Summary

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.

Marine species with relatively low migratory capacity are threatened by habitat alterations derived from human activities. In November 2002 the tanker Prestige sank off the Spanish northwest coast releasing 70,000 tons of fuel and damaging biota in the area. Despite efforts to clean the damaged areas, fuel remnants have affected marine species over the last nine years. This study is focused on two flatfish, Lepidorhombus boscii (four-spotted megrim) and L. whiffiagonis (megrim), whose spawning areas are located at the edge of the continental platform. We have analyzed megrim samples from North Spanish and French waters obtained before and after the oil spill. Genotypes at the nuclear marker 5S rDNA indicate a significant increase in interspecific hybridization after the Prestige accident, likely due to forced spawning overlap. The mitochondrial D-Loop region was employed for determining the direction of hybrid crosses, which were most frequently L. boscii female x L. whiffiagonis male. Reduced ability of L. boscii females to select conspecific mates would explain such asymmetric hybridization. To our knowledge this is the first time that increased hybridization between fish species can be associated to an oil spill. These results illustrate the potential long-term effect of petrol wastes on wild fish species.

Background

Know the causes of nasal and bronchial symptoms persistence in patients with Samter's syndrome under treatment in a period of time.

Methods

Cohort study. Inclusion criteria: Patients with asthma diagnoses, hypersensitivity to aspirin and nasal polyps. Exclusion criteria: Other kind of asthma, COPD. Twelve patients were followed from June 2009 to June 2011. Nasal and bronchial symptoms were assessed every 6 months using the Visual Analogue Scale of severity (VAS) from EPOS guidelines and spirometry from GINA. All were treated with intranasal mometasone furoate 200 mcg at day, montelukast 10 mg at day, salmeterol plus fluticasone 50/100 powder 2 inhalation every 12 hours, fluticasone spray 150 mg every 12 hours, loratadine tablet 10 mg if was necessary, with modifications of doses every 3 months.

Patients diagnosed at 6 months with sinusitis and nasal polyposis were administered amoxicillin plus clavulanate 1.5 g daily for 5 weeks. The patients without response at 6 and 18 months were prescribed clarithromycin 400 mg daily for 4 weeks. All patients underwent CT of the sinuses through the Lund-Mackay system, chest CT scan, skin prick test. Evaluated by otolaryngology at the 6, 12, and 18 months.

Results

In the 98, 2% had negative skin prick tests. At 6 months, 58.3% had nasal symptoms with VAS <7. At 33.3% reported bronchial relapses with FEV1 <80. At year nasal symptoms increased, with WAS> 7 in 66.6%. The bronchial relapse decreased to 16.6%. At year and a half it increased nasal symptoms in 75% of patients, with VAS> 7. At 41.6% had obstruction of 100% and pansinusitis. They needed antibiotic scheme. At 2 years in 83.3% had a VAS>7. At 58.3% had pansinusitis. The bronchial relapse did not increase. We determined the presence of VAS> 7 and pansinusitis (OR = 4). The bronchial relapse did not influence with increasing VAS (OR = 1).

Conclusions

Nasal symptoms persistent were secondary to the nasal polyps and pansinusitis with higher levels of VAS. It was determinated a 4-fold risk over pansinusitis with a VAS> 7 (OR = 4). It should be stressed the palliative surgical treatment in earlier stages and desensitization protocols.

Background

Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaVα2δ-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects.

Results

We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaVα2δ-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaVα2δ-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaVα2δ-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaVα2δ-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation.

Conclusions

Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaVα2δ-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.

Past studies in rodent models identified the suppression of primary humoral immune responses as one of the most sensitive sequela associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Yet, the sensitivity of humoral immunity to TCDD in humans represents an important toxicological data gap. Therefore, the objectives of this investigation were two-fold. The first was to assess the induction of known aryl hydrocarbon receptor (AHR)–responsive genes in primary human B cells as a measure of early biological responses to TCDD. The second was to evaluate the direct effect of TCDD on CD40 ligand–induced immunoglobulin M (IgM) secretion by human primary B cells. The effects of TCDD on induction of AHR-responsive genes and suppression of the IgM response were also compared with B cells from a TCDD-responsive mouse strain, C57BL/6. AHR-responsive genes in human B cells exhibited slower kinetics and reduced magnitude of induction by TCDD when compared with mouse B cells. Evaluation of B-cell function from 12 donors identified two general phenotypes; the majority of donors exhibited similar sensitivity to suppression by TCDD of the IgM response as mouse B cells, which was not attributable to decreased B-cell proliferation. In a minority of donors, no suppression of the IgM response by TCDD was observed. Although donor-to-donor variation in sensitivity to TCDD was observed, human B cells from the majority of donors evaluated showed impairment of effector function by TCDD. Collectively, data presented in this series of studies demonstrate that TCDD impairs the humoral immunity of humans by directly targeting B cells.

The concomitant presence of three histopathologically different entities in the pituitary gland is a rare occurrence. Most publications identify at least two distinct pathologies, mainly, a pituitary adenoma coexisting with a second intrasellar lesion. We present a case of a 71-year-old female referred for evaluation and treatment of acromegaly. Questioning revealed she was experiencing facial palsy, visual disturbances, and syncopal spells for several weeks. When laboratory evaluation showed elevated somatomedin (IGF-I) levels and an oral glucose tolerance test failed to demonstrate any suppression of her growth hormone (GH) values, an MRI of the pituitary revealed a sellar mass. A presumptive diagnosis of pituitary adenoma was established. The patient underwent transsphenoidal resection of the sellar mass, which proved to be a large B-cell lymphoma (Stage I-E) associated with areas of adenoma and lymphocytic hypophysitis.

Background

Deep brain stimulation (DBS) of the anterior limb of the internal capsule (ALIC) may be effective in treating depression. Parental verbal abuse has been linked to decreased fractional anisotropy (FA) of white matter and reduced FA correlated with depression and anxiety scores. Utilizing a nonhuman primate model of mood and anxiety disorders following disrupted mother-infant attachment, we examined whether adverse rearing conditions lead to white matter impairment of the ALIC.

Methods

We examined white matter integrity using Diffusion Tensor Imaging (DTI) on a 3T-MRI. Twenty-one adult male Bonnet macaques participated in this study: 12 were reared under adverse [variable foraging demand (VFD)] conditions whereas 9 were reared under normative conditions. We examined ALIC, posterior limb of the internal capsule (PLIC) and occipital white matter.

Results

VFD rearing was associated with significant reductions in FA in the ALIC with no changes evident in the PLIC or occipital cortex white matter.

Conclusion

Adverse rearing in monkeys persistently impaired frontal white matter tract integrity, a novel substrate for understanding affective susceptibility.

The role of the prefrontal cortex as an executive oversight of posterior brain regions raises the question of the extent to which the anterior regions of the brain interconnect with the posterior regions. The aim of this study is to test the complexity of rostral white matter tracts, which connect anterior and posterior brain regions, in comparison to caudal white matter tracts and the corpus callosum. Diffusion tensor imaging (DTI) is a modality that measures fractional anisotropy (FA). Higher white matter complexity could result in a decrease of FA, possibly through denser intersection of fiber tracts. DTI was used to determine regional FA in 9 healthy bonnet macaques (Macaca radiata). Four regions of interest were included: anterior and posterior limbs of the internal capsule, the occipital lobe white matter, and the corpus callosum. FA of the anterior limbs of the internal capsule was lowest compared to all other regions of interest (Newman-Keuls (N-K); p < 0.0001), whereas FA of the corpus callosum was highest (N-K; p < 0.0001). The posterior limbs of the internal capsule and the occipital white matter were not distinguishable but exhibited intermediate FA in comparison to the former (N-K; p < 0.0001) and the latter (N-K; p < 0.0001). The current study demonstrates that FA, a measure of white matter complexity, can vary markedly as a function of region of interest. Moreover, validation of these findings using neurohistological studies and replication in human samples appears warranted.

Wildlife on isolated oceanic islands is highly susceptible to the introduction of pathogens. The recent establishment in the Galápagos Islands of the mosquito Culex quinquefasciatus, a vector for diseases such as avian malaria and West Nile fever, is considered a serious risk factor for the archipelago's endemic fauna. Here we present evidence from the monitoring of aeroplanes and genetic analysis that C. quinquefasciatus is regularly introduced via aircraft into the Galápagos Archipelago. Genetic population structure and admixture analysis demonstrates that these mosquitoes breed with, and integrate successfully into, already-established populations of C. quinquefasciatus in the Galápagos, and that there is ongoing movement of mosquitoes between islands. Tourist cruise boats and inter-island boat services are the most likely mechanism for transporting Culex mosquitoes between islands. Such anthropogenic mosquito movements increase the risk of the introduction of mosquito-borne diseases novel to Galápagos and their subsequent widespread dissemination across the archipelago. Failure to implement and maintain measures to prevent the human-assisted transport of mosquitoes to and among the islands could have catastrophic consequences for the endemic wildlife of Galápagos.

Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-β-lactamase (MBL). The identified blaVIM-19 gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.

SYNOPSIS

Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are well documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the free fatty acid docosahexaenoic acid (DA), but not oleic acid (OA), is decreased and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may alone, or in conjunction with Ly, exert antibacterial action against P. aeruginosa. We found that DA and Ly synergistically inhibit the metabolic activity of P. aeruginosa, in contrast to OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of Ly. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA leading to bacterial cell death. Our study elucidates a molecular basis for the synergistic action of free fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.

OBJECTIVE

We hypothesized that intranasal insulin (I-I) delivery targets the nervous system while avoiding potential adverse systemic effects when compared with subcutaneous insulin (S-I) for experimental streptozotocin-induced diabetic peripheral neuropathy (DPN).

RESEARCH DESIGN AND METHODS

I-I or S-I at 0.87 IU daily or placebo were delivered in separate cohorts of diabetic and nondiabetic CD1 mice during 8 months of diabetes. Radiolabeled insulin detection was used to compare delivery and biodistribution for I-I and S-I. Biweekly behavioral testing and monthly electrophysiological and quantitative studies assessed progression of DPN. At and before end point, morphometric analysis of DRG, peripheral nerve, distal epidermal innervation, and specific molecular markers were evaluated.

RESULTS

Radiolabeled I-I resulted in more rapid and concentrated delivery to the spinal cord and DRG with less systemic insulin exposure. When compared with S-I or intranasal placebo, I-I reduced overall mouse mortality and sensory loss while improving neuropathic pain and electrophysiological/morphological abnormalities in diabetic mice. I-I restored mRNA and protein levels of phosphoinositide 3-kinase/Akt, cyclic AMP response element–binding protein, and glycogen synthase kinase 3β to near normal levels within diabetic DRGs.

CONCLUSIONS

I-I slows the progression of experimental DPN in streptozotocin mice, avoids adverse effects associated with S-I treatment, and prolongs lifespan when compared with S-I. I-I may be a promising approach for the treatment of DPN.

Antibiotics are among the most valuable compounds used for fighting human diseases. Unfortunately, pathogenic bacteria have evolved towards resistance. One important and frequently forgotten aspect of antibiotics and their resistance genes is that they evolved in non-clinical (natural) environments before the use of antibiotics by humans. Given that the biosphere is mainly formed by micro-organisms, learning the functional role of antibiotics and their resistance elements in nature has relevant implications both for human health and from an ecological perspective. Recent works have suggested that some antibiotics may serve for signalling purposes at the low concentrations probably found in natural ecosystems, whereas some antibiotic resistance genes were originally selected in their hosts for metabolic purposes or for signal trafficking. However, the high concentrations of antibiotics released in specific habitats (for instance, clinical settings) as a consequence of human activity can shift those functional roles. The pollution of natural ecosystems by antibiotics and resistance genes might have consequences for the evolution of the microbiosphere. Whereas antibiotics produce transient and usually local challenges in microbial communities, antibiotic resistance genes present in gene-transfer units can spread in nature with consequences for human health and the evolution of environmental microbiota that are largely ignored.

Administration of doxapram hydrochloride, a respiratory stimulant, is experienced by panic disorder patients to be similar to panic attacks but has reduced emotional effect in normal volunteers thus providing a laboratory model of panic for functional imaging. Six panic patients and seven normal control subjects underwent positron emission tomography with 18F-deoxyglucose imaging after a single-blinded administration of either doxapram or a placebo saline solution. Saline and doxapram were administered on separate days in counterbalanced order. Patients showed a greater heart rate increase on doxapram from saline than controls, indicating differential response. On the saline placebo day, patients had greater prefrontal relative activity than controls. In response to doxapram, patients tended to decrease prefrontal activity more than controls, and increased cingulate gyrus and amygdala activity more than controls. This suggests that panic disorder patients activate frontal inhibitory centers less than controls which may lower the threshold for panic.

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.

Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.

Author Summary

There is a new population of HIV+ men who are developing AIDS over months instead of years as typical. It has recently become popular among gay and bisexual men to consume very high levels of Meth. Unsafe sex together with Meth abuse has been suspected to lead to rapid disease progression. While studies show exacerbated AIDS symptoms and disease progression in HIV+ Meth abusers, the molecular mechanism is yet unknown. It was postulated, yet unproven, that the rapid disease progression might be due to a mutant “superstrain” of HIV that was extremely virulent. It was also assumed that the effects of the drug on behavior may lead to unsafe sex, although this would not explain the more rapid time course of the disease. We now demonstrate the first direct evidence that Meth is an immunosuppressive agent, and that the molecular mechanism of this immunosuppression is due to the collapse of acidic organelle pH in cells of the immune system, inhibiting the functions of antigen presentation, as well as phagocytosis. These effects compromise the immune response to opportunistic infections and HIV. These findings could have a major impact on public health, as there are over 35 million Meth abusers worldwide

Structural and promoter MBL2 gene polymorphisms responsible for low MBL levels are associated with increased risk of infection. The objective of this study was to assess the possible association between polymorphisms of the MBL2 gene and the incidence of septic shock and bacteremia in patients with acute pyelonephritis due to Escherichia coli. The study included 62 female patients with acute pyelonephritis due to E. coli who required hospital admission, as well as 133 healthy control subjects. Six single-nucleotide polymorphisms (−550 G/C, −221 C/G, +4 C/T, codon 52 CGT/TGT, codon 54 GGC/GAC, and codon 57 GGA/GAA) in the MBL2 gene were genotyped by using a sequence-based typing technique. No significant differences were observed in the frequencies for low-expression MBL2 genotypes (O/O and LXA/O) between patients with acute pyelonephritis and healthy controls. Patients with acute pyelonephritis and septic shock had a higher incidence of low-expression MBL2 genotypes than patients with acute pyelonephritis without septic shock (odds ratio = 9.019, 95% confidence interval = 1.23 to 65.93; P = 0.03). No association was found between bacteremic acute pyelonephritis and low-expression MBL2 genotypes. We found that low-expression MBL2 genotypes predispose to septic shock but not to bacteremia in patients with E. coli-induced acute pyelonephritis. Determination of MBL2 polymorphisms could be useful for assessing the risk of septic shock in women undergoing acute pyelonephritis.

The CR1 element defined by an orf513 gene encoding a putative recombinase and a recombination crossover site has been identified upstream of several antibiotic resistance genes in Enterobacteriaceae. This CR1 element was shown to bring promoter sequences that play a role in the expression of unrelated antibiotic resistance genes.

A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.

The migration of neutrophils through infected tissues is mediated by the CXC chemokines and its receptors (CXCR1 and CXCR2). It has been proposed that a CXCR1 deficiency could confer susceptibility to acute pyelonephritis in children. The objective of the study is to assess the surface expression of CXCR1 and CXCR2 and the existence of polymorphisms in the CXCR1 gene in premenopausal women with recurrent urinary tract infections. The study included 20 premenopausal women with recurrent urinary infections, with normal urinary tracts, and without diseases potentially associated with relapsing urinary infections and 30 controls without previous urinary infections. The levels of CXCR1 and CXCR2 expression on neutrophils were measured and analyzed by flow cytometry by measuring the mean fluorescence intensity (MFI) channel. The promoter and coding regions of the CXCR1 gene were analyzed for the presence of polymorphisms by a sequence-based typing method. Patients with recurrent urinary tract infections exhibited median levels of CXCR1 expression, determined from MFI values, similar to those of the controls. The analysis of CXCR2 showed that patients with recurrent urinary infections had lower median levels of expression, determined from the MFI values, than the controls (P = 0.002, Mann-Whitney U test). No polymorphisms were detected at the promoter or at the exon 1 region of the CXCR1 gene either in the patients or in the controls. Polymorphisms were detected at the exon 2 of CXCR1, but their frequencies did not differ between patients and controls. We have found a low level of CXCR2 expression in patients with recurrent urinary tract infections. These results suggest that a low level of CXCR2 expression may increase the susceptibilities of premenopausal women to urinary tract infections.

Plasmid-mediated resistance to quinolones is increasingly reported in studies of Enterobacteriaceae. Using a PCR-based strategy, a series of gram-negative species were screened for qnrA-like genes. Shewanella algae, an environmental species from marine and fresh water, was identified as its reservoir. This is a one of the very few examples of progenitor identification of an acquired antibiotic resistance gene.

The possibility that triclosan selects Stenotrophomonas maltophilia mutants overexpressing the multidrug resistance pump SmeDEF is analyzed. Five out of 12 triclosan-selected mutants were less susceptible to antibiotics than the wild-type strain and overproduced SmeDEF. Results are discussed in relation to current debates on the potential selection of antibiotic-resistant bacteria by household biocides.

The smeT-smeDEF region and the smeT gene, which encodes the smeDEF repressor, are highly polymorphic. Few changes in smeT might be associated with smeDEF overexpression. The results obtained with cellular extracts suggest that mutant SmeT proteins cannot bind to the operator and that other transcription factors besides SmeT are involved in the regulation of smeDEF expression.

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14ARF. Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O2 concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.