids, as well as the expression of the gene during the development. Here we discuss the structure of the coding region and the regulatory sequences in the promoter region. The comparison of the amino acid sequence of TBA6 with other alpha-tubulin isotypes of C. elegans, suggests that these proteins are highly conserved in most of the N-terminal and intermediate sequence, but they have highly divergent C-terminal sequences. TBA6 has also high homology with other alpha-tubulin families (e.g. human, mouse, Drosophila melangaster). The in situ experiment results suggest that the tba-6 alpha-tubulin gene is required during the entire embryonic development, therefore it is required during the early cell division stages. Further, we determined the 3D structure of C. elegans TBA6 alpha-tubulin by altering (computationally) the crystal structure of the alpha-tubulin (TBA_pig) from porcine alpha- beta tubulin dimer. We discuss structural conservation and changes in the pattern of interactions between secondary structure elements of TBA_pig and TBA6, respectively.

C. elegans KLP-11/OSM-3/KAP-1: orthologs of the sea urchin kinesin-II, and mouse KIF3A/KIFB/KAP3 kinesin complexes.

Kinesins are intracellular multimeric transport motor proteins that move cellular cargo on microtubule tracks. It has been shown that the sea urchin KRP85/95 holoenzyme associates with a KAP115 non-motor protein, forming a heterotrimeric complex in vitro, called the Kinesin-II. Here we describe isolation of a cDNA clone corresponding to the klp-11 kinesin in C. elegans. Our sequence analysis of the encoded KLP-11 shows that it shares high homology with the OSM-3 kinesin. We also describe a nematode cDNA encoding KAP-1 that shares extensive homology with the sea urchin KAP115 kinesin associated protein. Sequence-based structural analysis of the OSM-3, KLP-11, and KAP-1, presented here suggests that these may form a heterotrimeric complex. We also describe the presence of a Drosophila armadillo consensus motif in CeKAP-1, first found in spKAP115, that suggests a possible role for the KAP-1 in signal transduction.

A novel C-terminal kinesin subfamily may be involved in chromosomal movement in caenorhabditis elegans.

C-terminal kinesin motor proteins, such as the Drosophila NCD and yeast KAR3, are involved in chromosomal segregation. Previously we have described two orthologs of NCD in Caenorhabditis elegans, KLP-3 and KLP-17, which also participate in chromosome movement. Here we report cDNA cloning of klp-15 and klp-16, and the expression pattern of the genes encoding C-terminal motor kinesins including klp-15 and klp-16. Interestingly KLP-15 and KLP-16 form a unique class of C-terminal kinesins, distinct from the previously known C-terminal motors in other organisms. Using in situ hybridization and RNA interference assay, we show that although all of these motors mediate chromosome segregation, they do so in a combination of unique and overlapping manners, suggesting a complex hierarchy of kinesin motor function in metazoans.

A role of ADF chemosensory neurones in dauer formation behaviour in C. elegans.

In Caenorhabditis elegans, mutants in osmotic avoidance behaviour (osm), which fail to avoid high concentrations of salts and sugars, have been previously identified. These osm mutants are also defective in dauer larva formation, and fail to take up fluorescein dye in six pairs of amphid neurones (ADF, ADL, ASH, ASI, ASJ, and ASK) and two pairs of phasmid neurones. Analysis of the FITC dye uptake by osm-3 mutants show that seven of the eight osm-3 alleles can take up FITC dyes in one pair of amphid neurones, ADF. Comparison of dauer larva formation behaviour in different osm-3 alleles shows a direct correlation between improved behaviour and FITC dye uptake. Therefore, these allelic strains are useful in defining the role of ADF neurones in dauer larva formation.

C. elegans osm-3 gene mediating osmotic avoidance behaviour encodes a kinesin-like protein.

In the nematode Caenorhabditis elegans, mutants in osm-3 gene are known to be defective in osmotic avoidance, chemotaxis and dauer formation behaviours. To study the molecular basis of these pleiotropic defects we have cloned the osm-3 gene by germline transformation of osm-3 (p802) mutants through microinjection of the wild type genomic DNA. Northern analysis reveals a 3.0 kb transcript corresponding to osm-3. DNA sequencing of the transforming 4.3 kb fragment revealed a kinesin heavy chain-like protein, which contains conserved ATPase and microtubule binding domains. Our results are consistent with the previous EM data on osm-3 (p802) mutants that show an accumulation of dense matrix material in the amphid sheath cytoplasm and a shortened distal segment of the amphid channel cilium. These data suggest a kinesin-like role of the osm-3 product in axonal transport.

Computational prediction of the three-dimensional structures for the Caenorhabditis elegans tubulin family.

In this article we characterize, from a structural point of view, all 16 members of the tubulin gene family of Caenorhabditis elegans (9 alpha-tubulins, 6 beta-tubulins, and 1 gamma-tubulin). We obtained their tertiary structures by computationally modifying the X-ray crystal structure of the pig brain alpha/beta-tubulin dimer published by Nogales et al. [Nature (London) 1998;391:199-203]. Our computational protocol involves changing the amino acids (with MIDAS; Jarvis et al., UCSF MIDAS. University of California, San Francisco, 1986) in the 3D structure of pig brain alpha/beta-tubulin dimer followed by geometry optimization with the AMBER force field (Perlman et al., AMBER 4. University of California, San Francisco, 1990). We subsequently analyze and compare the resulting structures in terms of the differences in their secondary and tertiary structures. In addition, we compare the pattern of hydrogen bonds and hydrophobic contacts in the guanosine triphosphate (GTP)-binding site for all members of the tubulin family. Our computational results show that, except for gamma-tubulin, all members of the C. elegans tubulin family have similar secondary and 3D structures and that the change in the pattern of hydrogen bonds in the GTP-binding site may be used to assess the relative stability of different alpha/beta-tubulin dimers formed by monomers of the tubulin family.

NMR evidence of formation of cyclocystine loops in peptide models of the high sulfur proteins from wool.

We report that in peptide models of the high sulfur proteins of the matrix from wool a disulfide bond forms between two sequential Cys-residues as a result of a simple oxidation procedure. This tiny cyclocystine loop manifests itself in many ways in 1H NMR spectra. The formation of the loop is accompanied, as expected, by conversion of the Cys-Cys peptide bond from its usual trans-configuration into the energetically less favorable cis-configuration. Possible consequences of the formation of cyclocystine loops for the elasticity of the network of the matrix are discussed.

Mutations affecting axonal growth and guidance of motor neurons and mechanosensory neurons in the nematode Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, each of its 302 individual neurons is an identified neuron. We have screened more than 100 mutations affecting locomotion in C. elegans immunocytochemically, using monoclonal antibodies that recognize specific subsets of neurons. Mutations in 25 genes affect the axonal outgrowth and guidance of a set of 6 mechanosensory receptor neurons (ALML, ALMR, AVM, PVM, PLML and PLMR). Similarly, mutations in 14 genes alter the axonal growth and process placement of two classes of inhibitory motor neurons (DD and VD). Most of these genes affect both embryonic and postembryonic development of the C. elegans nervous system, and have variable expressivity. Our results suggest that different neuron types are specified by a combination of genes that are activated in different cell types. Molecular characterization of such genes could lead to the identification of molecules critical in axonal outgrowth and guidance in higher organisms.

Examination of neurons in wild type and mutants of Caenorhabditis elegans using antibodies to horseradish peroxidase.

Antibodies to horseradish peroxidase (HRP) recognize 27 of 302 neurons and several non-neuronal cells in adult hermaphrodites of the soil nematode Caenorhabditis elegans and can be used to label these cells for cytological analysis in whole animals. The antibodies bind to the anterior members, but not to the posterior members of a set of mechanosensory neurons in wild type animals. Binding to one of the posterior mechanosensory neurons (PVM) occurs when this neuron migrates to an abnormal anterior position in mab-5 mutant animals, suggesting that expression of the epitope recognized by these antibodies is position dependent or that mab-5 mutations transform PVM into AVM intrinsically. The antibodies were used to characterize morphologies of two pairs of lumbar neurons (PHC and PVN) in uncoordinated mutants representing 95 unc genes. PHC and PVN morphologies were normal in most of the unc mutants examined, however, in mutants of 9 unc genes (unc-6, unc-13, unc-33, unc-44, unc-51, unc-61, unc-71, unc-73, and unc-98), misdirected PHC and/or PVN processes were observed at a high frequency. The morphologies of 2 other lumbar neurons, PHA and PHB, were determined previously in these mutants (Hedgecock et al., 1985). Mutations in most, but not all of these 9 unc genes affect the growth of the embryonic lumbar neurons PHA and PHB differently than they affect the growth of the postembryonic lumbar neurons PHC and PVN, indicating that these neurons require different, but overlapping sets of genes for different stages of normal growth and guidance.

Effect of the dpy-20 and rol-6 cotransformation markers on alpha-tubulin gene expression in C. elegans transformants.

An alpha-1 tubulin::lacZ fusion gene was introduced into the germline of Caenorhabditis elegans, using either rol-6 or dpy-20 genomic DNA as a cotransformation marker. Distinct patterns in cellular specificity of the alpha-1 tubulin::lacZ fusion gene expression were observed, depending on the cotransformation marker used. For the rol-6 marker, the tubulin fusion gene was expressed in several neurons in the head and tail ganglia and a set of 38-39 ventral cord motor neurons along the body length of the animal during larval and adult development. In contrast, for the dpy-20 marker system, not only were fewer neurons stained in the head and tail region, but also the staining of ventral cord motor neurons was extremely reduced both in number and intensity. The dpy-20 marked-mediated suppression of the alpha-1 tubulin gene expression was observed both in the cis and trans configurations. Similar down-regulation in the ventral cord motor neurons was observed when the alpha-2 tubulin::lacZ fusion gene construct was tested in these experiments using the dpy-20 marker. In controls, where the tubulin fusion gene was directly injected to obtain transformants without any marker DNA, the cellular staining pattern was close to the fusion gene expression observed with the rol-6 marker DNA. These results underline the importance of the choice of transformation marker system in generation of the transgenic animals, and reveal a down-regulation of the alpha-tubulin fusion gene expression in the ventral cord motor neurons in transgenic animals when the dpy-20 gene was used as a cotransformation marker.

cDNA cloning and expression of a C-terminus motor kinesin-like protein KLP-17, involved in chromosomal movement in Caenorhabditis elegans.

Members of the kinesin protein family transport intracellular cargo to their correct cellular destination. Previously we have characterized the klp-3 gene from Caenorhabditis elegans, which encodes an ortholog of the retrograde C-terminus kinesin motors, such as Drosophila NCD, and yeast KAR3, involved in the chromosomal movement. Here we report the cloning of a full-length klp-17 cDNA in C. elegans, encoding a C-terminus kinesin of 605 amino residues. KLP-17 sequence defines a novel phylogenetic group, distinct from the NCD/KAR3 family. Interestingly, the klp-17 gene transcript is restricted to the nuclear compartment, as deduced by the RNA in situ hybridization in embryos. The klp-17::gfp-expressing transgenic animals do not display any GFP fluorescence signal, but expression of the extra chromosomal arrays cause production of abnormal males, and embryos with morphological defects and lethality in the progeny. Similarly, the klp-17 RNA interference assay results in embryonic death, arrested embryos, and polyploid cells. Thus, KLP-17 represents a new motor protein that mediates chromosome movement, essential for cell divisions during metazoan development.

Kynurenine hydroxylase mutants of the nematode Caenorhabditis elegans.

The relation of intestinal autofluorescence to tryptophan catabolism in the free-living nematode Caenorhabditis elegans has been investigated. L-Kynurenine hydroxylase (EC 1.14.13.9) activity has been detected in normal (wild-type) individuals. Mutants in the gene flu-1 which are characterized by an altered autofluorescence of the intestine cells, i.e., more intense than wild type and bluish purple instead of light blue have also been examined. They show a markedly reduced activity of kynurenine hydroxylase. The finding supports the previously proposed model for altered fluorescence based on chromatographic identification of tryptophan catabolites present.