Enhancing axial localization with wavefront control.

Enhancing the ability to resolve axial details is crucial in three-dimensional optical imaging. We provide experimental evidence showcasing the ultimate precision achievable in axial localization using vortex beams. For Laguerre-Gauss (LG) beams, this remarkable limit can be attained with just a single intensity scan. This proof-of-principle demonstrates that microscopy techniques based on LG vortex beams can potentially benefit from the introduced quantum-inspired superresolution protocol.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

APOE-mimetic peptides reduce behavioral deficits, plaques and tangles in Alzheimer's disease transgenics.

BACKGROUND: After age, the second largest risk factor for Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype, where APOE4 is associated with lower apoE protein levels, more severer brain pathology, enhanced inflammation and disease. Small peptides corresponding to the receptor-binding region of apoE mimic the anti-inflammatory activity of the apoE holoprotein. These apoE mimetics greatly improve behavioral outcomes and neuronal survival in head trauma models that display AD pathology and neuronal loss. OBJECTIVE: To determine whether apoE mimetics change behavior, inflammation and pathology in CVND-AD (SwDI-APP/NOS2(-/-)) transgenic mice. METHODS: Starting at 9 months, apoE peptides were subcutaneously administered 3 times per week for 3 months followed by behavioral, histochemical and biochemical testing. RESULTS: Treatment with apoE mimetics significantly improved behavior while decreasing the inflammatory cytokine IL-6, neurofibrillary tangle-like and amyloid plaque-like structures. Biochemical measures matched the visible pathological results. CONCLUSIONS: Treatment with apoE mimetics significantly improved behavior, reduced inflammation and reduced pathology in CVND-AD mice. These improvements are associated with apoE-mimetic-mediated increases in protein phosphatase 2A activity. Testing in additional AD models showed similar benefits, reinforcing this novel mechanism of action of apoE mimetics. These data suggest that the combination of anti-inflammatory and neuroprotective activities of apoE mimetics represents a new generation of potential therapeutics for AD.

[Potential role of presenilin 1 in regulation of synaptic function].

One of the earliest neuropathological symptoms of Alzheimer's disease is the loss of synapses, which preceed the formation of amyloidosis and neurodegeneration. Although most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic disfunction are not yet completely understood. In this paper we analysed of the intracellular and extracellular distribution of PS1 in the cultures of mouse cortical embryonic neurons. We found that PS1 is concentrated on the surface of the growth cone and at neurite contact sites. PS1 was also found in synapses where it is co-localized with synaptophysin. Independent evidense of involvement of PS1 in synaptic function we obtained by transfection of neurons with GFP-PS1 cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained processed N-cadherin. This result presents an additional proof of involvment PS1 in synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare synaptic density in neuronal cell cultures from PS1 knockout mice PS1 (-/-) and wild type mice PS1 (+/+). Our results clearly show that PS1 (-/-) displayed a low number of morphological synapses in comparing with wild type culture PS1 (+/+). In summary, our results indicate a role of PS1 in synaptic function.

Spotlight on measles 2010: a cluster of measles in a hospital setting in Slovenia, March 2010.

After ten years of being measles free, Slovenia experienced a cluster with secondary transmission in a hospital setting in March 2010. The index case, a resident of Ireland, was hospitalised on the day after his arrival to Slovenia and diagnosed with measles two days later. After his discharge, two cases of measles were notified, a hospital staff member and a visitor to the clinic, suggesting transmission in a hospital setting.

[Familial Alzheimer's disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts].

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer's disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial Hep2 cells. It is assumed that mutations in familial Alzheimer's disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.

[Studying pathogenesis of Alzheimer's disease in a Drosophila melanogaster model: human APP overexpression in the brain of transgenic flies leads to deficit of the synaptic protein synaptotagmin].

Alzheimer's disease (AD) is a progressive neurodegenerative disease whose main pathomorphological sign is synapse degeneration in the cortex and hippocampus. Abnormal synaptogenesis precedes amyloidosis and neurodegeneration and correlates with memory impairment during the early clinical phase. Mutations in the amyloid precursor protein (APP) gene cause familial AD and enhance the secretion of amyloid-beta-protein (Abeta). However, it remains unclear in what way APP and Abeta are involved in synaptic disorder in the absence of visible amyloid structures. In this study, the role of the human APP gene in synaptogenesis in transgenic lines of Drosophila melanogaster whose nerve cells express the human APP695 isoform, truncated APPs, and the presynaptic marker synaptotagmin driving the sequence of the green fluorescent protein. The expression of APP and its truncated forms caused a decrease in the synaptotagmin content of antennal lobes and mushroom lobes of the D. melanogaster brain, as well as neurodegeneration that progressed with age. The results suggest that that abnormal synaptogenesis and neurodegeneration occur in the Drosophila brain in the absence of Abeta. It is assumed that impaired cellular functions of APP and secretion of Abeta independently contribute to the pathogenesis of AD.

The burden of genital warts in Slovenia: results from a national probability sample survey.

The objective of this study was to estimate the lifetime age-specific cumulative incidence of self-reported genital warts diagnosis in Slovenia and to explore the association with demographic characteristics and self-reported sexual behaviour. Data were collected in the period from November 1999 to February 2001 from a national probability sample of the general population aged 18-49 years through a combination of face-to-face interviews at the respondents' homes and anonymous self-completed questionnaires. In total, 849 men and 903 women were interviewed (response: 63.3% men, 70.9% women). Among sexually experienced respondents with available information (752 men and 842 women), previous diagnosis of genital warts was reported by 0.3% of men (95% confidence interval (CI): 0.0%-1.3%) and 0.4% of women (95% CI: 0.1%-1.1%), and in the age group of 40-49 year-olds by 0.5% of men (95% CI:0.0-3.2) and 0.7% of women (95% CI: 0.2%-2.9%). In comparison to women with fewer than 10 lifetime male partners, those who reported to have had at least 10 male partners were more likely to have a previous diagnosis of genital warts (adjusted odds ratio: 7.2 (95% CI: 1.1%-47.8%). The lifetime cumulative incidence of self-reported genital warts diagnosis among Slovenians was relatively low in comparison to other published estimates from probability sample surveys in the general population in European countries. Our findings will inform the Slovenian vaccination policy against human papillomaviruses (HPV) and contribute to a better understanding of the differences between European countries regarding the burden of genital warts.

[Degeneration of growth cones in a culture of embryonic neurons of mouse with presenilin 1 gene knockout].

A comparative study of growth cone morphology in cultured embryonic neurons derived from wild type PS 1(+/+) and knockout PS 1(-/-) mice has been performed. Growth cones from wild type PS 1(+/+) mice were well spread and usually formed radially continuous and regular lamellar extensions with numerous filopodia. In contrast, most growth cones from knockout PS 1(-/-) mice exhibited small lamellar extensions, short filopodia, and pure adhesion. A significant amount of growth cones from knockout PS 1(-/-) mice collapsed after 3-4 days in culture. It was suggested that PS 1 plays an important role in growth cone structure by stabilizing the integrity of the cytoskeleton. The growth cone collapse may be the main reason for abnormal neuronal migration and impaired synaptic function in PS 1(-/-) mice.

An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction.

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

Apolipoprotein E-derived peptides reduce CNS inflammation: implications for therapy of neurological disease.

The apolipoprotein E4 isoform (apoE4) was initially identified as a susceptibility gene for the development of Alzheimer's disease, and has also recently been associated with poor outcome after acute traumatic and ischemic brain injury. One mechanism by which apoE may influence outcome in acute and chronic neurological disease is by downregulating glial activation and the neuroinflammatory response. Because it does not readily cross the blood-brain barrier (BBB), the apoE holoprotein has limited therapeutic potential. However, smaller peptides derived from the receptor binding region of apoE have been developed that mimic the functional anti-inflammatory and neuroprotective effects of the intact apoE protein. These apoE-derived therapeutic peptides cross the BBB and have been demonstrated to improve functional and histological outcomes in murine models of brain injury. Thus, the development of apoE-derived peptides represent a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

[Expression of presenilin 1 on the cell surface in motile polarized cells].

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.

Disrupted spermine homeostasis: a novel mechanism in polyglutamine-mediated aggregation and cell death.

Our data suggest a novel mechanism whereby pathological-length polyglutamine (polyQ) proteins promote the spermine synthetic pathway, increasing polyQ-aggregation and cell death. As detected in a cell-free turbidity assay, spermine promotes aggregation of thio-polyQ62 in a dose-dependent manner. Using a stable neuronal cell line expressing pathological-length [polyQ57-yellow fluorescent protein (YFP) (Q57)] or non-pathological-length [polyQ19-YFP (Q19)] polyglutamine protein, we show that multiple steps in the production of polyamines are affected in Q57 cells, suggesting dysfunctional spermine homeostasis. As the building block for spermine synthesis, arginine transport is significantly increased in neuronal cell lines stably expressing Q57. Q57 lines displayed upregulated basal and inducible arginase I activities that were not seen in polyQ19-YFP lines. Normal induction of spermidine/spermine N-acetyltransferase in Q19 lines regulating back-conversion of spermine, thereby reducing spermine levels, however, was not observed in Q57 lines. Pharmacological activation of ornithine decarboxylase (ODC), a key enzyme of the polyamine synthetic pathway, increased cellular aggregates and increased cell death in Q57 cells not observed in Q19 cells. Inhibition of ODC by difluoromethylornithine prevented basal and induced cell death in Q57 cells, demonstrating a central role for polyamines in this process.

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport.

Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.

Steroid and high-temperature induction of the small heat-shock protein genes in Drosophila.

Transcription of the four small heat-shock protein genes of Drosophila melanogaster can be induced in cultured cells by high-temperature shock, or by physiological doses of the moulting hormone, ecdysterone. We have characterized and compared the two induction events, focusing on hsp22 and hsp23, in terms of rates of heat-shock protein synthesis, transcription rate, messenger RNA abundance and mRNA half-life. The results indicate that relative to hsp22, the rate of hsp23 synthesis is significantly greater during recovery from heat shock and during ecdysterone induction. This difference is not due to differences in transcription rate, but rather reflects differences in mRNA stability and translational efficiency. One intriguing finding is that hsp message stability is temperature-dependent; hsp transcripts are two to three times more stable at 35 degrees C than at 25 degrees C. The possible mechanism and significance of this phenomenon are discussed.

Transcript length heterogeneity at the small heat shock protein genes of Drosophila.

Expression of the small heat shock protein (hsp) genes can be induced in cultured Drosophila cells by high temperature shock and by exposure to physiological doses of the insect molting hormone ecdysterone. Northern blot analysis was performed in order to compare the size of small hsp transcripts synthesized in response to these two stimuli. Transcripts from several other genes were also examined. Two types of length heterogeneity were observed for the small hsp gene transcripts. One involved the synthesis of what are designated as long form transcripts during heat shock; small hsp messenger RNAs extended at the 3' end by some 1.5 X 10(3) base-pairs. The second type of size heterogeneity observed is based on differences in the length of the poly(A) tail. The results of S1 nuclease protection analysis provided evidence that different initiation sites are not used for hsp 22 mRNA transcription in response to the two stimuli.

Increasing amyloid peptide precursor production and its impact on Alzheimer's disease.

Amyloid peptide precursor (APP) gene expression increases in the brains of patients with Alzheimer's disease (AD). Regulation of expression at the transcriptional and posttranscriptional levels contributes significantly to these increases. Polypeptide hormones stimulate the promoter of the APP gene resulting in increased levels of heterogeneous nuclear RNAs (hnRNA). Through splicing, these hnRNAs are processed into 3 or more mature APP RNAs. The amounts of each of these similar but nonidentical APP RNAs may yield ratios of APP proteins which are required for neuronal survival, pathogenesis of neurons and/or produce phenocopies of learning and memory mutants of Drosophila.

Focusing on IL-1-promotion of beta-amyloid precursor protein synthesis as an early event in Alzheimer's disease.

A rationale for increased synthesis of beta-amyloid peptide percursor (APP) protein in Alzheimer's disease (AD) is developed in which Interleukin-1 (IL-1) plays a key role. This cytokine is elevated in AD, its receptors are on APP mRNA positive cells and it promotes APP gene expression. Potential involvement of the protease inhibitor (PI) activity of certain APP proteins in the activation process for IL-1 and Nerve Growth Factor (NGF) are proposed. The possibility of feedback loops among IL-1, APP and NGF and the implications for neuronal survival and function are discussed.