Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures

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.

The Dark Side of the Moon: Meta-analytical Impact of Recruitment Strategies on Risk Enrichment in the Clinical High Risk State for Psychosis

Background: The individual risk of developing psychosis after being tested for clinical high-risk (CHR) criteria (posttest risk of psychosis) depends on the underlying risk of the disease of the population from which the person is selected (pretest risk of psychosis), and thus on recruitment strategies. Yet, the impact of recruitment strategies on pretest risk of psychosis is unknown. Methods: Meta-analysis of the pretest risk of psychosis in help-seeking patients selected to undergo CHR assessment: total transitions to psychosis over the pool of patients assessed for potential risk and deemed at risk (CHR+) or not at risk (CHR−). Recruitment strategies (number of outreach activities per study, main target of outreach campaign, and proportion of self-referrals) were the moderators examined in meta-regressions. Results: 11 independent studies met the inclusion criteria, for a total of 2519 (CHR+: n = 1359; CHR−: n = 1160) help-seeking patients undergoing CHR assessment (mean follow-up: 38 months). The overall meta-analytical pretest risk for psychosis in help-seeking patients was 15%, with high heterogeneity (95% CI: 9%–24%, I 2 = 96, P < .001). Recruitment strategies were heterogeneous and opportunistic. Heterogeneity was largely explained by intensive (n = 11, β = −.166, Q = 9.441, P = .002) outreach campaigns primarily targeting the general public (n = 11, β = −1.15, Q = 21.35, P < .001) along with higher proportions of self-referrals (n = 10, β = −.029, Q = 4.262, P = .039), which diluted pretest risk for psychosis in patients undergoing CHR assessment. Conclusions: There is meta-analytical evidence for overall risk enrichment (pretest risk for psychosis at 38monhts = 15%) in help-seeking samples selected for CHR assessment as compared to the general population (pretest risk of psychosis at 38monhts=0.1%). Intensive outreach campaigns predominantly targeting the general population and a higher proportion of self-referrals diluted the pretest risk for psychosis.

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods.

Sodium/hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H+) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na(+)-dependent H+ fluxes (JH+) are equivalent to currents >20 pA for NHE1 in Chinese hamster ovary fibroblasts, >200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pA for NHE3 in opossum kidney cells. The fluxes are blocked by an NHE inhibitor, ethylisopropylamiloride, and are absent in NHE-deficient AP-1 cells. NHE1 activity is stable with perfusion of nonhydrolyzable ATP [adenosine 5'-(beta,gamma-imido)triphosphate], is abolished by ATP depletion (2 deoxy-D-glucose with oligomycin or perfusion of apyrase), can be restored with phosphatidylinositol 4,5-bisphosphate, and is unaffected by actin cytoskeleton disruption (latrunculin or pipette perfusion of gelsolin). NHE3 (but not NHE1) is reversibly activated by phosphatidylinositol 3,4,5-trisphosphate. Both NHE1 and NHE3 activities are disrupted in giant patches during gigaohm seal formation. NHE1 (but not NHE3) is reversibly activated by cell shrinkage, even at neutral cytoplasmic pH without ATP, and inhibited by cell swelling. NHE1 in Chinese hamster ovary fibroblasts (but not NHE3 in opossum kidney cells) is inhibited by agents that thin the membrane (L-alpha-lysophosphatidylcholine and octyl-beta-D-glucopyranoside) and activated by cholesterol enrichment, which thickens membranes. Expressed in AP-1 cells, however, NHE1 is insensitive to these agents but remains sensitive to volume changes. Thus, changes of hydrophobic mismatch can modulate NHE1 but do not underlie its volume sensitivity.