Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.     

Malignant glioma progression and nitric oxide

Glioblastoma multiforme, the most common of the malignant gliomas, carries a dismal prognosis in spite of the most aggressive therapy and recent advances in molecular pathways of glioma progression. Although it has received relatively little attention in the setting of malignant gliomas, nitric oxide metabolism may be intimately associated with the disease process. Interestingly, nitric oxide has both physiological roles (e.g., neurotransmitter-like activity, stimulation of cyclic GMP), and pathophysiological roles (e.g., neoplastic transformation, tumor neovascularization, induction of apoptosis, free radical damage). Moreover, whether nitric oxide is neuroprotective or neurotoxic in a given disease state, or whether it enhances or diminishes chemotherapeutic efficacy in malignant neoplasia, is unresolved. This review discusses the multifaceted activity of nitric oxide with particular reference to malignant gliomas.     

Genetic exchange of the S2 and S3 subunits in pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, produces a complex hetero-oligomeric exotoxin, named pertussis toxin (PTX), which is responsible for several of the clinical manifestations associated with whooping cough. The toxin is composed of five dissimilar subunits, named S1 through S5 and arranged in a hexameric structure with a 1S1:1S2:1S3:2S4:1S5 stoichiometry. Although S2 and S3 share 70% amino acid identity, these two subunits were previously thought not to be able to substitute for each other in toxin assembly/secretion and the biological activities of PTX. Here, we show that toxin analogues containing two S3 subunits and lacking S2 (PTXdeltaS2), or containing two S2 subunits and lacking S3 (PTXdeltaS3), can be produced, assembled and secreted by B. pertussis strains, in which the S2-encoding cistron or the S3-coding cistrons have been inactivated by internal in-frame deletions that avoid downstream effects. In fact, PTXdeltaS3 was produced in higher amounts in the bacterial culture supernatants than natural PTX, whereas PTXdeltaS2 was produced in lower amounts than PTX. The action of the toxin analogues on the clustering of Chinese Hamster Ovary cells was also affected differentially by the S2-S3 substitution. These toxin analogues constitute thus interesting probes for the study of cellular functions, in particular immune cell functions, for which natural PTX has already shown its usefulness.     

The importance of quadriceps and hamstring muscle loading on knee kinematics and in-situ forces in the ACL

This study investigated the effect of hamstring co-contraction with quadriceps on the kinematics of the human knee joint and the in-situ forces in the anterior cruciate ligament (ACL) during a simulated isometric extension motion of the knee. Cadaveric human knee specimens (n = 10) were tested using the robotic universal force moment sensor (UFS) system and measurements of knee kinematics and in-situ forces in the ACL were based on reference positions on the path of passive flexion/extension motion of the knee. With an isolated 200 N quadriceps load, the knee underwent anterior and lateral tibial translation as well as internal tibial rotation with respect to the femur. Both translation and rotation increased when the knee was flexed from full extension to 30 of flexion; with further flexion, these motion decreased. The addition of 80 N antagonistic hamstrings load significantly reduced both anterior and lateral tibial translation as well as internal tibial rotation at knee flexion angles tested except at full extension. At 30 of flexion, the anterior tibial translation, lateral tibial translation, and internal tibial rotation were significantly reduced by 18, 46, and 30%, respectively (p<0.05). The in-situ forces in the ACL under the quadriceps load were found to increase from 27.8+/-9.3 N at full extension to a maximum of 44.9+/-13.8 N at 15 of flexion and then decrease to 10 N beyond 60 of flexion. The in-situ force at 15 was significantly higher than that at other flexion angles (p<0.05). The addition of the hamstring load of 80 N significantly reduced the in-situ forces in the ACL at 15, 30 and 60 of flexion by 30, 43, and 44%, respectively (p<0.05). These data demonstrate that maximum knee motion may not necessarily correspond to the highest in-situ forces in the ACL. The data also suggest that hamstring co-contraction with quadriceps is effective in reducing excessive forces in the ACL particularly between 15 and 60 of knee flexion.     

Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis

Background

Severe leptospirosis features bleeding and multi-organ failure, leading to shock and death. Currently it is assumed that both exaggerated inflammation and immune suppression contribute to mortality in sepsis. Indeed, several proinflammatory cytokines are reported to be induced during leptospirosis. Toll-like receptors, which play an important role in the initiation of an innate immune response, are inhibited by negative regulators including the membrane-bound ST2 (mST2) receptor. Soluble ST2 (sST2) has been implicated to inhibit signaling through mST2. The aim of this study was to determine the extent of sST2 and (pro-) inflammatory cytokine release in patients with severe leptospirosis.

Methodology and Principal Findings

In an observational study, 68 consecutive cases of severe leptospirosis were included. Soluble ST2 and cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-10) were repeatedly measured. To determine whether blood cells are a source of sST2 during infection, we undertook an in vitro experiment: human whole blood and peripheral blood mononuclear cells (PBMC) were stimulated with viable pathogenic Leptospira. All patients showed elevated sST2, IL-6, IL-8, and IL-10 levels on admission. Admission sST2 levels correlated with IL-6, IL-8, and IL-10. Thirty-four patients (50%) showed clinical bleeding. Soluble ST2 levels were significantly associated with bleeding overall (OR 2.0; 95%CI: 1.2–3.6) and severe bleeding (OR 5.1; 95%CI: 1.1–23.8). This association was unique, since none of the cytokines showed this correlation. Moreover, sST2 was associated with mortality (OR 2.4; 95%CI: 1.0–5.8). When either whole blood or isolated PBMCs were stimulated with Leptospira in vitro, no sST2 production could be detected.

Conclusions

Patients with severe leptospirosis demonstrated elevated plasma sST2 levels. Soluble ST2 levels were associated with bleeding and mortality. In vitro experiments showed that (white) blood cells are probably not the source. In this regard, sST2 could be an indicative marker for tissue damage in patients suffering from severe leptospirosis.     

Proteomic analysis of human nail plate

Shotgun proteomic analysis of the human nail plate identified 144 proteins in samples from Causcasian volunteers. The 30 identified proteins solubilized by detergent and reducing agent, 90% of the total nail plate mass, were primarily keratins and keratin associated proteins. Keratins comprised a majority of the detergent-insoluble fraction as well, but numerous cytoplasmic, membrane, and junctional proteins and histones were also identified, indicating broad use by transglutaminases of available proteins as substrates for cross-linking. Two novel membrane proteins were identified, also found in the hair shaft, for which mRNAs were detected only at very low levels by real-time polymerase chain reaction in other tissues. Parallel analyses of nail samples from volunteers from Inner Mongolia, China gave essentially the same protein profiles. Comparison of the profiles of nail plate and hair shaft from the latter volunteers revealed extensive overlap of protein constituents. Analyses of samples from an arsenic-exposed population revealed few proteins whose levels were altered substantially but raised the possibility of detecting sensitive individuals in this way.     

Genetic Affinities within a Large Global Collection of Pathogenic Leptospira: Implications for Strain Identification and Molecular Epidemiology

Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland.     

Predation Danger Can Explain Changes in Timing of Migration: The Case of the Barnacle Goose

Understanding stopover decisions of long-distance migratory birds is crucial for conservation and management of these species along their migratory flyway. Recently, an increasing number of Barnacle geese breeding in the Russian Arctic have delayed their departure from their wintering site in the Netherlands by approximately one month and have reduced their staging duration at stopover sites in the Baltic accordingly. Consequently, this extended stay increases agricultural damage in the Netherlands. Using a dynamic state variable approach we explored three hypotheses about the underlying causes of these changes in migratory behavior, possibly related to changes in (i) onset of spring, (ii) potential intake rates and (iii) predation danger at wintering and stopover sites. Our simulations showed that the observed advance in onset of spring contradicts the observed delay of departure, whereas both increased predation danger and decreased intake rates in the Baltic can explain the delay. Decreased intake rates are expected as a result of increased competition for food in the growing Barnacle goose population. However, the effect of predation danger in the model was particularly strong, and we hypothesize that Barnacle geese avoid Baltic stopover sites as a response to the rapidly increasing number of avian predators in the area. Therefore, danger should be considered as an important factor influencing Barnacle goose migratory behavior, and receive more attention in empirical studies.     

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.