Last Interglacial Iberian Neandertals as fisher-hunter-gatherers.

Marine food-reliant subsistence systems such as those in the African Middle Stone Age (MSA) were not thought to exist in Europe until the much later Mesolithic. Whether this apparent lag reflects taphonomic biases or behavioral distinctions between archaic and modern humans remains much debated. Figueira Brava cave, in the Arrábida range (Portugal), provides an exceptionally well preserved record of Neandertal coastal resource exploitation on a comparable scale to the MSA and dated to ~86 to 106 thousand years ago. The breadth of the subsistence base-pine nuts, marine invertebrates, fish, marine birds and mammals, tortoises, waterfowl, and hoofed game-exceeds that of regional early Holocene sites. Fisher-hunter-gatherer economies are not the preserve of anatomically modern people; by the Last Interglacial, they were in place across the Old World in the appropriate settings.

First evidence of an extensive Acheulean large cutting tool accumulation in Europe from Porto Maior (Galicia, Spain).

We describe a European Acheulean site characterised by an extensive accumulation of large cutting tools (LCT). This type of Lower Paleolithic assemblage, with dense LCT accumulations, has only been found on the African continent and in the Near East until now. The identification of a site with large accumulations of LCTs favours the hypothesis of an African origin for the Acheulean of Southwest Europe. The lithic tool-bearing deposits date back to 293-205 thousand years ago. Our chronological findings confirm temporal overlap between sites with clear "African" Acheulean affinities and Early Middle Paleolithic sites found elsewhere in the region. These complex technological patterns could be consistent with the potential coexistence of different human species in south-western Europe during the Middle Pleistocene.

DNA exhibits multi-stranded binding recognition on glass microarrays.

In the course of exploring the hybridization properties of glass DNA microarrays, multi-stranded DNA structures were observed that could not be accounted for by classical Watson-Crick base pairing. Non-denatured double-stranded DNA array elements were shown to hybridize to single-stranded (ss)DNA probes. Similarly, ssDNA array elements were shown to bind duplex DNA probes. This led to a series of experiments demonstrating the formation of multi-stranded DNA structures on the surface of microarrays. These structures were observed with a number of heterogeneous sequences, including both purine and pyrimidine bases, with shared sequence identity between the ssDNA and one of the duplex strands. Furthermore, we observed a strong binding preference near the ends of duplexes containing a 3'-homologous strand. We suggest that such binding interactions on cationic solid surfaces could serve as a model for a number of biological processes mediated through multi-stranded DNA.

Effect of commercial saccharin preparations on urethan-induced lung tumorigenesis in strain A mice.

The effect of commercial saccharin preparations on urethan-induced mouse lung tumorigenesis was assessed by gavaging groups of male strain A mice with 1-g/kg doses of each saccharin preparation on a daily basis 5 days/week. Gavage was initiated 1 week before i.p. injection of either a low (0.1 mg/g) or a high (1 mg/g) dose of urethan and continued until the mice were sacrificed 16 weeks after urethan administration. The average number of surface lung tumors per mouse for each group of mice was determined and was compared statistically with the appropriate control group. The commercial saccharin preparations did not produce an elevated lung tumor response when administered alone. One of the four saccharin preparations enhanced the lung tumor response to urethan when given in conjunction with the low dose of urethan, but this enhancement was not statistically significant. At the high urethan dose, all saccharin preparations produced a statistically significant enhancement of the lung tumor response to urethan.

Inhibition by magnesium and calcium acetates of lead subacetate- and nickel acetate-induced lung tumors in strain A mice.

The ability of the physiologically essential divalent metals calcium and magnesium to inhibit the tumorigenic activities of lead and nickel towards the lungs of strain A mice was investigated. The tumorigenic salts lead(II) subacetate and nickel(II) acetate were injected i.p. at their maximal tolerated doses (0.04 mmol/kg/injection of each metal) for a total of 24 injections, whenever possible. Calcium(II) acetate and magnesium(II) acetate were administered in the same preparation along with the lead and nickel salts at molar doses of approximately 1, 3, 10, and 30 times the maximal tolerated dose of the tumorigen. The animals were sacrificed 30 weeks after the first injection, and the lung tumors were counted. The lead and nickel salts, administered alone, each produced a significant increase in the observed number of lung adenomas per mouse. When administered with any of the doses of calcium acetate or magnesium acetate tested, neither lead subacetate nor nickel acetate showed any significant tumorigenic activity. Calcium acetate alone (total dose, 11 mmol/kg of body weight) appeared to yield a significant rise in lung adenomas observed. The results indicate an antagonism between magnesium and calcium and the tumorigenic metals nickel and lead.

Optimized T7 amplification system for microarray analysis.

Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides.

The methylphosphonate oligonucleotide synthesis methods described here give the desired products in good yield. Superior amounts of product are achieved by modifying both the DNA synthesis program and the reagent to compensate for the unstable methylphosphonite intermediate. Deprotection conditions have also been altered to maximize the recovery of oligonucleotide from DNA synthesis supports and to minimize the amount of base modification. Mass-spectrometry analysis of our oligonucleotides has verified their purity and confirmed the absence of modified bases. When compared to standard DNA synthesis methods, this procedure uses only about one-third the usual amount of monomer. Using these procedures, it should be possible to synthesize reliably methylphosphonate oligonucleotides at 1- and 15-mumol scales.

A scale model study of the effects of meteorological, soil, and house parameters on soil gas pressures.

To assess the contribution of various environmental parameters to the entry of Rn into basements, it is advantageous to simplify and control the important variables present in the field situation. A scale model system, simulating a house in soil, has been constructed to investigate the effect of meteorological parameters and house and soil characteristics on soil gas flow around houses. The house walls and soil are of variable permeability. Wind is simulated by applying a static pressure distribution to the soil surface. The effect of temperature differences and appliances is simulated by depressurizing the model house. Soil gas pressures at various locations around the house are measured under different conditions. The results show that the ratio of wall to soil permeability is the determining factor in soil gas flow patterns. For a wind of 8.94 m s-1 (20 mph), the horizontal pressure gradients are about 99 Pa m-1 in the model when the wall is at least as permeable as the soil. This corresponds to 3.3 Pa m-1 in the field. When the soil is two or more orders of magnitude more permeable than the wall, the gradient is about 19.8 Pa m-1 in the model, or 0.66 Pa m-1 in the field. There is a logarithmic dependence of pressure gradient on the ratio of wall to soil permeability in the range -2 less than log (kw/ks) less than 0. Conversely, it takes a large temperature difference of 27 degrees C to cause a 99 Pa m-1 horizontal pressure gradient in model systems with wall permeability greater than soil permeability. The effects of changes in the model system on soil gas flow patterns are investigated for the cases of lowered soil surface permeability, partial surface capping, and presence of a subfloor gravel bed. Partial surface capping, as would occur with driveways and patios, was found to have a minor effect on soil gas pressures. However, lowered surface permeability, caused by precipitation, can significantly change soil gas flow patterns. The only change in soil pressure gradients or pressure differences in the presence of a gravel bed is in the system with the highest wall-to-soil permeability ratio. In this system, under all conditions (house depressurization, wind, and wind with house depressurization), there is an increase in the absolute value of both upwind and downwind pressure differences and pressure gradients with the addition of a gravel bed.

Neandertal roots: Cranial and chronological evidence from Sima de los Huesos.

Seventeen Middle Pleistocene crania from the Sima de los Huesos site (Atapuerca, Spain) are analyzed, including seven new specimens. This sample makes it possible to thoroughly characterize a Middle Pleistocene hominin paleodeme and to address hypotheses about the origin and evolution of the Neandertals. Using a variety of techniques, the hominin-bearing layer could be reassigned to a period around 430,000 years ago. The sample shows a consistent morphological pattern with derived Neandertal features present in the face and anterior vault, many of which are related to the masticatory apparatus. This suggests that facial modification was the first step in the evolution of the Neandertal lineage, pointing to a mosaic pattern of evolution, with different anatomical and functional modules evolving at different rates.

Field gamma dose-rate assessment in natural sedimentary contexts using LaBr3(Ce) and NaI(Tl) probes: a comparison between the "threshold" and "windows" techniques.

Results are presented for a series of replicate in situ gamma spectrometry measurements (n=20) made in natural sedimentary contexts using LaBr3(Ce) and NaI(Tl) probes. For both types of detectors, gamma dose rates were calculated using the "threshold" technique (Murray et al., 1978), and compared with results obtained previously by Arnold et al. (2012) using the "windows" technique (Aitken, 1985). Our results show that gamma dose rates obtained using these two techniques are consistent at 1σ for a given probe, and that the threshold technique yields reproducible results for the LaBr3(Ce) and NaI(Tl) probes. In comparison with the energy windows approach, the threshold approach offers an improvement in the precision with which gamma dose rates can be determined using the LaBr3(Ce) probe. The potential of an alternative threshold approach (the "energy threshold" approach of Guérin and Mercier, 2011) was also tested for both probe types, and the resultant gamma dose rates were found to be in agreement with those obtained using the standard threshold and energy windows techniques. Our results provide new insights into methods and instrumentation used for assessing in situ gamma dose rates in Electron Spin Resonance (ESR) and Luminescence dating. We conclude that LaBr3(Ce) probes can reliably be used for portable gamma dosimetry in low level activity sedimentary environments (500-1500µGy/a) when using the threshold approach, provided that their non-negligible internal background activities (equivalent to ∼758µGy/a for our probe) are accurately assessed and subtracted from gamma ray spectra measured in the field. Our results also suggest that there may be some minor merit in applying an internal background-subtraction procedure to NaI(Tl) gamma ray spectra when using the threshold technique, in spite of the lower intrinsic activities of NaI(Tl) detectors.

Safety and pharmacokinetics of repeat-dose micafungin in young infants.

Given the risk of central nervous system infection, relatively high weight-based echinocandin dosages may be required for the successful treatment of invasive candidiasis and candidemia in young infants. This open-label study assessed the safety and pharmacokinetics (PK) of micafungin in 13 young infants (>48 h and <120 days of life) with suspected candidemia or invasive candidiasis. Infants of body weight > or =1,000 and <1,000 g received 7 and 10 mg/kg/day, respectively, for a minimum of 4-5 days. In the 7-mg/kg/day group, the mean baseline weight and gestational age were 2,101 g and 30 weeks, respectively; in the 10-mg/kg/day group, they were 688 g and 25 weeks, respectively. The median pharmacokinetic values for the 7- and 10-mg/kg/day groups, respectively, were as follows: area under the concentration-time curve from 0 to 24 h (AUC(0-24)), 258.1 and 291.2 microg x h/ml; clearance at steady state adjusted for body weight, 0.45 and 0.57 ml/min/kg; maximum plasma concentration, 23.3 and 24.9 micro g/ml; and volume of distribution at steady state adjusted for body weight, 341.4 and 542.8 ml/kg. No deaths or discontinuations from treatment occurred. These data suggest that micafungin dosages of 7 and 10 mg/kg/day are well tolerated and provide exposure levels that have been shown (in animal models) to be adequate for central nervous system coverage.

A structure of pyridine nucleotides in solution.

Re-examination of the structure of pyridine coenzymes in solution by use of the 220-MHz high-frequency nuclear magnetic resonance spectrometer indicates that there is primarily one folded structure that is in rapid equilibrium with an open form. Reduced DPN(+) and reduced analogs of DPN(+) exist predominantly with the B side of the dihydropyridine ring folded against the adenine moiety. (The oxidized coenzymes appear to exist in the same folded structure.) Furthermore, the ribose protons undergo very little conformational change upon reduction of the pyridine ring; this observation strongly suggests a considerable similarity between the folded forms of the oxidized and reduced coenzymes. A model of the folded structure is presented.

Assay formats involving acridinium-ester-labeled DNA probes.

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.

Proton nuclear magnetic resonance study of the conformation and configuration of the cyclized pyridine nucleotide adducts.

We have closely examined by high-frequenty 1H nuclear magnetic resonance spectroscopy the structure of the adducts which form when various carbonyl compounds react with pyridine nucleotides at elevated pH. These studies show that the adducts of N-(2,6-dichlorobenzyl)nicotinamide-acetone, n-(2,6-dichlorobenzyl)nicotinamide-pyruvate, NMN-pyruvate, NAD-pyruvate, NAD-acetaldehyde, and NAD-oxaloacetate form with identical structural features as well as configuration. The following structural features are observed: (1) the adducts are pyridine N-4-substituted compounds; (2) a second six-membered ring forms by addition of the nicotinamide amido to the carbonyl group of the compound forming the addition complex; (3) cyclization occurs stereospecifically, indicating that the stereochemistry is predetermined by the initial attack at the N-4 position; (4) two diastereomeric forms are observed for each nucleotide adduct. Finally, the determination of configuration at all symmetric carbon atoms in these adducts will be discussed.

Antineoplastic activity of poly(L-lysine) with some ascites tumor cells.

We have found that poly(L-lysine) can be a very effective agent in preventing the growth of Ehrlich ascites tumors in mice. When given optimal doses of poly(L-lysine) (Mr 60 x 10(3)) intraperitoneally for 5 consecutive days, beginning on day 1 after inoculation with Ehrlich ascites cells. White Swiss mice show nearly a 100% remission from subsequent tumor growth. Rechallenge of "cured" animals with tumor cells, however shows no long-term immunological protection. In tissue culture, poly(L-lysine) shows a related potent cytotoxicity with HeLa cells; interestingly, the D isomer. In addition, there is a strong molecular weight dependence in that the small polylysine (Mr 3 x 10(3)) possesses less than 1/20th the cytotoxicity of large polymers (Mr 70 x 10(3)) on a weight basis in both cell culture and animal studies. At the same time, none of these lysine polymers gives any significant increase in life span to BDF1 mice infected with L1210 murine leukemia cells. We have also further explored the mechanism by which the polylysines express their cytotoxicity. These data indicate that lysine polymers show cell specificity in their action and in some cases they may be beneficial as potent antineoplastic agents, particularly when molecular weight is taken into consideration.