Implementation and evaluation of a new automated interactive image analysis system

OBJECTIVE: To compare automated interactive screening using the ThinPrep Imaging System with independent manual primary screening of 12,000 routine ThinPrep slides. STUDY DESIGN: With the first 6,000 cases, the Review Scopes (RS) screening results from the 22 fields of view (FOV) only were compared to independent manual primary screening. In the next 6,000 cases, any abnormality detected in the 22 FOV resulted in full manual screening on the cytotechnologist's own microscope. Sensitivity and specificity together with their 95% CIs were calculatedfor each method. RESULTS: In the first set of 6, 000 cases, diagnostic sensitivity and specificity of the imager were 85.19% and 96.67%, respectively. The diagnostic sensitivity and specificity of manual primary screening were 89.38% and 98.42%. This highersensitivity and specificity of manual primary screening were found to be statistically significant. The second set of 6,000 cases demonstrated no significant statistical difference in sensitivity or specificity between the sets of data. CONCLUSION: The results from our study show that the sensitivity and specificity of the imager technology are equivalent to those of manual primary screening. The system is ideally suited to the rapid screening of negative cases, allowing increased laboratory productivity and greater throughput of cases on a daily basis.     

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.     

IRAK-2 participates in multiple toll-like receptor signaling pathways to NFkappaB via activation of TRAF6 ubiquitination

Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFkappaB signaling axis, recent data have shown that it is dispensable for NFkappaB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFkappaB activation pathway. The poxviral TLR antagonist A52 inhibited NFkappaB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFkappaB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFkappaB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFkappaB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFkappaB.     

The Glycobiome of the Rumen Bacterium Butyrivibrio proteoclasticus B316T Highlights Adaptation to a Polysaccharide-Rich Environment

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316^T is a Gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.     

Hereditary dentine disorders: dentinogenesis imperfecta and dentine dysplasia

Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life), skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum), hearing impairment (moderate-to-severe sensorineural hearing loss), gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12). Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating manifestations. Infections must be treated frequently. Otolaryngological treatment of fluid in the middle ear is often required and use of hearing aids is invariably required. Early educational intervention for development of social skills is needed and physiotherapy is important to improve bodily function. Orthopedic surgery may be necessary. The long-term prognosis is poor. There is an insidiously slow progression of neuromuscular and skeletal deterioration over several decades, making most patients wheel-chair dependent. No patients manage to be completely socially independent. Many patients are over 50 years of age.     

Metapopulation structure,population trends,and status of Florida Grasshopper Sparrows

ABSTRACT The federally Endangered Florida Grasshopper Sparrow (FGSP, Ammodramus savannarum floridanus) is endemic to dry prairie habitat in central Florida and is currently only found at three public management areas (Avon Park Air Force Range, APAFR; Kissimmee Prairie Preserve State Park, KPPSP; and Three Lakes Wildlife Management Area, TLWMA). We analyzed long‐term (1991–2008) point‐count data to compare population trends of FGSPs at these management areas to determine if they function as independent populations by using Spearman's rank correlation to test for independence between annual trends. We also examined banding and resighting data to infer metapopulation structure. Populations fluctuated across years at all three sites, declining significantly at APAFR (r_s=?0.89, P < 0.001, N= 13) and KPPSP (r_s=?0.78, P= 0.004, N= 11) and remaining stable at TLWMA (r_s= 0.19, P= 0.46, N= 17). Population trends among the three management areas appeared independent (absolute value of r_s≤ 0.50, P≥ 0.12). Previous studies indicated that sparrows in the three areas were not genetically differentiated, and two cases of dispersal between APAFR and KPPSP have been documented. However, dispersal rates among areas appear to be too low to influence demographic dynamics within individual areas. Within APAFR, FGSPs are aggregated into three spatially distinct habitat patches previously considered separate populations, but dispersal among these patches is more frequent than previously reported and population trends among these patches are correlated (r_s≥ 0.91, P < 0.001). These patterns suggest that a single metapopulation of FGSPs exists consisting of three distinct populations (APAFR, KPPSP, and TLWMA), and the spatially distinct aggregations at APAFR constitute a single population using several habitat patches. The population at APAFR is at risk of extirpation, and immediate action is needed if that population is to recover. Taking broader metapopulation dynamics into account will be useful for guiding management efforts aimed at conserving the FGSP in the broader central Florida landscape.     

M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis

M-cells are specialized cells found in the follicle-associated epithelium of intestinal Peyer's patches of gut-associated lymphoid tissue and in isolated lymphoid follicles, appendix and in mucosal-associated lymphoid tissue sites outside the gastrointestinal tract. In the gastrointestinal tract, M-cells play an important role in transport of antigen from the lumen of the small intestine to mucosal lymphoid tissues, where processing and initiation of immune responses occur. Thus, M-cells act as gateways to the mucosal immune system and this function has been exploited by many invading pathogens. Understanding the mechanism by which M-cells sample antigen will inform the design of oral vaccines with improved efficacy in priming mucosal and systemic immune responses. In this review, the origin and morphology of M-cells, and their role in mucosal immunity and pathogenesis of infections are discussed.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.