The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease.

MicroRNA in situ hybridization in tissue microarrays.

In situ hybridization is used to visualize nucleic acids in microscopic tissue sections and has in recent years been used successfully to detect microRNAs. We have further optimized a technique to detect and semiquantitatively assay microRNA expression in tissue microarrays derived from formalin-fixed paraffin-embedded archival tumor tissue.

Dysregulation of protein synthesis and disease.

The regulation of protein synthesis plays as important a role as transcriptional control in the control of gene expression. Once thought solely to act globally, translational control has now been shown to be able to control the expression of most genes specifically. Dysregulation of this process is associated with a range of pathological conditions, notably cancer and several neurological disorders, and can occur in many ways. These include alterations in the expression of canonical initiation factors, mutations in regulatory mRNA sequence elements in 5' and 3' untranslated regions (UTRs), such as upstream open reading frames (uORFs), internal ribosome entry segments (IRESs) and micro-RNA (miR) target sites, and the altered expression of trans-acting protein factors that bind to and regulate these elements. Translational control is increasingly open for study in both fresh and fixed tissue, and this rapidly developing field is yielding useful diagnostic and prognostic tools that will hopefully provide new targets for effective treatments.

Distal tunnel placement improves scaphoid flexion with the Brunelli tenodesis procedure for scapholunate dissociation.

PURPOSE: Treatment of scapholunate dissociation remains difficult. The modified Brunelli procedure, a flexor carpi radialis tenodesis through the scaphoid and secured with dorsal wrist ligaments, has shown promising results. This study compares the biomechanical effects on scaphoid flexion and scapholunate gap between proximal and distal tunnel placement in the modified Brunelli procedure. METHODS: Eight fresh-frozen cadaveric forearms were used. A dorsal approach to the wrist through the floor of the fourth compartment was used. Metallic markers were implanted into the scaphoid and lunate. Tunnels were drilled through the proximal and distal poles of the scaphoid. Wrists were positioned in neutral and loaded to 100 N through the wrist flexor and extensor tendons. Posteroanterior and lateral radiographs were taken with the scapholunate interval intact, with the scapholunate interval sectioned, and after the modified Brunelli tenodesis was performed through the proximal and then distal tunnels using Mersilene tape. Radiographs were analyzed for change in scapholunate angle and scapholunate gap. Multivariate analysis of variance was performed to assess statistical significance for each state compared with the intact wrist. RESULTS: In the intact wrist, the mean scapholunate gap was 1.6 mm +/- 0.1. With the scapholunate interval sectioned, the scapholunate angle increased by 26 degrees +/- 12 and gap increased to 4.2 mm +/- 1.2. With a proximal tunnel for the modified Brunelli procedure, the change in scapholunate angle decreased to 15 degrees +/- 10 and gap decreased to 1.8 mm +/- 0.3. With a distal tunnel for the modified Brunelli procedure, the change in scapholunate angle decreased to 4 degrees +/- 7 and gap decreased to 1.3 mm +/- 0.2. CONCLUSIONS: These biomechanical data suggest that a tunnel exiting in the distal pole of the scaphoid results in better correction of scaphoid flexion when performing the modified Brunelli procedure.

Heparin therapy for complications of placental dysfunction: a systematic review of the literature.

OBJECTIVE: To assess the benefits and harms of antenatal antithrombotic therapy for women at risk of adverse pregnancy outcomes associated with placental dysfunction. SEARCH STRATEGY: PUBMED and the Cochrane Controlled Trials Register (CENTRAL) were searched. Reference lists of retrieved studies were searched by hand. No date or language restrictions were placed. Date of last search February 2008. SELECTION CRITERIA: Randomized controlled trials comparing antenatal antithrombotic therapy (alone or combined with other agents) with placebo or no treatment were considered. Cohort studies with an appropriate control group were also considered. Studies were evaluated independently for appropriateness for inclusion and methodological quality without consideration of their results. Our search strategy identified five case series, two cohort studies with a control group, and one randomized controlled trial. All of the case series and one of the cohort studies were excluded. DATA COLLECTION AND ANALYSIS: The methodological quality of the included studies was poor. There was considerable variation in methodology and the interventions. It was not appropriate to combine results in meta-analysis. MAIN RESULTS: From the randomized trial, heparin was not associated with a reduction in preterm birth less than 37 weeks gestation (Heparin 5/68 versus Control 7/39; relative risk (RR) 0.41; 95% confidence intervals (CIs) 0.14-1.20), or birth weight below 10th centile (Heparin 4/68 versus Control 6/39; RR 0.38; 95% CI 0.11-1.27). CONCLUSION: There is insufficient information to recommend the use of heparin during pregnancy for women at risk of complications due to placental dysfunction. Further information from randomized trials is required.

Synthetic chemerin-derived peptides suppress inflammation through ChemR23.

Chemerin is a chemotactic protein that binds to the G protein-coupled receptor, ChemR23. We demonstrate that murine chemerin possesses potent antiinflammatory properties that are absolutely dependent on proteolytic processing. A series of peptides was designed, and only those identical to specific C-terminal chemerin sequences exerted antiinflammatory effects at picomolar concentrations in vitro. One of these, chemerin15 (C15; A(140)-A(154)), inhibited macrophage (MPhi) activation to a similar extent as proteolyzed chemerin, but exhibited reduced activity as a MPhi chemoattractant. Intraperitoneal administration of C15 (0.32 ng/kg) to mice before zymosan challenge conferred significant protection against zymosan-induced peritonitis, suppressing neutrophil (63%) and monocyte (62%) recruitment with a concomitant reduction in proinflammatory mediator expression. Importantly, C15 was unable to ameliorate zymosan-induced peritonitis in ChemR23(-/-) mice, demonstrating that C15's antiinflammatory effects are entirely ChemR23 dependent. In addition, administration of neutralizing anti-chemerin antibody before zymosan challenge resulted in a significant exacerbation of peritoneal inflammation (up to 170%), suggesting an important endogenous antiinflammatory role for chemerin-derived species. Collectively, these results show that chemerin-derived peptides may represent a novel therapeutic strategy for the treatment of inflammatory diseases through ChemR23.

Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (beta-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic-pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54- and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic-pituitary-gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.

Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry.

We have identified a novel motif which consists of the sequence (CCU)(n) as part of a polypyrimidine-rich tract and permits internal ribosome entry. A number of constructs containing variations of this motif were generated and these were found to function as artificial internal ribosome entry segments (AIRESs) in vivo and in vitro in the presence of polypyrimidine tract-binding protein (PTB). The data show that for these sequences to function as IRESs the RNA must be present as a double-stranded stem and, in agreement with this, rather surprisingly, we show that PTB binds strongly to double-stranded RNA. All the cellular 5' untranslated regions (UTRs) tested that harbor this sequence were shown to contain internal ribosome entry segments that are dependent upon PTB for function in vivo and in vitro. This therefore raises the possibility that PTB or its interacting protein partners could provide a bridge between the IRES-RNA and the ribosome. Given the number of putative cellular IRESs that could be dependent on PTB for function, these data strongly suggest that PTB-1 is a universal IRES-trans-acting factor.

Changes in the centrin and microtubule cytoskeletons after metaphase arrest of the Chlamydomonas reinhardtii met1 mutant.

The temperature-sensitive conditional met1 Chlamydomonas reinhardtii mutant arrests in metaphase at the restrictive temperature (33 degrees C) with an intact spindle and high cell division kinase levels. In this study, met1 was investigated with respect to changes in the microtubule and centrin-based cytoskeletons after arrest at 33 degrees C. Immunofluorescence microscopy revealed that, initially on arrest, the microtubule spindle and centrin-based cytoskeleton appeared as previously reported for wild-type metaphase cells; crescent-shaped spindles were seen with two brightly labelled centrin foci at each spindle pole in the basal body region at the cell surface. Observation of met1 held at the restrictive temperature reveals spindles can detach from one spindle pole and chromosomes eventually detach from the spindles. Moreover, a pseudo-anaphase event of spindle and nucleus elongation occurs in the absence of chromosome separation. Electron microscopy confirms that cytokinesis is initiated, the nuclei maintain a crescent shape but are distended and multiple pyrenoids are detected, suggesting chloroplast division also continues. Interestingly, prolamellar-like bodies usually present in etioplasts of dark-grown plants appear at the nuclear envelope. These results are discussed in relation to the coordination of division events in Chlamydomonas reinhardtii and the loss of viability in arrested cells of this mutant.

Stella is a maternal effect gene required for normal early development in mice.

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.

Cyclin/Cdk complexes: their involvement in cell cycle progression and mitotic division.

DNA replication and mitosis are dependent on the activity of cyclin-dependent protein kinase (CDK) enzymes, which are heterodimers of a catalytic subunit with a cyclin subunit. Cyclin binding to specific individual proteins is thought to provide potential substrates to Cdk. Protein binding by cyclins is assessed in terms of its mechanisms and biological significance, using evidence from diverse organisms including substrate specificity in animal Cdk enzymes containing D-, A-, and B-type cyclins and extensive cyclin gene manipulations in yeasts. Assembly of protein complexes with cyclin/Cdk is noted and the capacity of the cyclin-dependent kinase subunit Cks, in such complex, to extend the range of Cdk substrates is documented and discussed in terms of cell cycle regulation. Cell cycle progression involves changing abundance of individual cyclins, due to changing rates of their transcription or proteolysis, with consequent changes in the substrates of CDK through the cell cycle. Some overlap of the functions of individual cyclins in vivo has been identified by cyclin deletions and is suggested to follow a pattern in which cyclins can commonly complete functions initiated by the preceding cyclins well enough to preserve viability as groups of cyclins are removed by proteolysis. Cyclin accumulation is particularly important in terminating the G1 phase, when it raises CDK activity and starts events leading to DNA replication. It is suggested that plants share this mechanism. The distribution of cyclins and Cdk in maize root tip cells during mitosis and cytokinesis indicates the presence of Cdk1 (Cdc2a) and cyclin CycB1zm;2 at the mature and disassembling preprophase band and the presence of CycB1zm;2 at condensing and condensed chromosomes. Both observations correlate with the earlier-reported capacity of injected metaphase cyclin/CDK to accelerate preprophase band disassembly and chromosome condensation and with observations of the location of Cdk and cyclins in other laboratories. Additionally CycB1zm;2 is seen at the nuclear envelope during its breakdown, which correlates with an acceleration of the process by injected metaphase cyclin B/CDK. A phenomenon possibly unique to the plant kingdom is the persistence of mitotic cyclins after anaphase. Participation of cyclins in cytokinesis is indicated by the concentration of the mitotic cyclin CycA1;zm;1 at the phragmoplast. It is suggested that cyclins have a general function of spatially focusing Cdk activity and that in the plant cell the concentrations of cyclins are important mediators of CDK activity at the cytoskeleton, chromosomes, spindle, nuclear envelope, and phragmoplast.

Merit of mobilization technique in transfer of non-autotransferable R-plasmids.

A total of 200 urinary isolates of Esch.coli received at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli during the years 1995 to 1997 were studied for transferable drug resistance. Out of 188 strains, 134 strains showing resistance to either Nalidixic acid or Rifampicin were subjected to autotransferable resistance studies. Of these 134 strains 131 showed either partial or enbloc transfer of R-factor. Mobilization experiment successfully transferred resistance marker in 14 of the 68 isolates in which resistance to one or more drugs could not be transferred during conjugation experiment.

Prevalance and resistance pattern of Salmonella serotypes in India.

During the period 1990-91, 3222 Salmonella strains were identified at the National Salmonella and Escherichia Centre (NSEC) at Central Research Institute, Kasauli. Of these, 2894 were from humans, 226 from poultry, 84 from animals and remaining 18 from reptiles, birds and other sources. These strains belonged to 53 different serotypes. These include 4 serotypes reported for the first time in India, namely S. kedogou, S. VP. bornheim, S. kisarawe and S. madras. Drug resistance studies revealed that 573 strains were sensitive to all the antibiotics commonly used, 1351 single drug resistant, 594 resistant to two drugs and 704 were multidrug resistant. One strain from human stool was resistant to all the antibiotics used. Prevalence of various Salmonella serotypes and their response to various drugs is discussed.

Interaction of atomic wave packets with four-wave mixing: detection of rubidium and potassium wave packets by coherent ultraviolet emission.

The observation of an atomic wave packet by use of a coherent, nonlinear-optical process is reported. Wave packets formed in K or Rb vapor by two-photon excitation of ns and (n-2)dstates (n=8 for K; n=11 , 12 for Rb) with red (~620-nm) , 80-100-fs pulses were detected by four-wave mixing in pump-probe experiments. The temporal behavior of the wave packet is observed by monitoring the coherent UV radiation generated near the alkali mp(2)P? (2)S(1/2) (7

Adjuvant effect of DEAE-dextran and tetanus toxoid on whole cell heat inactivated phenol preserved typhoid vaccine.

Active mouse protection test (AMPT) and enzyme linked immunosorbent assay (ELISA) were used to determine the immunogenicity of whole cell typhoid vaccine when administered in conjunction with either tetanus toxoid (TT) or DEAE-Dextran (DD). Immunization of mice with whole cell typhoid vaccine showed enhanced potency either when administered in conjunction with TT or DD and values were statistically significant (p < 0.05) in comparison to conventional or standard typhoid vaccines. For ELISA, the mice were immunized with 2 different schedules, one in which a single dose of 0.25 ml subcutaneously (s/c) was administered and in another two doses of 0.25 ml each s/c, 14 days apart. In case of single dose schedule of immunization D vaccine (Whole cell typhoid + 5 mg/ml DD) showed significant increase of immune response (3.201 log10) as compared to plain vaccine (2.550 log10). Two dose schedule further increased the titres to 3.856 log10. DD adjuvanted vaccine showed higher potency by AMPT as compared to the TT adjuvanted vaccine or plain vaccine. The present study clearly demonstrates that a single dose of 0.25 ml which is equivalent to half of the conventionally used single human dose of typhoid vaccine adjuvanted with DD can significantly improve the immunogenicity of the vaccine.

Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia.

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant of Chlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells of Tradescantia virginiana in which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase.

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34cdc2-like protein, recoverable in the p13suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.