Evolutionarily conserved dual lysine motif determines the non-chaperone function of secreted Hsp90alpha in tumour progression.

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and ß) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90α and Hsp90ß was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90ß leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90ß, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90ß subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90ß with lysines convert Hsp90ß to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90α, instead of the intracellular Hsp90α and Hsp90ß.

Flexible high-conductivity carbon-nanotube interconnects made by rolling and printing.

Applications of carbon nanotubes (CNTs) in flexible and complementary metal-oxide-semiconductor (CMOS)-based electronic and energy devices are impeded due to typically low CNT areal densities, growth temperatures that are incompatible with device substrates, and challenges in large-area alignment and interconnection. A scalable method for continuous fabrication and transfer printing of dense horizontally aligned CNT (HA-CNT) ribbon interconnects is presented. The process combines vertically aligned CNT (VA-CNT) growth by thermal chemical vapor deposition, a novel mechanical rolling process to transform the VA-CNTs to HA-CNTs, and adhesion-controlled transfer printing without needing a carrier film. The rolling force determines the HA-CNT packing fraction and the HA-CNTs are processed by conventional lithography. An electrical resistivity of 2 mOmega . cm is measured for ribbons having 800-nm thickness, while the resistivity of copper is 100 times lower, a value that exceeds most CNT assemblies made to date, and significant improvements can be made in CNT structural quality. This rolling and printing process could be scaled to full wafer areas and more complex architectures such as continuous CNT sheets and multidirectional patterns could be achieved by straightforward design of the CNT growth process and/or multiple rolling and printing sequences.

Healthcare evaluation of the use of atosiban and fibronectin for the management of pre-term labour.

Our objective was to assess the effectiveness, safety and efficiency of a protocol combining fetal fibronectin diagnostic testing and atosiban tocolysis for the management of pre-term labour, using a combination of narrative review and economic modelling. We compared the proposed protocol to alternatives using nifedipine with or without fetal fibronectin. We have found that the proposed protocol may be safer and more acceptable by women than the alternatives, and its use can result in significant cost-savings.

Prenatal iron supplements impair zinc absorption in pregnant Peruvian women.

Prenatal iron supplements may adversely influence zinc absorption during pregnancy. To examine the impact of prenatal iron supplements on supplemental zinc absorption, fractional zinc absorption was measured in 47 pregnant Peruvian women during the third trimester of pregnancy (33 +/- 1 wk gestation). Of these 47 women, 30 received daily prenatal supplements from wk 10-24 of pregnancy until delivery. Supplements contained 60 mg of Fe and 250 microg of folate without [iron group (Fe), n = 16] or with [iron and zinc supplemented group (Fe + Zn), n = 14] 15 mg of Zn. The remaining 17 women [unsupplemented control group (C)] received no prenatal supplementation. Zinc concentrations were measured in plasma, urine and cord blood and percentage zinc absorption was determined following dosing with oral ((67)Zn) and intravenous ((70)Zn) stable zinc isotopes. Percentage zinc absorption was significantly lower than controls in fasting women receiving iron- containing prenatal supplements (20.5 +/- 6.4 vs. 20.2 +/- 4.6 vs. 47.0 +/- 12.6%, Fe, Fe + Zn and C groups, respectively, P: < 0.0001, n = 40). Plasma zinc concentrations were also significantly lower in the Fe group compared to the C group (8.2 +/- 2.2 vs. 9.2 +/- 2.2 vs. 10.9 +/- 1. 8 micromol/L, Fe, Fe + Zn and C groups, respectively, P: = 0.002), and cord zinc concentrations were significantly related to maternal plasma Zn levels (y = 6.383 + 0.555x, r = 0.486, P: = 0.002). The inclusion of zinc in prenatal supplements may reduce the potential for iron supplements to adversely influence zinc status in populations at risk for deficiency of both these nutrients.

Plasma paracetamol concentrations and pharmacokinetics following rectal administration in neonates and young infants.

BACKGROUND: Despite widespread use in children pharmacokinetic data about paracetamol are relatively scarce, not the least in the youngest age groups. This study aimed to describe plasma paracetamol concentrations and pharmacokinetics of a single rectal paracetamol dose in neonates and young infants. METHODS: Perioperatively, 17 neonates and infants < or =160 days of age received one rectal paracetamol dose (mean 23.9 mg/kg (+/-4.2 mg/kg)). Blood samples were drawn at 60, 120, 180, 240, 300 and 360 min, according to the infants' weights. Plasma paracetamol concentrations were measured by a Colorometric Assay, Ectachem Clinical Chemistry Slides (Johnson & Johnson Clinical Diagsnostics). RESULTS: The plasma paracetamol concentrations were mainly below the therapeutic (i.e. antipyretic) range of 66-132 micromol/l and did not exceed 160 micromol/l in any infant. The mean maximum plasma concentration (Cmax) was 72.4 micromol/l (+/-33.5 micromol/l) and the time to Cmax, i.e. the mean Tmax was 102.4 min (_+59.1 min). The mean "apparent" terminal half-life (n=10) was 243.6 min (+/-114.1 min). CONCLUSION: The absorption of rectal paracetamol (mean dose 23.9 mg/kg, +/-4.2mg/kg) in young infants <160 days is variable and often prolonged and achieves mainly subtherapeutic plasma concentrations.

Murine phospholipid hydroperoxide glutathione peroxidase: cDNA sequence, tissue expression, and mapping.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), also known as glutathione peroxidase 4 (GPX4), is a 19-kDa, monomeric enzyme that protects cells from lipid peroxide-mediated damage by catalyzing the reduction of lipid peroxides. PHGPx is synthesized in two forms, as a 194-amino acid peptide that predominates in gonadal tissue and localizes to mitochondria, and as a 170-amino acid protein that predominates in most somatic tissues and localizes to the cytoplasm. With the rapid amplification of cDNA ends (RACE) procedure, an 876-bp PHGPx cDNA was amplified from mouse testis, and a 767-bp PHGPx cDNA was amplified from mouse heart. The cDNA sequences were identical except that the testis cDNA contained an additional 109 bp at its 5' end. With a partial cDNA with complete homology to both the testis and myocardial PHGPx cDNAs, the murine tissue distribution of PHGPx mRNA expression was determined by Northern blotting. Highest level of PHGPx expression was found in the testis, followed by the kidney, heart and skeletal muscle, liver, brain, lung, and spleen. Northern blotting performed with a cDNA specific for the longer PHGPx transcript demonstrated that this longer PHGPx transcript was present only in the testis. A 1.4-kb PHGPx genomic fragment was amplified from murine kidney DNA and used to map the PHGPx gene by linkage analysis of restriction fragment length variants (RFLVs). The murine PHGPx gene (Gpx4) was mapped to a region of murine Chromosome (Chr) 10, located 43 cM from the centromere, that is syntenic with the human locus, which is located at the terminus of the short arm of human Chr 19. This information may be valuable in characterizing the role of PHGPx in modulating susceptibility to lipid peroxide-mediated injury in inbred murine strains and for targeted disruption of the gene.

Influence of prenatal iron and zinc supplements on supplemental iron absorption, red blood cell iron incorporation, and iron status in pregnant Peruvian women.

BACKGROUND: It is estimated that 60% of pregnant women worldwide are anemic. OBJECTIVE: We aimed to examine the influence of iron status on iron absorption during pregnancy by measuring supplemental iron absorption, red blood cell iron incorporation, and iron status in pregnant women. DESIGN: Subjects were 45 pregnant Peruvian women (33+/-1 wk gestation), of whom 28 received daily prenatal supplements containing 60 mg Fe and 250 microg folate without (Fe group, n = 14) or with (Fe+Zn group, n = 14) 15 mg Zn, which were were consumed from week 10 to 24 of gestation until delivery. The remaining 17 women (control) received no prenatal supplementation. Iron status indicators and isotopes were measured in maternal blood collected 2 wk postdosing with oral (57Fe) and intravenous (58Fe) stable iron isotopes. RESULTS: Maternal serum ferritin and folate concentrations were significantly influenced by supplementation (P < 0.05). Serum iron was also significantly higher in the Fe than in the Fe+Zn (P < 0.03) or control (P < 0.001) groups. However, the supplemented groups had significantly lower serum zinc concentrations than the control group (8.4+/-2.3 and 10.9+/-1.8 micromol/L, respectively, P < 0.01). Although percentage iron absorption was inversely related to maternal serum ferritin concentrations (P = 0.036), this effect was limited and percentage iron absorption did not differ significantly between groups. CONCLUSIONS: Because absorption of nonheme iron was not substantially greater in pregnant women with depleted iron reserves, prenatal iron supplementation is important for meeting iron requirements during pregnancy.

PIP: The influence of iron status on iron absorption during pregnancy was examined among pregnant Peruvian women. This was done by measuring supplemental iron absorption, red blood cell iron incorporation and iron status. The subjects were 45 pregnant Peruvian women (33 +or- 1 week gestation) who were divided into 2 groups. The first group of 28 pregnant women received daily prenatal supplements containing 60 mg of iron and 250 mcg of folate with or without 15 mg of zinc, from week 10 to 24 of gestation until delivery. The second group of 17 women served as the control group. The control group was not given prenatal supplementation. The iron status indicators and isotopes were measured in maternal blood collected 2 weeks postdosing with oral iron-57 and intravenous iron-58 stable isotopes. The results showed that supplementation significantly influenced the maternal serum ferritin and folate concentrations (P 0.05). The serum iron of the iron group was significantly higher than that of the iron + zinc group (P 0.03) or control group (P 0.001). However, the serum zinc concentrations were lower in the supplemented group than in the control group. Even though the percentage of iron absorption was inversely related to maternal serum ferritin concentration, the effect was limited and the percentage of iron absorption did not differ significantly between the two groups. Considering that the absorption of nonheme iron was not substantially greater in pregnant women with depleted iron reserves, it was concluded that prenatal iron supplementation is essential for meeting iron requirements, especially during pregnancy.

Regulation of mineral metabolism from fetus to infant: metabolic studies.

Optimal mineral intake is crucial, especially during the period of rapid growth that occurs during infancy and childhood. Two minerals that have been found to play key roles during this period are iron and zinc. Supplementation studies have shown that these minerals have significant effects on birth weight as well as on weight and height increase during childhood. However, because a myriad of nutritional factors influence growth, it has often been difficult to characterize the role of any given mineral on fetal and early childhood growth. Stable iron and zinc isotopes can be used to study how the mineral status of iron- and zinc-deficient pregnant women affects their ability to absorb and transfer iron to the fetus. Furthermore, these isotopic tracers can be employed to examine the ability of infants to modify mineral absorption over time as the mineral stores of birth are depleted. Further studies using stable mineral isotopes during gestation, infancy and childhood will provide additional information on the regulation of mineral absorption and transport during these key periods of growth.

Dietary protein affects intestinal calcium absorption.

BACKGROUND: Changes in dietary protein in adults are associated with changes in urinary calcium excretion. The mechanisms underlying this effect are not completely understood, but alterations in intestinal absorption of calcium are not thought to be involved. OBJECTIVE: We reexamined this mechanism by evaluating the effect of 2 amounts of dietary protein (low: 0.7 g/kg; and high: 2.1 g/kg) on fractional calcium absorption in 7 healthy, young women. DESIGN: The experiment consisted of 2 wk of a well-balanced diet containing moderate amounts of calcium, sodium, and protein followed by 5 d of an experimental diet that contained 1 of 2 amounts of protein and constant amounts of other nutrients known to influence calcium metabolism. Seven subjects received both amounts of dietary protein in random order. Blood and urine were sampled at baseline and on day 4. Fractional calcium absorption was measured by dual-stable calcium isotopes on day 5. In a second study of 5 additional women, we evaluated the effects of dietary fiber on calcitropic hormones. RESULTS: Subjects developed hypocalciuria and secondary hyperparathyroidism on day 4 of the low-protein diet. Urinary calcium excretion and the glomerular filtration rate were elevated significantly by day 4 of the high-protein compared with the low-protein diet. Fractional calcium absorption after the low-protein diet was 0.19+/-0.03, which was significantly lower than that after the high-protein diet (0.26+/-0.03, P=0.05). CONCLUSION: These data provide evidence that depressed intestinal calcium absorption explains, in part, low-protein-induced secondary hyperparathyroidism.

Combined calcium and vitamin D supplementation reduces bone loss and fracture incidence in older men and women.

A recent supplementation study of 389 men and women, over the age of 65 years was conducted to address the impact of combined calcium and vitamin D supplementation on nonvertebral fracture incidence and maintenance of bone mass. Daily supplementation with 500 mg calcium and 700 IU vitamin D for 3 years moderately reduced bone loss at several sites and significantly decreased the rate of nonvertebral fractures, compared with a placebo group. Optimal intake of both calcium and vitamin D may be an easily implemented strategy to maintain existing bone mass and reduce the risk of fracture in older men and women.

Absorption by 1-year-old children of an iron supplement given with cow's milk or juice.

A paucity of data are available on toddlers for the evaluation of optimal strategies of Fe supplementation. In this study, we used a two-tracer stable isotope technique to determine Fe absorption from a 5-mg dose of stable isotopically enriched (57Fe or 58Fe) ferrous sulfate given with cow's milk (CM) compared with the same dose given with apple juice. Ten children (age 13 +/- 1 mo, weight 10.8 +/- 1.1 kg) who had recently discontinued formula feeding and begun on CM were studied. Red blood cell (RBC) iron incorporation of the isotope was determined 14 d after dosing with 57Fe and 58Fe. Fe absorption was calculated based on the assumption that 90% of absorbed Fe is incorporated into RBC. Absorption of Fe was significantly greater (13.7 +/- 6.4%) when given with juice than with milk (5.7 +/- 4.0%), p < 0.01 by paired t tests. Fe absorption from the dose given with juice was significantly negatively correlated with serum ferritin (n = 9, r = -0.70, p < 0.05). These results indicate that 1) a small supplement of Fe is better absorbed when given with juice than with CM, and 2) a large variability in Fe absorption exists in healthy 1-y-old infants, which is related to their existing Fe stores.

Osteoporosis workup: evaluating bone loss and risk of fractures.

Osteoporosis afflicts millions of Americans, causing an age-related increase in the incidence of fractures of the hip, wrist, spine, and other bones. Older patients require assessment of their fracture risk, including evaluation of skeletal fragility and risk of falls. Secondary causes of bone loss should be sought in individuals with bone density lower than expected for age. Preventive measures, such as supplementary calcium and vitamin D, and environmental changes to decrease the risk of falls in the home are recommended. Physical therapy may decrease the risk of falls and help maintain bone mass. Antiresorptive therapy should be considered for any individual at high risk for fracture, regardless of age.

Application of magnetic sector thermal ionization mass spectrometry to studies of erythrocyte iron incorporation in small children.

The optimal evaluation of iron metabolism requires the administration of two isotopes of iron. However, high-precision measurement of isotopic ratios from blood samples obtained after administration of two stable isotopes of iron to human subjects has not previously been reported. Using a cation-exchange system to isolate iron from blood samples, we found that high-precision (< 0.2%) measurements of 58Fe/56Fe and 57Fe/56Fe could be performed using magnetic sector thermal ionization mass spectrometry. Clinical studies in four 1-year-old infants showed that this technique could be used to demonstrate a lower rate of iron absorption in small children given an iron supplement (57Fe) with milk compared to those given iron (58Fe and ferrous sulfate) with ascorbic acid. This technique will enable the evaluation of iron metabolism in populations in whom the use of radioactive iron tracers is not appropriate.

Calcium kinetics in lactating women with low and high calcium intakes.

Absorption of calcium and its mobilization from bone during lactation are important for delivery of calcium to breast-feeding infants; whether calcium intake offsets bone resorption is not known. We hypothesized that calcium absorption is increased in lactation and greater in women on low calcium diets, resulting in similar rates of bone resorption and accretion. Calcium absorption and kinetic indexes were calculated by using two stable isotopic tracers in 8 women; 6 were studied both during lactation and nonlactation. Women consumed low calcium diets, with half receiving supplemental calcium. Intestinal absorption was related to serum 1,25-dihydroxyvitamin D and did not increase during lactation. Despite decreased urinary calcium excretion during lactation, especially in women with low calcium intake, net balance tended to be lower during lactation. Mean residence time decreased and bone resorption exceeded accretion in almost all lactating women. Calcium need for milk production appears to be met by decreased urinary excretion and increased bone resorption, and not by increased intestinal absorption.

Dose rate determination for 125I seeds.

Dose rates in water have been determined for the two types of 125I seed currently used in brachytherapy. The need for such determinations became evident when water/air ratios measured with a silicon diode were found to be lower than expected. Extensive measurements using lithium fluoride thermoluminescent dosimeters (TLD's) have been performed in a solid water phantom, at distances from 0.1 to 10 cm from the seed center and at angular increments of 10 degrees, 15 degrees, or 30 degrees within a plane through the seed axis. Dose calibration of the TLD's was accomplished by irradiation in air with 125I seeds of the same type and of strengths traceable to a calibration at the National Institute of Standards and Technology (NIST). Relative calibration of TLD's was monitored by irradiation, in an oven-type x-ray machine, of control dosimeters simultaneously and all dosimeters intercurrently with the 125I irradiations. Values obtained for the dose rate constant, i.e., dose rate per unit air-kerma strength at 1 cm on the transverse axis, were 0.853 and 0.932 cGy h-1 U-1 (1.08 and 1.18 cGy h-1 mCi-1) for the 6711 and 6702 seeds, respectively. Measured data were supplemented with Monte Carlo-calculated relative dose rate data generated using the MORSE code. These calculations used 100 energy groups from 10 to 35.4 keV and involved energy collection bins ranging from 0.025 to 1.2 cm on an edge. Normalized at 1 cm, transverse axis calculated data are not significantly different from measured data (ours or cited literature) at distances either less than 2.5 or greater than 8 cm. Normalized at different distances along the transverse axis, our off-axis calculated and measured distributions agree closely at all angles but differ from literature measured distributions at small (less than or equal to 1 cm) distances and, for small angles, increasingly at larger distances (greater than or equal to 5 cm).

The role of protein breakdown in growth, quiescence, and starvation of vascular smooth muscle cells.

Protein accumulation in growing cells may be due in part to a reduction in the rate of protein breakdown. Previous studies of the relation of cell proliferation to protein degradation often produced growth arrest by conditions that may involve nutritional deprivation. However, nutrient lack can itself accelerate proteolysis and produce negative protein balance. We therefore reexamined the relation between growth and protein breakdown using a more selective method for limiting cell growth. We produced quiescent cell cultures using a chemically defined, serum-free medium supplemented with hormones and nutrients. Such media can maintain viability and near neutral protein balance in cultured vascular smooth muscle cells, in part because of reduced breakdown of cellular protein. We then compared rates of protein degradation in these quiescent but not starving cells, to those of cultures stimulated to grow by addition of mitogenic substances. Platelet-derived growth factor, fibroblast growth factor, or fetuin added to insulin-containing medium stimulated growth of smooth muscle cells, but further reduced protein breakdown only slightly. Contrary to the implications of certain previous studies, our results show that proliferating cells can accumulate protein without an appreciable reduction in the rates of protein breakdown. Thus, while accelerated proteolysis appears to be an important adaptation to adverse nutritional conditions, growth of smooth muscle cells does not require changes in overall protein breakdown, but occurs primarily through an increase in protein synthesis.

Culture of quiescent arterial smooth muscle cells in a defined serum-free medium.

An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10(-6) M) and transferrin (5 micrograms/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10(-4) M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.