The genus Prevotella in cystic fibrosis airways

Airway disease resulting from chronic bacterial colonization and consequential inflammation is the leading cause of morbidity and mortality in patients with Cystic Fibrosis (CF). Although traditionally considered to be due to only a few pathogens, recent re-examination of CF airway microbiology has revealed that polymicrobial communities that include many obligate anaerobes colonize lower airways. The purpose of this study was to examine Prevotella species in CF airways by quantitative culture and phenotypic characterization. Expectorated sputum was transferred to an anaerobic environment immediately following collection and examined by quantitative microbiology using a variety of culture media. Isolates were identified as facultative or obligate anaerobes and the later group was identified by 16S rRNA sequencing. Prevotella spp. represented the majority of isolates. Twelve different species of Prevotella were recovered from 16 patients with three species representing 65% of isolates. Multiple Prevotella species were often isolated from the same sputum sample. These isolates were biochemically characterized using Rapid ID 32A kits (BioMérieux), and for their ability to produce autoinducer-2 and β-lactamases. Considerable phenotypic variability between isolates of the same species was observed. The quantity and composition of Prevotella species within a patients’ airway microbiome varied over time. Our results suggest that the diversity and dynamics of Prevotella in CF airways may contribute to airway disease.     

Carbonaceous preservation of Cambrian hexactinellid sponge spicules

Early fossil sponges offer a direct window onto the evolutionary emergence of animals, but insights are limited by the paucity of characters preserved in the conventional fossil record. Here, a new preservational mode for sponge spicules is reported from the lower Cambrian Forteau Formation (Newfoundland, Canada), prompting a re-examination of proposed homologies and sponge inter-relationships. The spicules occur as wholly carbonaceous films, and are interpreted as the remains of robust organic spicule sheaths. Comparable sheaths are restricted among living taxa to calcarean sponges, although the symmetries of the fossil spicules are characteristic of hexactinellid sponges. A similar extinct character combination has been documented in the Burgess Shale fossil Eiffelia. Interpreting the shared characters as homologous implies complex patterns of spicule evolution, but an alternative interpretation as convergent autapomorphies is more parsimonious. In light of the mutually exclusive distributions of these same characters among the crown groups, this result suggests that sponges exhibited an early episode of disparity expansion followed by comparatively constrained evolution, a pattern shared with many other metazoans but obscured by the conventional fossil record of sponges.     

Wheat mitochondrial proteomes provide new links between antioxidant defense and plant salinity tolerance

The mitochondrial proteome and differences associated with salt tolerance have been investigated in Australian commercial varieties of wheat. Mitochondria isolated from shoots were used to generate a wheat mitochondrial reference map; 68 unique wheat mitochondrial proteins were identified from 192 gel spots using 2D PAGE and LC-MS/MS. This analysis also provided MS/MS spectra for 199 proteotypic peptides as a foundation for the development of targeted proteomics to study the respiratory apparatus in wheat. Using this reference map and 2D DIGE, we have found quantitative differences in the shoot mitochondrial proteomes of v. Wyalkatchem and v. Janz, two commercially important wheat varieties that are known from a range of experiments to differ in salinity tolerance. These proteins included Mn-superoxide dismutase (Mn-SOD), cysteine synthase, nucleotide diphosphate kinase, and the voltage dependent anion channel (VDAC). Antibodies to the mitochondrial alternative oxidase (AOX), previously linked to reduced ROS formation from the electron transport chain and salt tolerance in Arabidopsis, also showed a commensurate higher abundance in v. Wyakatchem in both control and salt-treated conditions. Together, the data presented here suggest that differences in mitochondrial ROS defense pathways in the mitochondrial proteomes of key Australian wheat varieties correlate with whole-plant salinity tolerance.     

Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis

PURPOSE: Systemic juvenile idiopathic arthritis is a chronic pediatric disease. The initial clinical presentation can mimic other pediatric inflammatory conditions, which often leads to significant delays in diagnosis and appropriate therapy. SJIA biomarker development is an unmet diagnostic/prognostic need to prevent disease complications. EXPERIMENTAL DESIGN: We profiled the urine peptidome to analyze a set of 102 urine samples, from patients with SJIA, Kawasaki disease (KD), febrile illnesses (FI), and healthy controls. A set of 91 plasma samples, from SJIA flare and quiescent patients, were profiled using a customized antibody array against 43 proteins known to be involved in inflammatory and protein catabolic processes. RESULTS: We identified a 17-urine-peptide biomarker panel that could effectively discriminate SJIA patients at active, quiescent, and remission disease states, and patients with active SJIA from confounding conditions including KD and FI. Targeted sequencing of these peptides revealed that they fall into several tight clusters from seven different proteins, suggesting disease-specific proteolytic activities. The antibody array plasma profiling identified an SJIA plasma flare signature consisting of tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin (IL)-18, regulated upon activation, normal T cell expressed and secreted (RANTES), P-Selectin, MMP9, and L-Selectin. CONCLUSIONS AND CLINICAL RELEVANCE: The urine peptidomic and plasma protein analyses have the potential to improve SJIA care and suggest that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9058-8) contains supplementary material, which is available to authorized users.     

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

Background

The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.

Methodology/Principal Findings

We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1^Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1^Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.

Conclusions/Significance

These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.     

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.     

Genetic covariation of the marine fungal symbiont Haloguignardia irritans (Ascomycota, Pezizomycotina) with its algal hosts Cystoseira and Halidrys (Phaeophyceae, Fucales) along the west coast of North America

The fungal endophyte Haloguignardia irritans induces gall formation on the brown algal genera Cystoseira and Halidrys occurring from Oregon to Baja California, Mexico. Here we examine genetic covariation and compare rDNA phylogenies to investigate the coevolutionary histories of H. irritans and its algal hosts. Despite recognition of H. irritans as a single morphological species, internal transcribed spacer rDNA sequences representative of its geographic range are characterized by sequence variation at the intraspecific to intrageneric levels. An assessment of parallel cladogenesis between endophyte and host phylogenies provides evidence for a combination of independent fungal divergence and host jumping, similar to that observed in terrestrial lichens. Our results suggest that reduced gene flow due to geographic isolation is a major contributing factor to more concerted covariation observed at one island site, rather than to differences among algal host species alone. Because geography and its effects on gene flow can create heterogeneous mosaics of coevolution for symbioses in terrestrial environments, our results support the notion that conservation efforts toward the maintenance of genetic diversity in marine environments should likewise consider geographic complexity and its effects on coevolving marine species.     

JNK Deficiency Enhances Fatty Acid Utilization and Diverts Glucose From Oxidation to Glycogen Storage in Cultured Myotubes

Although germ‐line deletion of c‐Jun NH₂‐terminal kinase (JNK) improves overall insulin sensitivity in mice, those studies could not reveal the underlying molecular mechanism and the tissue site(s) in which reduced JNK activity elicits the observed phenotype. Given its importance in nonesterified fatty acids (NEFA) and glucose utilization, we hypothesized that the insulin‐sensitive phenotype associated with Jnk deletion originates from loss of JNK function in skeletal muscle. Short hairpin RNA (shRNA)–mediated gene silencing was used to identify the functions of JNK subtypes in regulating energy metabolism and metabolic responses to elevated concentrations of NEFA in C2C12 myotubes, a cellular model of skeletal muscle. We show for the first time that cellular JNK2‐ and JNK1/JNK2‐deficiency divert glucose from oxidation to glycogenesis due to increased glycogen synthase (GS) activity and induction of Pdk4. We further show that JNK2‐ and JNK1/JNK2‐deficiency profoundly increase cellular NEFA oxidation, and their conversion to phospholipids and triglyceride. The increased NEFA utilization was coupled to increased expressions of selective NEFA handling genes including Cd36, Acsl4, and Chka, and enhanced palmitic acid (PA)‐dependent suppression of acetyl‐CoA carboxylase (Acc). In JNK‐intact cells, PA inhibited insulin signaling and glycogenesis. Although silencing Jnk1 and/or Jnk2 prevented PA‐induced inhibition of insulin signaling, it did not completely block decreased insulin‐mediated glycogenesis, thus indicating JNK‐independent pathways in the suppression of glycogenesis by PA. Muscle‐specific inhibition of JNK2 (or total JNK) improves the capacity of NEFA utilization and glycogenesis, and is a potential therapeutic target for improving systemic insulin sensitivity in type 2 diabetes (T2D).     

Administration and interpretation of the Trail Making Test

Measurement of cognitive functions is an increasingly important goal for clinicians and researchers. Many neuropsychological test batteries are comprehensive and require specialized training to administer and interpret. The Trail Making Test is an accessible neuropsychological instrument that provides the examiner with information on a wide range of cognitive skills and can be completed in 5-10 min. Its background, psychometric properties, administration procedures and interpretive guidelines are provided in this protocol.     

Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix

The interactions of ATP inside plant mitochondria were investigated by identifying the soluble nucleotide binding proteome captured using immobilized ATP. Selected proteins were separated by 1D SDS-PAGE and 2D IEF-SDS-PAGE and identified by ESI-Q-TOF MS/MS. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA binding proteins, as well as proteins known to contain nucleotide binding domains and/or to be inhibited or stimulated by ATP.