Covalent bound sensing layers on surface acoustic wave (SAW) biosensors

This paper reports on the development of immunosensors based on commercially available surface acoustic wave (SAW) devices working at 380 MHz. Approaches for coating the sensor surface with a sensing layer of receptive biomolecules are presented and discussed. It was found that the sensitivity strongly relates to the immobilization method. Additionally, the sensitivity can be influenced by the density of accessible biomolecules on the active sensing area. Usually, by most of the standard immobilization procedures, two-dimensional layers of receptive biomolecules are obtained. We present a three-dimensional layer, which provides a higher absolute amount of recognition molecules. A dextran layer is photoimmobilized to the sensor surface and the recognition molecules are covalently embedded into the dextran matrix. The feasibility of specific immunosensing is investigated using SAW sensors connected to a fluid handling system.     

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

Air quality improvement and cognitive decline in community-dwelling older women in the United States: A longitudinal cohort study

BackgroundLate-life exposure to ambient air pollution is a modifiable risk factor for dementia, but epidemiological studies have shown inconsistent evidence for cognitive decline. Air quality (AQ) improvement has been associated with improved cardiopulmonary health and decreased mortality, but to the best of our knowledge, no studies have examined the association with cognitive function. We examined whether AQ improvement was associated with slower rate of cognitive decline in older women aged 74 to 92 years.Methods and findingsWe studied a cohort of 2,232 women residing in the 48 contiguous US states that were recruited from more than 40 study sites located in 24 states and Washington, DC from the Women’s Health Initiative (WHI) Memory Study (WHIMS)-Epidemiology of Cognitive Health Outcomes (WHIMS-ECHO) study. They were predominantly non-Hispanic White women and were dementia free at baseline in 2008 to 2012. Measures of annual (2008 to 2018) cognitive function included the modified Telephone Interview for Cognitive Status (TICSm) and the telephone-based California Verbal Learning Test (CVLT). We used regionalized universal kriging models to estimate annual concentrations (1996 to 2012) of fine particulate matter (PM_2.5) and nitrogen dioxide (NO₂) at residential locations. Estimates were aggregated to the 3-year average immediately preceding (recent exposure) and 10 years prior to (remote exposure) WHIMS-ECHO enrollment. Individual-level improved AQ was calculated as the reduction from remote to recent exposures. Linear mixed effect models were used to examine the associations between improved AQ and the rates of cognitive declines in TICSm and CVLT trajectories, adjusting for sociodemographic (age; geographic region; race/ethnicity; education; income; and employment), lifestyle (physical activity; smoking; and alcohol), and clinical characteristics (prior hormone use; hormone therapy assignment; depression; cardiovascular disease (CVD); hypercholesterolemia; hypertension; diabetes; and body mass index [BMI]). For both PM_2.5 and NO₂, AQ improved significantly over the 10 years before WHIMS-ECHO enrollment. During a median of 6.2 (interquartile range [IQR] = 5.0) years of follow-up, declines in both general cognitive status (β = −0.42/year, 95% CI: −0.44, −0.40) and episodic memory (β = −0.59/year, 95% CI: −0.64, −0.54) were observed. Greater AQ improvement was associated with slower decline in TICSm (β_{PM2.5improvement} = 0.026 per year for improved PM_2.5 by each IQR = 1.79 μg/m³ reduction, 95% CI: 0.001, 0.05; β_{NO2improvement} = 0.034 per year for improved NO₂ by each IQR = 3.92 parts per billion [ppb] reduction, 95% CI: 0.01, 0.06) and CVLT (β_{PM2.5 improvement} = 0.070 per year for improved PM_2.5 by each IQR = 1.79 μg/m³ reduction, 95% CI: 0.02, 0.12; β_{NO2improvement} = 0.060 per year for improved NO₂ by each IQR = 3.97 ppb reduction, 95% CI: 0.005, 0.12) after adjusting for covariates. The respective associations with TICSm and CVLT were equivalent to the slower decline rate found with 0.9 to 1.2 and1.4 to 1.6 years of younger age and did not significantly differ by age, region, education, Apolipoprotein E (ApoE) e4 genotypes, or cardiovascular risk factors. The main limitations of this study include measurement error in exposure estimates, potential unmeasured confounding, and limited generalizability.ConclusionsIn this study, we found that greater improvement in long-term AQ in late life was associated with slower cognitive declines in older women. This novel observation strengthens the epidemiologic evidence of an association between air pollution and cognitive aging.

Diana Younan and colleagues investigate whether air quality improvement is associated with rate of cognitive decline in community-dwelling older women in the United States.     

Productivity and nutrient uptake in a holm oak (Quercus ilex L.) stand and during regeneration after clearcut

The effect of clearcutting on biomass production and nutrient consumption was examined during the two years in a clearcut and a mature Quercus ilex L. coppice. The comparison was limited to the leaf compartment and to the annual shoots, which were the only comparable compartments in the two situations. At the same time, the dynamics of the nutrient pool (N, P, K, Ca, Mg), monthly uptake and recycling from the foliage, were studied.The second year after cutting, the leaf production was roughly the same at the two sites. However, the photosynthates appear to be used differently: in the young stand the perennial wood production was greater than in the mature stand, where the maintenance costs are higher. This is corroborated by the patterns of nutrient use.     

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.     

CAP, a new human suspension cell line for influenza virus production

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log₁₀ HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log₁₀ HA units/100 μL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production.     

Variations in cell surfaces of estrogen treated breast cancer cells detected by a combined instrument for far-field and near-field microscopy.

The response of single breast cancer cells (cell line T-47D) to 17beta-estradiol (E(2)) under different concentrations was studied by using an instrument that allows to combine far-field light microscopy with high resolution scanning near-field (AFM/SNOM) microscopy on the same cell. Different concentrations of E(2) induce clearly different effects as well on cellular shape (in classical bright-field imaging) as on surface topography (atomic force imaging) and absorbance (near-field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle-like shape at un-physiologically high concentrations. The surface topography of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E(2) concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un-physiological high E(2) concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near-field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co-localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers.