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Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Pulp and paper mill effluent can cause changes in the morphology and energy metabolism in the zebrafish (Danio rerio) testis. Betulin, a naturally occurring triterpene is commonly present in this type of effluent and is suspected of being involved in these effects. The aim of this study was to compare the effects pulp and paper mill effluent and betulin on various aspects of testicular physiology in the zebrafish. This included the in vitro effects of effluent and betulin on testicular lactate content and lactate dehydrogenase (LDH) activity. In addition, the effects of betulin on glucose uptake, glycogen, alanine aminotransferase (ALT), reactive oxygen and nitrogen species formation and oxidative damage in the testes were determined. Furthermore, we compared the effects and mechanism of action of betulin and effluent on calcium homeostasis in testes. In vitro exposure to both effluent and betulin decreased lactate and calcium influx, possibly due to the activation of the sodium‑calcium exchanger (NCX) pump. Additionally, betulin-treated testes had higher reactive oxygen species (ROS) and reduced glutathione (GSH) content, as well as increased glutathione transferase (GST) activity and a tendency towards decreased catalase (CAT) activity. Thus, this study shows that alterations in testis physiology caused by the pulp and paper mill effluent in the testis may be due in part to the actions of betulin.

Deacidification is an established conservation treatment for the prolongation of the acidic paper stability. Several different deacidification systems are in use today, such as the dispersions of alkaline nanoparticles in organic solvents. The aim of the study was to compare the effects of different calcium nanoparticle dispersions in alcohols on lignocellulosic paper. Commercially available nano calcium hydroxide dispersions for paper deacidification, and laboratory prepared nano calcium carbonate dispersions in ethanol and 2-propanol were investigated and compared as to their effectiveness in increasing the stability of paper. The FE-SEM analyses were used to determine the size of Ca(OH)2 and CaCO3 particles in the dispersions. The SEM–EDS analyses were performed both on the paper surface and its cross-section in order to ascertain the distribution of calcium ions following the deacidification treatment. An evaluation of the changes of color, molecular weight, pH and alkaline reserve on different lignocellulosic papers was performed. In comparison to untreated samples, our results indicate all the investigated deacidification treatments decrease the degradation rate constant of cellulose, as determined by accelerated degradation. However, the treatments involving nano calcium hydroxides in both alcohols noticeably affect the color of the treated lignocellulosic papers. According to the obtained results, nano calcium carbonate is therefore the more suitable deacidification agent for the lignin containing papers.

Modulation of brain state, e.g., by anesthesia, alters the correlation structure of spontaneous activity, especially in the delta band. This effect has largely been attributed to the ∼1 Hz slow oscillation that is characteristic of anesthesia and nonrapid eye movement (NREM) sleep. However, the effect of the slow oscillation on correlation structures and the spectral content of spontaneous activity across brain states (including NREM) has not been comprehensively examined. Further, discrepancies between activity dynamics observed with hemoglobin versus calcium (GCaMP6) imaging have not been reconciled. Lastly, whether the slow oscillation replaces functional connectivity (FC) patterns typical of the alert state, or superimposes on them, remains unclear. Here, we use wide-field calcium imaging to study spontaneous cortical activity in awake, anesthetized, and naturally sleeping mice. We find modest brain state-dependent changes in infraslow correlations but larger changes in GCaMP6 delta correlations. Principal component analysis of GCaMP6 sleep/anesthesia data in the delta band revealed that the slow oscillation is largely confined to the first three components. Removal of these components revealed a correlation structure strikingly similar to that observed during wake. These results indicate that, during NREM sleep/anesthesia, the slow oscillation superimposes onto a canonical FC architecture.

Lime mud is a kind of solid waste in the papermaking industry, which has been a source of serious environmental pollution. Ceramsites containing anorthite and gehlenite were prepared from lime mud and fly ash through the solid state reaction method at 1050°C. The objective of this study was to explore the efficiency of Ca 2 + and OH − release and assess the phosphorus and copper ion removal performance of the ceramsites via batch experiments, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results show that Ca 2 + and OH − were released from the ceramsites due to the dissolution of anorthite, gehlenite and available lime. It is also concluded that gehlenite had stronger capacity for Ca 2 + and OH − release compared with anorthite. The Ca 2 + release could be fit well by the Avrami kinetic model. Increases of porosity, dosage and temperature were associated with increases in the concentrations of Ca 2 + and OH − released. Under different conditions, the ceramsites could maintain aqueous solutions in alkaline conditions (pH = 9.3–10.9) and the release of Ca 2 + was not affected. The removal rates of phosphorus and copper ions were as high as 96.88% and 96.81%, respectively. The final pH values of both phosphorus and copper ions solutions changed slightly. The reuse of lime mud in the form of ceramsites is an effective strategy.

In this communication a prototype of paper-based analytical device designed for simultaneous determination of orthophosphate and calcium ions, which levels are significant for hyperphosphatemia diagnostics, is presented. The laboratory-on-paper structure for two analytes detection was wax-printed on the surface of filter paper. These two-analyte disposable paper strips are combined with two paired LED-based fluorescence detectors and simple voltmeter used as recorder of analytical signal, what makes the developed device miniature, extremely low-cost, portable and user-friendly. Thus the developed device allows usage outside of specialized clinical laboratory. Moreover, each paper strip is disposable and its utilization is easy and fast and, additionally, burnt strip tests ensure waste non-infectious. The presented LED&Paper-based analytical device provides low detection limits: 1.4 μmol L−1 and 7.4 μmol L−1 for orthophosphate and calcium ions, respectively. The practical utility of the developed device for calcemia/phosphatemia diagnostics is demonstrated using control serum standards and real human serum.

Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology. Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light. Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature. Results: A specific combination of wavelengths at low irradiances (250 to 400 mW/cm2 for 1064 nm and 55 to 65 mW/cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes. Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

Background: Development of cost-effective platforms for identification of biomarkers is of paramount importance in low-income settings. The present work focuses on the development of a microfluidic paper-based analytical device (μPADs) for the diagnosis of osteoporosis by measuring three important bone biomarkers – calcium, alkaline phosphatase and vitamin D. Methods: Antibody-based detection is realized in μPAD reservoirs based on principle of colorimetric sensing, where μPAD images are captured using a smartphone. The results are compared with the calibration curves to give proper quantification of analytes. Conclusion: This device can be potentially developed into a point-of-care diagnostic device for low-income settings. To the best of our understanding, a μPAD platform for simultaneous diagnosis of multiple osteoporosis markers is not reported yet.

Clinical and subclinical hypocalcaemia are common in dairy cows, yet evidence in the literature assessing their impact on fertility is inconsistent. The aim of this prospective cohort study was to examine associations between blood ionised calcium concentration at calving and fertility outcomes in dairy cattle. Blood samples were taken from 137 Holstein cows from four commercial dairy herds within 24 hours of calving and analysed for blood ionised calcium using an Epocal Point of Care Analyser (Epocal, Ottawa, Canada). Data collected from routine veterinary fertility examinations and herd records were used to investigate the association of ionised calcium with the outcomes: time to first service, time to conception and endometritis. There were significant negative associations between blood ionised calcium concentration and time to first service (HR 1.33, P=0.001) and blood ionised calcium concentration and time to conception (HR 1.16, P=0.04). There was no significant association between blood ionised calcium concentration and endometritis. The results of this study imply that management policies that minimise the reduction in blood ionised calcium concentration in the periparturient dairy cow are likely to improve reproductive outcomes and should be considered as part of the multifactorial approach to optimising dairy cow fertility.

Lignosulfonates are important by-products of the paper industry and may be transformed into different commodities. We studied the molecular properties of ammonium (LS-AM) and calcium Lignosulfonates (LS-C) and evaluated their bioactivity towards the early development of maize plantlets. The FT-IR, 13C NMR and 1H-13C-HSQC-NMR spectra showed that the two lignosulfonates varied in hydroxyl, sulfonate and phenolic content, while DOSY-NMR spectroscopy suggested a similar diffusivity. High Performance Size Exclusion Chromatography (HPSEC) was used to simulate the effects of root-exuded acids and describe the conformational dynamics of both LS substrates in acidic aqueous solutions. This technique showed that LS-C was stabilized by the divalent Ca2+ counterion, thus showing a greater conformational stability than LS-AM, whose components could not be as efficiently aggregated by the monovalent NH4+ counter-ion. The plant bioassays revealed that LS-AM enhanced the elongation of the root system, whereas LS-C significantly increased both total and shoot plant weights. We concluded that the lignosulfonate bioactivity on plant growth depended on the applied concentrations, their molecular properties and conformational stability.

We employ transcranial wide-field single-photon imaging to compare genetically encoded calcium sensors under transgenic or viral vector expression strategies. Awake, head-fixed animals and brief visual flash stimuli are used to assess function. The use of awake transcranial imaging may reduce confounds attributed to cranial window implantation or anesthesia states. We report differences in wide-field epifluorescence brightness and peak ΔF/F0 response to visual stimulation between expression strategies. Other metrics for indicator performance include fluctuation analysis (standard deviation) and regional correlation maps made from spontaneous activity. We suggest that multiple measures, such as stimulus-evoked signal-to-noise ratio, brightness, and averaged visual ΔF/F0 response, may be necessary to characterize indicator sensitivity and methods of expression. Furthermore, we show that strategies using blood brain barrier-permeable viruses, such as PHP.eB, yield comparable expression and function as those derived from transgenic mice. We suggest that testing of new genetically engineered activity sensors could employ a single-photon, wide-field imaging pipeline involving visual stimulation in awake mice that have been intravenously injected with PHP.eB.

Recycled paper mills produce large quantities of fibrous rejects and fines which are usually sent to landfills as solid waste. These cellulosic materials can be enzymatically hydrolyzed into sugars for the production of biofuels and biomaterials. Paper mill wastes also contain large amounts of calcium carbonate which inhibits cellulase activity. The calcium carbonate (30%, w/w) decreased 40–60% of sugar yield of unbleached softwood kraft pulp. The prime mechanisms for this are by pH variation, competitive and non-productive binding, and aggregation effect. Addition of acetic acid (pH adjustment) increased the sugar production from 19 to 22 g/L of paper mill waste fibers. Strong affinity of enzyme—calcium carbonate decreased free enzyme in solution and hindered sugar production. Electrostatic and hydrogen bond interactions are mainly possible mechanism of enzyme—calcium carbonate adsorption. The application of the nonionic surfactant Tween 80 alleviated the non-productive binding of enzyme with the higher affinity on calcium carbonate. Dissociated calcium ion also inhibited the hydrolysis by aggregation of enzyme.

In this work, we applied scanning electron microscopy (SEM), microanalysis and Raman spectroscopy to study the fungi inhabiting a richly illuminated parchment document and the damage induced by their activity. To that aim, we collected samples of fungal mycelium from the deteriorated areas on a removable adhesive tape specifically intended for lifting fungi without damaging the support. SEM analysis of the adhesive tape samples showed the co-occurrence of several species of fungi. One strain closely resembling Acremonium species was observed only in the tape micrographs but no agar cultures were obtained. Its fungal structures showed the production of abundant oxalates with an outstanding leaching of the calcium-based materials of parchment (typically manufactured with gypsum and lime). Needle-like crystals of calcium oxalate produced by the fungus forming a uniform and quite regular grid around conidial slimy heads were documented. As a result, the areas affected by moulds were weakened, stained and characterised by a powdery patina rich in calcium. Confocal μ-Raman confirmed the presence of oxalates while EDS showed the presence of calcium in crystals. We conclude that the defacement of the parchment was due to both collagenolytic activity, and to the biotransformation of calcium-based minerals by fungi.

A sample set of model graded papers containing known organic and inorganic components was used to culture an Aspergillus terreus strain able to produce acidic metabolites and cellulolytic enzymes. This study set out to document the biogenic formation of calcium oxalate crystals, as a consequence of fungal activity and growth, on a range of paper samples. Scanning electron microscopy and FTIR spectroscopy techniques were employed to achieve this end. Bioleaching processes induced by fungi on carbonate used in the manufacture of paper, together with the capacity of fungi to produce - very rapidly - significant biogenesis of monohydrate and dihydrate calcium oxalate crystals on the surface of papers, were investigated. SEM images of calcium containing paper samples disclosed a very clear difference in the pattern of inorganic material distribution when areas spoiled by fungi and those not affected by fungal growth were compared. In addition, the FTIR spectrum of CaCO(3) sized paper clearly demonstrated, following fungal growth, the FTIR absorbance of fungus and calcium oxalate, together with the disappearance of carbonate bands. (C) 2010 Elsevier Ltd. All rights reserved.

We used toxicity identification evaluation (TIE) method to confirm the combinational effects of identified toxic metals in a paper recycling mill effluent in inducing the decreased lifespan in nematode Caenorhabditis elegans. Exposure to Ca + Al caused more severely decreased lifespan than that exposed to Ca, or Al; and exposure to Ca + Fe induced more severely decreased lifespan than that exposed to Ca, or Fe. Exposure to Ca+Al+Fe caused more severely decreased lifespan than that exposed to Ca, or Ca+Fe. Moreover, the baseline toxicity on lifespan was doubled by doubling the concentration of combined metals (Ca+Al+Fe) in spiking test in original effluent (oe), and lifespan defects in oe+Ca+Al+Fe exposed nematodes were more severe than that in Ca+Al+Fe exposed nematode. Therefore, Ca+Al+Fe exposure may largely explain the formation of decreased lifespan induced by the examined industrial effluent. Furthermore, the observed reduction of lifespan induced by the combination of high level of Ca with other metals may be at least partially independent of the insulin-like pathway.

The objective of this paper is to identify deflocculation under short-term disturbances of dissolved oxygen (DO) in a continuous system, and to explore the strategies for minimizing the carry-over of biosolids. Sludge deflocculation was examined in four parallel sequencing batch reactors (SBRs) fed with primary treated effluents from a bleached kraft pulp and paper mill. Results show that the DO transients caused a reduction of soluble COD (SCOD) removal efficiency by 50–70%, increases in suspended solid (SS) concentration and turbidity by more than 100%. Under the DO disturbances, the ratio of bulk Ca2 + /K+ decreased by 10% in the treated effluents. All these changes were reversible within 24 hrs after the DO concentrations were restored up to 4 mg/L, indicating a physicochemical response of microorganisms to the DO transients. The sludge deflocculation is correlated with the decreasing ratio of Ca2 + to K+ in the extracellular environment. Addition of calcium chloride, tetraethylammonium chloride or glibenclamide promotes the formation of bioflocs bigger than 12.5 μm, but the deflocculation of biosolids under the DO transients wasn't completely prevented.

The main objective of this work is to study the technical viability of using the effluent generated in paper machines (white water) in the wash presses of the bleaching stage, reducing fresh water consumption. As a case study, the industrial process of Ripasa S.A. Celulose e Papel was evaluated. White water rate is about 700 m3/h and it is not possible to reuse all this volume in the bleaching stage without causing operational problems (fouling in tubes and clogging in the screens). A mass balance of the bleaching unit was developed in an electronic spreadsheet in order to evaluate the possibility of reducing fresh water consumption, using only a fraction of the available white water in the wash presses. To achieve this objective some physical-chemistry properties of the white water stream and of other streams of the process were determined. The maximum concentration of some non-process elements (Si, Ca, Mn and Fe), which could accumulate in the process, were determined in order to establish some parameters to allow process integration of the streams involved, considering operational constraints. The results obtained have shown that it is possible to reduce approximately by 13% the consumption of fresh water and this methodology has been satisfactory.

The objective of this study was to investigate the synthesis of zeolites from paper sludge ash, and to evaluate their applications for water purification to simultaneously remove NH4+ and PO43- in aqueous solution. Paper sludge ash had a low abundance of Si and a significant Ca content, due to the presence of calcite that was used as paper filler. Na2SiO3 was added to the NaOH solution to increase Si content in order to synthesize zeolites with high cation exchange capacity. The original ash without addition of Si yielded hydroxysodalite with a cation exchange capacity of ca. 50 cmol/kg. Addition of Si to the solution yielded Na-P1 (zeolite-P) with a higher cation exchange capacity (ca. 120 cmol/kg). The observed concentrations of Si and Al in the solution during the reaction explain crystallizations of these two phases. The product with Na-P1 could simultaneously remove NH4+ and PO43- from the solution between pH 4 and 9.

Paper sludge ash was partially converted into zeolites by reaction with 3M NaOH solution at 90 degrees C for 24 h. The paper sludge ash had a low abundance of Si and significant Ca content, due to the presence of calcite that was used as a paper filler. Diatomite was added to the NaOH solution to increase its Si content in order to synthesize zeolites with high cation exchange capacity. Diatomite residue was filtered from solution before addition of ash. The original ash without addition of diatomite yielded hydroxysodalite with a cation exchange capacity ca. 50 cmol/kg. Addition of Si to the solution yielded Na-P1 (zeolite-P) with a higher cation exchange capacity (ca. 130 cmol/kg). The observed concentrations of Si and Al in the solution during the reaction explain the crystallization of these two phases. The reaction products were tested for their capacity for PO(4)(3-) removal from solution as a function of Ca(2+) content, suggesting the formation of an insoluble Ca-phosphate salt. The product with Na-P1 exhibits the ability to remove NH(4)(+) as well as PO(4)(3-) from solution in concentrations sufficient for application in water purification. Both NH(4)(+) and PO(4)(3-) removal showed little variation with pH between 5 and 9. Alternative processing methods of zeolite synthesis, including the addition of ash to an unfiltered Si-NaOH solution and addition of a dry ash/diatomite mixture to NaOH solution, were tested. The third process yielded materials with lower cation exchange capacity due to formation of hydroxysodalite. The second process results in a product with relatively high cation exchange capacity, and reduces the number of processing steps necessary for zeolite synthesis.

Objective: To assess the correlation of serum parathyroid hormone (PTH) and vitamin D (25-OHD) levels based on different assays for measuring 25-OHD in healthy Saudi Arabians living along the east coast. Patients and Methods: A cross-sectional study was conducted in 200 patients (150 women and 50 men aged between 18-69 years) between January 2011 and December 2012, attending outpatient clinic at King Fahd Hospital of the University, Al Khobar. The first 200 patients seen without vitamin D supplementation at clinic were enrolled in the study. Serum calcium, phosphorous, alkaline phosphatase, parathormone, and 25-OHD tests were performed. 25-OHD was assessed using:chemiluminescence immunoassay (CLIA)radioimmunoassay (RIA) using Wallac 1470 Gamma CounterHPLC –LC.MS (high performance liquid chromatography-liquid chromatography with mass spectrometry. The data was collected, entered into a database and analysed using SPSS, Inc., version 14. Results: The mean age was 45.8±15.8 (18-74) years, and calcium level was 2.27±0.15 mmol/l. (range 2.125 to 2.62 mmol/l). Alkaline phosphatase was 88.91±35.94 (34-302) IU, parathormone 6.7±3.06 (1.35-21.2) (1.3-6.8 pmol/l). Of the participants, 188 were either vitamin D insufficient or deficient as measured by CLIA 11.85±6.14 (2-29.6), and 91 (48.4%) of them had secondary hyperparathyroidism 9.48±4.55 pc/l. Those with normal CLIA-measured 25-OHD levels had normal PTH levels. Of those with insufficiency, 4/21 (19%) had raised PTH levels; and of those with deficiency, 81/166 (48.79%) had raised levels, whereas with HPLC-LC.MS, 156 were shown to be insufficient and 97 deficient (with PTH level of 7.41±4.2). Thirteen of 41 patients (31.7%) with insufficiency were shown, by HPLC-LC.MS, to have raised PTH. All patients with vitamin D deficiency as diagnosed by HPLC-LC.MS had secondary hyperparathyroidism. Conclusions: The above results suggest that the method of measurement strongly influences vitamin D levels and that previous reports suggesting no association between vitamin D deficiency and secondary hyperparathyroidism should be viewed with caution.

Quantitative determination of Ca, Mg, and Zn in fingernails was performed with laser-induced breakdown spectroscopy. Two different methods of producing solid standards for calibration were explored – preparation of keratin pellets and deposition of aqueous solutions to filter papers. Measurements of the temperature and electron density of the plasma produced on keratin pellets, filter paper, and nails were performed, and it was determined that the standards prepared on filter paper gave plasma temperatures and electron densities closer to those observed on the nails. The ablation rate of the filter paper was also more similar to that of the nails. Using calibration curves produced using these filter paper standards, Ca, Mg, and Zn were determined in fingernails of 11 subjects. For comparison, the same samples were digested and atomic absorption was used to determine the same three elements. The differences in results are discussed in light of sample homogeneity and instrumental precision; the best agreement was obtained for determination of Zn. The work suggests that the filter paper method of standard preparation may be appropriate for LIBS analysis of other samples that give relatively low temperature, low electron density plasmas (i.e., polymers).

This paper describes the results of laboratory experiments using a thermophilic aerobic MBR (TMBR) at 50 °C. An innovative use of submerged flat-sheet MBR modules to treat circuit wastewater from the paper industry was studied. Two experiments were conducted with a flux of 8–13 L/m 2 /h without chemical membrane cleaning. COD and BOD 5 elimination rates were 83% and 99%, respectively. Calcium was reduced from 110 to 180 mg/L in the inflow to 35–60 mg/L in the permeate. However, only negligible membrane scaling occurred. The observed sludge yield was very low and amounted to 0.07–0.29 g MLSS/g COD eliminated . Consequently, the nutrient supply of ammonia and phosphate can be lower compared to a mesophilic process. Molecular-biological FISH analysis revealed a likewise high diversity of microorganisms in the TMBR compared to the mesophilic sludge used for start-up. Furthermore, ammonia-oxidising bacteria were detected at thermophilic operation.

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (–P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(–P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Simultaneous saccharification and co-fermentation (SSCF) of waste paper sludge to ethanol was investigated using two recombinant xylose-fermenting microbes: Zymomonas mobilis 8b and Saccharomyces cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper sludge at 37 degrees C, while similar titers and yields were achieved by Z. mobilis 8b at 30 degrees C. Both S. cerevisiae RWB222 and Z. mobilis 8b exhibited decreasing cell viability at 37 degrees C when producing over 40 g/L ethanol. A high ethanol concentration can account for S. cerevisiae RWB222 viability loss, but ethanol concentration was not the only factor influencing Z. mobilis 8b viability loss at 37 degrees C. Over 3 g/L residual glucose was observed at the end of paper sludge SSCF by Z. mobilis 8b, and a statistical analysis revealed that a high calcium concentration originating from paper sludge, a high ethanol concentration, and a high temperature were the key interactive factors resulting in glucose accumulation. The highest ethanol yields were achieved by SSCF of paper sludge with S. cerevisiae RWB222 at 37 degrees C and Z. mobilis 8b at 30 degrees C. With good sugar consumption at 37 degrees C, S. cerevisiae RWB222 was able to gain an improvement in the polysaccharide to sugar yield compared to that at 30 degrees C, whereas Z. mobilis 8b at 30 degrees C had a lower polysaccharide to sugar yield, but a higher sugar to ethanol yield than S. cerevisiae. Both organisms under optimal conditions achieved a 19% higher overall conversion of paper sludge to ethanol than the non-xylose utilizing S. cerevisiae D5A at its optimal process temperature of 37 degrees C.

The aim of the present study was to evaluate the significance of diffusion barriers in the development of artificial caries-like lesions. A 30-mm2 enamel surface area was covered with a layer of filter paper and exposed to a gently agitated 50 mM acetate buffer, pH 4.5, containing either 0.1 or 1.0 ppm fluoride for up to 48 h at 20 degrees C. Similar enamel surfaces, but unprotected by filter paper, served as controls. It was found that below the filter paper an approximately 60-microns-deep caries-like lesion was developed exhibiting a preserved surface layer, the mineral content of which depended on the fluoride concentration of the buffer. The lesions of the control teeth without a filter paper coverage were erosion-like surface lesions. In the filter paper calcium and phosphate released from the enamel combined with fluoride from the bulk solution had established a supersaturation with respect to fluorapatite while the undersaturation with respect to hydroxyapatite was maintained, which caused a caries lesion to develop. The lesions produced in noncovered enamel by bulk solution unsaturated with respect to both apatites were erosion-like. It was concluded that a diffusion barrier can significantly favor the establishment of supersaturation with respect to fluorapatite and thus facilitate the development of a subsurface caries-like lesion at the expense of the erosion.

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

Les ions corrosifs de fer et de cuivre ainsi que les acides dans les encres ferro-galliques ferriques ainsi que dans le vert-de-gris et la malachite de certains pigmenst tendent a intensifier la degradation de papier. Un recent projet europeen commun denomme INKCor propose l'utilisation de bromures d'amonium tetraalkyles combines a des ethoxudes de magnesium dans de l'ethanol afin de mieux stabiliser ces composants. Dans l'article suivant l'effet potentiellement stabilisateur d'un autre type d'antioxydants - les bromures imides d'alkyles - est compare a celui obtenu a l'aide de traitement non aqueux de bromure d'amonium tetrabutyle et lors d'un traitement aqueux a base de phytate de calcium. Les deux nouveaux antioxydants objets de cette etude a savoir: bromure imidazole 1 de methyle 3 (EMINBr) et bromure imidazole 1 dimithyle 2,3 (BDMINBr) combines avec l'alcali magnesium ethoxide dans l'ethanol ont effectivement stabilise l'encre ferro-gallique du papier traite. L'effet de stabilisation obtenu a ete meilleur que celui antoxydants deja connus, comme par exemple les bromures d'amonium tetraalkyles et les deux phytates. On n'y a pas observe d'effets secondaires nocifs sur la couleur de l'encre, ce qui fait de ces deux antioxydants des alternatives interessantes au traitement aqueux au phytate de calcium couramment employe. On a pousuivi cette etude sur des echantillons de papier presentant une pigmentation due a la corrosion par le vert-de-gris et la malachite. Le traitement le plus efficace a ete dans ce cas celui a base de bromures d'amonium tetraalkyles et d'oxyde de magnesium en suspension dans une solution d'ethanol. Bien qu'on n'ait pas observe d'effets negatifs sur le papier contenant de l'encre ferro-gallique ou une pigmentation cuivree, il reste capital d'etudier les effets de ce traitement sur un certain nombre d'echantillons historiques avant de pouvoir passer a leur utilisation dans la restauration d'objets historiques.

Physiological parameters were measured after experimental infection of roach (Rutilus rutilus L.) with Rhipidocotyle fennica Gibson, Valtonen et Taskinen, 1992 (Digenea) cercariae. The fish were caught from two lakes: a eutrophic bleached kraft mill effluent (BKME)-contaminated lake and an oligotrophic unpolluted lake. The intensity of infection was followed up to 10 days post infection (p.i.) and physiological parameters indicating non-specific stress responses and the condition of fish were examined simultaneously. The mean abundance, the number of parasites per fish, of R. fennica was significantly higher in the fish from the contaminated water during the first two days p.i., probably reflecting the decreased resistance of these fish to infection. The decrease of leukocrit, as well as the increase of the activity of transaminases (GOT and GPT) in infected fish of both groups are suggestive of pathological processes caused by cercariae penetrating the fish. A significantly lower leukocrit value, as well as higher alkaline phosphatase activity and plasma chloride levels were noted in fish originating from the contaminated lake compared to those from the unpolluted lake. No significant differences were noted in haematocrit, plasma protein and calcium values between the fish from the uncontaminated and contaminated lakes, or between the infected and uninfected control fish.

Increasing system closure in paper mills and higher process water temperatures make the applicability of thermophilic treatment systems increasingly important. The use of activated sludge as a suitable thermophilic post-treatment system for anaerobically pre-treated paper process water from a paper mill using recycled wastepaper was studied. Two lab-scale plug flow activated sludge reactors were run in parallel for 6 months; a thermophilic reactor at 55 degrees C and a reference reactor at 30 degrees C. Both reactors were operated simultaneously at 20, 15 and 10 days SRT. The effects of temperature and SRT on sludge settleability and chemical oxygen demand (COD) removal efficiencies of different fractions were studied. Total COD removal percentages over the whole experimental period were 58+/-5% at 30 degrees C and 48 +/- 10% at 55 degrees C. The effect of the SRT on the total COD removal was negligible. Differences in total COD removal between both systems were due to a lesser removal of soluble and colloidal COD at 55 degrees C compared to the reference system. At 30 degrees C, colloidal COD removal percentages were 65+/-25%, 75+/-17% and 86+/-22% at 20, 15 and 10 days SRT, respectively. At 55 degrees C, these percentages were 48+/-34%, 40+/-28% and 70+/-25%, respectively. The effluent concentrations of colloidal COD in both systems were related to the influent concentration of colloidal material. The thermophilic sludge was not able to retain influent colloidal material as well as the mesophilic sludge causing a higher thermophilic effluent turbidity. Sludge settling properties were excellent in both reactor systems. These were neither temperature nor SRT dependent but were rather caused by extensive calcium precipitation in the aeration tanks creating a very dense sludge. For application in the board industry, a thermophilic in line treatment system seems feasible. The higher effluent turbidity is most likely offset by the energy gains of treatment under thermophilic conditions.

A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC50 of 612±10 μ and a Hill slope of 4.9±0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine. Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of 36Cl−. 1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca2+ in colonic epithelial cells. 1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in ‘apical membrane only' preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons. Additionally, 1,10 phenanthroline activated basolateral K+ channels, both Ca2+- and cyclic AMP-sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin. The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K+ channels, may constitute useful lead compounds for adjunct therapy in CF. Keywords: CFTR, cystic fibrosis, chloride channel openers, phenanthrolines, 7,8 benzoquinoline, phenanthridine, potassium channels, cyclic AMP, intracellular Ca2+ Introduction Increased interest in agents affecting epithelial chloride channels arose after it was shown that the cystic fibrosis transmembrane conductance regulator (CFTR) is also a chloride channel in many epithelial membranes (Anderson et al., 1991). Not only have blockers of CFTR been described but considerable interest has also focused on CFTR chloride channel openers (Schultz et al., 1999). The most common mutation in cystic fibrosis (CF) is ΔF508, in which phenylalanine is absent from position 508. This mutant protein (ΔF508CFTR) can function as a chloride channel (Drumm et al., 1991), however only a minute fraction of this protein is transported to the apical domain of epithelial cells (Ward & Kopito, 1994). Instead the quality control mechanisms of the cell cause the mutant protein to be degraded in the proteasome. Therefore attempts are being made to increase the delivery of ΔF508 CFTR to the membrane (Brodsky, 2001; Fischer et al., 2001; Fuller & Cuthbert, 2000). Thus agents which open CFTR channels are of interest, particularly so if they also activate ΔF508 CFTR. The present classes of CFTR openers are as follows; phosphatase inhibitors (Becq et al., 1998), xanthines, particularly CPX (Guay-Broder et al., 1995), benzimidazolones such as EBIO (Devor et al., 1996; Cuthbert et al., 1999), flavones, for example genistein (Illek et al., 1995), benzoxazolones like chlorzoxazone (Singh et al., 2000) and finally the MPB compounds described by Becq et al. (1999). The purpose of this study was to further investigate the actions of 1,10 phenanthroline on chloride secretion in the mouse colon following the observations made by Duszyk et al. (1999) that 1,10 phenanthroline, a powerful inhibitor of matrix metalloproteases (MMPs) activated a chloride conductance, measured as whole cell current, in human airway epithelial cells, Calu-3. No effect was found in airway cells lacking CFTR, namely A549 cells. Furthermore, an antibody to MMP2 caused a similar effect in Calu-3 cells as did 1,10 phenanthroline, suggesting metalloproteinase inhibition was responsible. A further aim was to explore structure activity relations in the phenanthroline isomers and to ask if both heterocyclic nitrogens in the tricyclic aromatic molecules were necessary for the effect. From various experimental approaches we conclude that the phenanthrolines and related structures constitute a new class of agents that stimulate chloride secretion.

Measurement of oxalate in urine is important for the diagnosis of primary hyperoxaluria (McKusick 259900) and the secondary forms produced by excessive intake or abnormal intestinal absorption of oxalate (1). Determination of glycolic acid is essential for the diagnosis of primary hyperoxaluria type 1. Finally, to estimate the risk of stone formation in calcium oxalate urolithiasis and nephrocalcinosis, simultaneous determination not only of calcium but also of citrate (a potent inhibitor of calcium oxalate and calcium phosphate crystallization) and other constituents (electrolytes, phosphate and sulfate) is required to calculate urinary calcium saturation (2). Ion-chromatography HPLC (3)(4) and specific enzymatic assays (5)(6) are available only in specialized laboratories. In addition, preservation and storage of liquid samples may influence the stability of oxalate and glycolate (7). Use of urinary filter spots is a practical alternative for the collection and safe transport of samples to be analyzed for many metabolic disorders. To evaluate the age-related changes of oxalate, glycolate, citrate, and sulfate in a pediatric population, we developed an automated ion-chromatography system for the simultaneous measurement of these anions in urine and established their reference values for liquid urine samples as well as for dried urine on filter paper. We studied 20 individuals from each age group ( 16 years), all without renal or metabolic disease, all on a routine food intake at the time of study. The urines were preserved with 6 mol/L HCl at pH 1–2 to prevent nonenzymatic conversion of ascorbate to oxalate. Filter paper strips (3 × 5 …

Homotypically conditioned media (Hom. C.M.) produced by adult Biomphalaria glabrata weighing about 600 mg and fed on pure cellulose under bacteriostatic conditions at a density of one snail per 100 milliliter of standard snail water for three days significantly enhanced the growth of juveniles. Hom. C.M. produced under the same conditions by fasting snails that were previously fed on lettuce, also had a growth promoting effect but was less potent. The growth promoting factors have a molecular weight of 500-104, and appear to act directly on the snails and not indirectly by influencing the food medium. The extent to which absolute growth rate is stimulated by the growth factors correlates well with decrements in the conductivity and calcium concentration of the media. Possible modes of action of the growth factors are discussed. They appear not to influence the relative weights of shell, or body and water content of the snails. It is suggested that the adaptive significance of the growth factors is to facilitate rapid increase in numbers of this gregarious and often fugitive species when environmental conditions are favorable.

An analog of adenosine triphosphate, adenylyl imidodiphosphate (AMP-PNP), in which a "-grouping replaces the terminal bridge oxygen of the triphosphate chain has been synthesized by the reaction of imidodiphosphate with PI-adenosine P2-diphenyl pyrophosphate (Michelson, A. (1964), Biochim. Biophys. Acta 9/, 1). AMP-PNP is stable (

Procedures are described for the isolation of lipoproteins from human serum by precipitation with polyanions and divalent cations. A mixture of low and very low density lipoproteins can be prepared without ultracentrifugation by precipitation with heparin and either MnCl(2) alone or MgCl(2) plus sucrose. In both cases the precipitation is reversible, selective, and complete. The highly concentrated isolated lipoproteins are free of other plasma proteins as judged by immunological and electrophoretic methods. The low density and very low density lipoproteins can then be separated from each other by ultracentrifugation. The advantage of the method is that large amounts of lipoproteins can be prepared with only a single preparative ultracentrifugation. Polyanions other than heparin may also be used; when the precipitation of the low and very low density lipoproteins is achieved with dextran sulfate and MnCl(2), or sodium phosphotungstate and MgCl(2), the high density lipoproteins can subsequently be precipitated by increasing the concentrations of the reagents. These lipoproteins, containing small amounts of protein contaminants, are further purified by ultracentrifugation at d 1.22. With a single preparative ultracentrifugation, immunologically pure high density lipoproteins can be isolated from large volumes of serum.

The reaction of Paramecium caudatum to solutions containing the ash of cigarette paper was consistent with the response of Paramecium aurelia previously reported. After this confirmation, both these species of Paramecium were used as test organisms during a search for the causative agents for the "Paramecium reaction". A qualitative spectrochemical analysis of cigarette paper ash showed that its chief constituent is calcium (97%) with 12 other detected elements present at levels too low to elicit any response from paramecia. Hydroxyl ions in concentrations equivalent to 0.001 M Ca(OH)2, 0.0008 M KOH, or 0.002 M NaOH simulated but did not duplicate the effect of standard cigarette paper ash solution (as of one cigarette paper in 20 ml water). While neutralization caused the three hydroxide solutions to lose their toxicity, the toxicity of cigarette paper ash solution was not appreciably affected by neutralization. Equivalent solutions of facial tissue, filter paper, and a paper towel, burned the same way as cigarette paper, were used as controls. Each of them affected paramecia differently and none simulated the Paramecium reaction. Further, a comparison between ashes from a paperless cigarette, a whole cigarette, and one with two extra roll-your-own wrappers demonstrated a dependence of the Paramecium reaction on cigarette paper ash as well as a direct correlation between the reaction intensity and the amount of cigarette paper ash present.