Deciphering the molecular machinery of stem cells: a look at the neoblast gene expression profile

Background  

Mammalian stem cells are difficult to access experimentally; model systems that can regenerate offer an alternative way to characterize stem cell related genes. Planarian regeneration depends on adult pluripotent stem cells - the neoblasts. These cells can be selectively destroyed using X-rays, enabling comparison of organisms lacking stem cells with wild-type worms.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.     

Distribution and Numbers of Pygmies in Central African Forests

Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. However, their numbers and actual distribution is not known precisely. Here, we undertake this task by using locational data and population sizes for an unprecedented number of known Pygmy camps and settlements (n = 654) in five of the nine countries where currently distributed. With these data we develop spatial distribution models based on the favourability function, which distinguish areas with favourable environmental conditions from those less suitable for Pygmy presence. Highly favourable areas were significantly explained by presence of tropical forests, and by lower human pressure variables. For documented Pygmy settlements, we use the relationship between observed population sizes and predicted favourability values to estimate the total Pygmy population throughout Central Africa. We estimate that around 920,000 Pygmies (over 60% in DRC) is possible within favourable forest areas in Central Africa. We argue that fragmentation of the existing Pygmy populations, alongside pressure from extractive industries and sometimes conflict with conservation areas, endanger their future. There is an urgent need to inform policies that can mitigate against future external threats to these indigenous peoples’ culture and lifestyles.     

Fibrin Sealants in Dura Sealing: A Systematic Literature Review

BackgroundFibrin sealants are widely used in neurosurgery to seal the suture line, provide watertight closure, and prevent cerebrospinal fluid leaks. The aim of this systematic review is to summarize the current efficacy and safety literature of fibrin sealants in dura sealing and the prevention/treatment of cerebrospinal fluid leaks.MethodsA comprehensive electronic literature search was run in the following databases: Cochrane Database of Systematic Reviews, Cochrane Central Resister of Controlled Trials, clinicaltrials.gov, MEDLINE/PubMed, and EMBASE. Titles and abstracts of potential articles of interest were reviewed independently by 3 of the authors.ResultsA total of 1006 database records and additional records were identified. After screening for duplicates and relevance, a total of 78 articles were assessed by the investigators for eligibility. Thirty-eight were excluded and the full-text of 40 articles were included in the qualitative synthesis. Seven of these included only safety data and were included in the safety assessment. The remaining 33 articles included findings from 32 studies that enrolled a total of 2935 patients who were exposed to fibrin sealant. Among these 33 studies there were only 3 randomized controlled trials, with the remaining being prospective cohort analysis, case controlled studies, prospective or retrospective case series. One randomized controlled trial, with 89 patients exposed to fibrin sealant, found a greater rate of intraoperative watertight dura closure in the fibrin sealant group than the control group (92.1% versus 38.0%, p<0.001); however, post-operative cerebrospinal fluid leakage occurred in more fibrin sealant than control patients (6.7% versus 2.0%, p>0.05). Other clinical trials evaluated the effect of fibrin sealant in the postoperative prevention of cerebrospinal fluid leaks. These were generally lower level evidence studies (ie, not prospective, randomized, controlled trials) that were not designed or powered to demonstrate a significant advantage to fibrin sealant use. Two small case series studies evaluated the effect of fibrin sealants in persistent cerebrospinal fluid leak treatment, but did not establish firm efficacy conclusions. Specific adverse reports where fibrin sealants were used for dura sealing were limited, with only 8 cases reported in neurosurgical procedures since 1987 and most reporting only a speculative relationship/association with fibrin sealant exposure.ConclusionsA major finding of this systematic literature review is that there is a paucity of randomized studies that have evaluated the effectiveness and safety of fibrin sealants in providing intraoperative watertight dura closure and post-operative cerebrospinal fluid leakage. Among the limited studies available, evidence from a single randomized, controlled trial indicates that fibrin sealants provide a higher rate of intraoperative watertight closure of the dura suture line than control, albeit with a higher rate of postoperative cerebrospinal fluid leakage. Evidence from non-randomized, controlled trials suggests that fibrin sealants may be effective in preventing cerebrospinal fluid leaks with an acceptable safety profile. There is a substantial need for randomized, controlled clinical trials or well-designed prospective observational trials where the conduct of a randomized trial is not feasible to fully assess the impact of fibrin sealant utilization on the rates of intraoperative dura closure, postoperative cerebrospinal leakage, and safety.     

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.