Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

Background aimsBone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees.MethodsBiopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren–Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores.ResultsThe expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05).ConclusionsThese data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.     

Solution structure of the complex formed by the two N-terminal RNA-binding domains of nucleolin and a pre-rRNA target

Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.     

A pre-processing pipeline to quantify,visualize, and reduce technical variation in protein microarray studies

Technical variation, or variation from non-biological sources, is present in most laboratory assays. Correcting for this variation enables analysts to extract a biological signal that informs questions of interest. However, each assay has different sources and levels of technical variation, and the choice of correction methods can impact downstream analyses. Compared to similar assays such as DNA microarrays, relatively few methods have been developed and evaluated for protein microarrays, a versatile tool for measuring levels of various proteins in serum samples. Here, we propose a pre-processing pipeline to correct for some common sources of technical variation in protein microarrays. The pipeline builds upon an existing normalization method by using controls to reduce technical variation. We evaluate our method using data from two protein microarray studies and by simulation. We demonstrate that pre-processing choices impact the fluorescent-intensity based ranks of proteins, which in turn, impact downstream analysis.     

Identification of phagocytic glial cells after lesion-induced anterograde degeneration using double-fluorescence labeling: combination of axonal tracing and lectin or immunostaining

 Retrograde and anterograde degeneration have been reported to be sufficient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic application of small crystals of biotinylated and tetramethylrhodamine (TRITC)-conjugated dextran amine Mini Ruby into the medial entorhinal cortex resulted in a stable rhodamine fluorescence confined to fibers and terminals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion of the entorhinal cortex induced transformation of rhodamine-fluorescent fibers and terminals into small granules. Incorporation of these granules by microglial cells [labeled by fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B₄] or astrocytes (labeled by FITC-coupled glial fibrillary acidic protein antibodies) resulted in phagocytosis-dependent labeling of these non-neuronal cells, which could be identified by double-fluorescence microscopy. Electron microscopical analysis revealed that, following lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- and biotin-conjugated dextran amines are versatile tracers leading to Phaseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemically identified glial cells can be directly observed by this technique on the light and electron microscopical levels. Accepted: 8 January 1997     

Time pressure modulates electrophysiological correlates of early visual processing

Background

Reactions to sensory events sometimes require quick responses whereas at other times they require a high degree of accuracy–usually resulting in slower responses. It is important to understand whether visual processing under different response speed requirements employs different neural mechanisms.

Methodology/Principal Findings

We asked participants to classify visual patterns with different levels of detail as real-world or non-sense objects. In one condition, participants were to respond immediately, whereas in the other they responded after a delay of 1 second. As expected, participants performed more accurately in delayed response trials. This effect was pronounced for stimuli with a high level of detail. These behavioral effects were accompanied by modulations of stimulus related EEG gamma oscillations which are an electrophysiological correlate of early visual processing. In trials requiring speeded responses, early stimulus-locked oscillations discriminated real-world and non-sense objects irrespective of the level of detail. For stimuli with a higher level of detail, oscillatory power in a later time window discriminated real-world and non-sense objects irrespective of response speed requirements.

Conclusions/Significance

Thus, it seems plausible to assume that different response speed requirements trigger different dynamics of processing.     

Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth

Background

An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection.

Methods and Findings

Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-α was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes.

Conclusions

Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.     

Life on the edge: hydrogen sulfide and the fish communities of a Mexican cave and surrounding waters

Most eucaryotic organisms classified as living in an extreme habitat are invertebrates. Here we report of a fish living in a Mexican cave (Cueva del Azufre) that is rich in highly toxic H₂S. We compared the water chemistry and fish communities of the cave and several nearby surface streams. Our study revealed high concentrations of H₂S in the cave and its outflow (El Azufre). The concentrations of H₂S reach more than 300 μM inside the cave, which are acutely toxic for most fishes. In both sulfidic habitats, the diversity of fishes was heavily reduced, and Poecilia mexicana was the dominant species indicating that the presence of H₂S has an all-or-none effect, permitting only few species to survive in sulfidic habitats. Compared to habitats without H₂S, P. mexicana from the cave and the outflow have a significantly lower body condition. Although there are microhabitats with varying concentrations of H₂S within the cave, we could not find a higher fish density in areas with lower concentrations of H₂S. We discuss that P. mexicana is one of the few extremophile vertebrates. Our study supports the idea that extreme habitats lead to an impoverished species diversity.     

Sexual harassment in live-bearing fishes (Poeciliidae): comparing courting and noncourting species

Sexual harassment by males has been reported from several live-bearingfishes (Poeciliidae) and has been shown to inflict costs onfemales. For example, poeciliid females have reduced feedingopportunities when accompanied by a male because females dedicateattention to avoiding male copulation attempts. Poeciliid speciesdiffer considerably in male mating behavior, such as the presenceor absence of courtship. Courting males display in front ofthe females, but males attempting to sneak-copulate approachfemales from behind, that is, in the blind portion of theirvisual field, and force copulations, which can be viewed asa male persistence trait. We predicted that poeciliid femalesneed to be more vigilant in the presence of noncourting males,and costs of harassment by noncourting males might be stronger.In a comparative approach we examined the costs of male sexualharassment for females as reduced feeding time in 9 speciesof live-bearing fishes, including courting (Poecilia latipinna,Poecilia reticulata, Xiphophorus cortezi, Xiphophorus variatus)and noncourting species (Poecilia mexicana [surface- and cave-dwellingform], Poecilia orri, Gambusia affinis, Gambusia geiseri, Heterandriaformosa). In all species examined except for the cave form ofP. mexicana, focal females spent significantly less time feedingin the presence of a male than when together with another female.The time females spent feeding was found to significantly declinewith increasing male mating activity (sum of all sexual behaviors),but there was no support for the idea that females would spendmore time feeding in the presence of courting males comparedwith noncourting ones.