Megakaryocyte interaction with subendothelial extracellular matrix is associated with adhesion, platelet-like shape change, and thromboxane A2 production

We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.     

Validation of an informant-reported web-based data collection to assess dementia symptoms

Background

The Web offers unprecedented access to the experience of people with dementia and their care partners, but data gathered online need to be validated to be useful.

Objective

To test the construct validity of an informant Web-based data collection to assess dementia symptoms in relation to the 15-point Dependence Scale (DS).

Methods

In an online survey posted on the DementiaGuide website, care partners of people with dementia built individualized profiles from the 60-item SymptomGuide and completed a questionnaire, which included the DS and a staging tool.

Results

In the 250 profilees (155, 62% women, mean age 77 years), increasing dependence was associated with a greater chance of institutionalization. For example, no one at the lowest levels of dependence (DS score < 5, n = 33) was in long-term care, compared with half (13/25) of the profilees at the highest levels of dependence (DS score > 12) being in institutions (χ² ₄ = 27.9, P < .001). The Web-based DS was correlated with the number of symptoms: higher DS scores were associated with a higher stage of dementia (F > 50, P < .001).

Conclusion

In an online survey, the Web-based DS showed good construct validity, potentially demonstrating how the Web can be used to learn more about dementia progression and how it relates to symptoms experienced by patients across the course of dementing illnesses. Even so, caution is needed to assure the validity of data collected online.     

From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH₂]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.     

Acceleromyography to assess neuromuscular recovery: is calibration before measurement mandatory?

Background: Acceleromyography has been shown to be an appropriate method in the detection of residual paralysis. However, the clinical importance of an individual calibration of the device in the single patient to improve reliability in detecting residual paralysis remains unclear. Methods: Observational study in 100 patients undergoing general anaesthesia with endotracheal intubation and a neuromuscular block with atracurium. In all patients, an individually calibrated acceleromyograph was used to estimate a possible residual block at the end of surgery. Immediately after finishing the calibrated measurements at the end of surgery, a non‐calibrated measurement was performed. Agreements between the two measurements were tested using Cohen's κ and a Bland–Altman analysis. Results: Data from 96 patients were analysed. At the end of surgery, a discordance in the calibrated and the non‐calibrated train‐of‐four ratio was found in 88 patients. Bland–Altman analysis showed a mean (bias) of 0.01, with limits of agreement of 0.15/?0.15. κ was calculated with κ=0.84 for the absence or presence of a potential residual block if defined as a train‐of‐four ratio of 1.0 as a threshold. Conclusions: The results imply a good agreement in the detection of the presence or absence of a residual neuromuscular block between calibrated and non‐calibrated acceleromyography if a train‐of‐four ratio of 1.0 has been chosen as the threshold. However, the estimated train‐of‐four values are not transferable between calibrated and non‐calibrated measurements.     

Nucleotide Excision Repair Capacity and XPC and XPD Gene Polymorphism Modulate Colorectal Cancer Risk

Background

Colorectal cancer (CRC) is leading malignant tumors to occur mainly in industrialized countries, where it exhibits one of the highest mortality rates. Up to 80% of all CRCs characterize a chromosomal instability (CIN) phenotype. The main challenge faced by scientist is to reveal the mechanism of CIN development. An often proposed model is defects in DNA repair in terms of efficiency and genetic variations that modulate the response to stimuli from the environment. The objectives of this research were to determine whether nucleotide excision repair (NER) might affect CRC risk.

Monoclonal antibodies demonstrate accessible HA2 epitopes in minor subpopulation of native influenza virus haemagglutinin molecules

Summary Haemagglutinin (HA) was detected on the surface of influenza virus infected cell with monoclonal antibodies (MAbs) against both HA glycopolypeptides, HA1 and HA2, however, the reactivity of HA2-specific MAbs was remarkably lower as compared with HA1-specific MAbs. Quantitative analysis with two MAbs, IB8 (anti-HA1) and IIF4 (anti-HA2) respectively, revealed that HA2 epitope was reachable for antibody only in minor subpopulation of the HA representing approximately 7% of all molecules (spikes). The basis of the HA heterogeneity is discussed.     

Radioimmunoassay of influenza A virus haemagglutinin. II. Antigenic cross-reactions of influenza A (H3 subtype) viruses as determined by radioimmunoassay and haemagglutination inhibition tests

Individual rabbits differed greatly in their antibody response to the "strain-specific" and "cross-reactive" antigenic determinants on the haemagglutinin (HA) subunit of influenza virus recombinant MRC11 (H3N2) and influenza virus Dunedin (H3N2), after immunization with whole virus or bromelain-released haemagglutinin (B-HA). Consequently, diverse cross-reactions between htese viruses and A/Hong Kong/68 virus were found in the haemagglutination inhibition (HI) test as well as in homologous radioimmunoassay (125I-B-HA from MRC11:anti MRC11 serum, and 125I-B-HA from Dunedin: anti Dunedin serum) when sera from different animals were employed. Radioimmunoassay (RIA), over and above to the HI test, was able to differentiate clearly the respective HAs also with antisera reacting to the same HI titre with both corresponding influenza virus strains. Thus it appeared that antigenic differences could be identified with higher sensitivity by homologous RIA than by the HI test and that multiple antigenic determinants were reactive on the 125I-B-HA in the RIA procedure employed. MRC11 and A/HK/68 viruses were also compared by heterologous RIA (125I-B-HA from MRC11: anti A/HK/68 serum). It was found that preferentially antigenic determinants with a high degree of cross-reactivity could be studied in the heterologous system.     

Single Nucleotide Polymorphisms and Long‐Term Clinical Outcome in Renal Transplant Patients: A Validation Study

Genome‐wide association studies (GWAS) are designed to investigate single nucleotide polymorphisms (SNPs) and the association with a clinical phenotype. A previous GWAS performed in 300 renal transplant recipients identified two SNPs (rs3811321 and rs6565887) associated with serum creatinine and clinical outcome. We sought to validate these findings. Genotyping of the two SNPs was performed using Taqman assays in 1638 Caucasians participating in the Assessment of LEscol in Renal Transplant (ALERT) study. Primary endpoint was death‐censored graft loss, and secondary endpoint was all‐cause mortality. Applying Cox regression, no crude association to graft loss was found for rs3811321 on chromosome 14 (hazard ratio [HR] 0.87, 95% CI 0.59–1.29, p = 0.50) or rs6565887 on chromosome 18 (HR 0.88, CI 0.62–1.25, p = 0.48). Multivariable adjustments did not change results, nor did evaluation of the number of risk alleles formed by the two SNPs. No association with mortality was detected. In conclusion, an impact of two SNPs on chromosomes 14 and 18 on death‐censored graft survival or all‐cause mortality was not confirmed. Our results emphasize the importance of validating findings from high‐throughput genetics studies and call for large collaborative research initiatives in the field of transplantation outcomes.