A protocol for using attenuated total reflection Fourier-transform infrared spectroscopy for pre-screening ancient bone collagen prior to radiocarbon dating.

RATIONALE: Pre-screening of bone collagen quality is important to reduce the cost for analyses such as radiocarbon (14 C) dating with accelerator mass spectrometry in archaeological studies. We developed a pre-screening protocol based on attenuated total reflection (ATR) Fourier-transform infrared spectroscopy (FTIR) for assessing the chemical composition and mineralogy of ancient bone samples. METHODS: ATR-FTIR measurements were performed on bulk bones of diverse origin and age before collagen extraction. The percentage nitrogen of bulk bones, as well as the weight percentage, and the percentage carbon and nitrogen of extracted organic matter were noted. Several machine learning algorithms were applied to the spectral data and compared for their efficacy in screening for well preserved collagen. RESULTS: The results showed that (a) the first derivative of the spectral data was better suited to screening than the raw FTIR data, especially for a wider spectral range and (b) certain classification algorithms [e.g. gradient boosting machine (GBM)] were able to efficiently predict the degree of preservation in bone samples. CONCLUSIONS: This pre-screening protocol can serve as a fast, concise and inexpensive pre-screening tool for determining relative degrees of preservation before collagen extraction and subsequent 14 C dating. The screening power based on the machine learning techniques can be further improved by accumulating the FTIR spectral data of bones.

Improved Method for Isolation and Purification of Underivatized Amino Acids for Radiocarbon Analysis.

We have improved a method for isolation and purification of individual amino acids for compound-specific radiocarbon analysis (CSRA). To remove high-performance liquid chromatography (HPLC) eluent blanks from isolated amino acid fractions prior to the radiocarbon (Δ14C) measurement, each fraction was filtered through a membrane filter and then washed with diethyl ether twice. Radiocarbon measurements on standard amino acids processed and purified with the above method using elemental analyzer-accelerator mass spectrometry resulted in Δ14C values that were in strong agreement ( R2 = 0.998) with the original Δ14C value of each amino acid standard. From these measurements, we calculate dead and modern carbon contamination contributions as 1.2 ± 0.2 and 0.3 ± 0.1 µgC, respectively, which are consistent with direct assessments of HPLC procedural blanks of 1.0 ± 0.8 µgC per sample. These contamination constraints allow correction of measured Δ14C values for accurate and precise CSRA and are widely applicable to future archeological and biogeochemical studies.

Widespread collapse of the Ross Ice Shelf during the late Holocene.

The stability of modern ice shelves is threatened by atmospheric and oceanic warming. The geologic record of formerly glaciated continental shelves provides a window into the past of how ice shelves responded to a warming climate. Fields of deep (-560 m), linear iceberg furrows on the outer, western Ross Sea continental shelf record an early post-Last Glacial Maximum episode of ice-shelf collapse that was followed by continuous retreat of the grounding line for ∼200 km. Runaway grounding line conditions culminated once the ice became pinned on shallow banks in the western Ross Sea. This early episode of ice-shelf collapse is not observed in the eastern Ross Sea, where more episodic grounding line retreat took place. More widespread (∼280,000 km(2)) retreat of the ancestral Ross Ice Shelf occurred during the late Holocene. This event is recorded in sediment cores by a shift from terrigenous glacimarine mud to diatomaceous open-marine sediment as well as an increase in radiogenic beryllium ((10)Be) concentrations. The timing of ice-shelf breakup is constrained by compound specific radiocarbon ages, the first application of this technique systematically applied to Antarctic marine sediments. Breakup initiated around 5 ka, with the ice shelf reaching its current configuration ∼1.5 ka. In the eastern Ross Sea, the ice shelf retreated up to 100 km in about a thousand years. Three-dimensional thermodynamic ice-shelf/ocean modeling results and comparison with ice-core records indicate that ice-shelf breakup resulted from combined atmospheric warming and warm ocean currents impinging onto the continental shelf.

Exposure age and ice-sheet model constraints on Pliocene East Antarctic ice sheet dynamics.

The Late Pliocene epoch is a potential analogue for future climate in a warming world. Here we reconstruct Plio-Pleistocene East Antarctic Ice Sheet (EAIS) variability using cosmogenic nuclide exposure ages and model simulations to better understand ice sheet behaviour under such warm conditions. New and previously published exposure ages indicate interior-thickening during the Pliocene. An ice sheet model with mid-Pliocene boundary conditions also results in interior thickening and suggests that both the Wilkes Subglacial and Aurora Basins largely melted, offsetting increased ice volume. Considering contributions from West Antarctica and Greenland, this is consistent with the most recent IPCC AR5 estimate, which indicates that the Pliocene sea level likely did not exceed +20 m on Milankovitch timescales. The inception of colder climate since ∼3 Myr has increased the sea ice cover and inhibited active moisture transport to Antarctica, resulting in reduced ice sheet thickness, at least in coastal areas.

Reorganization of Southern Ocean plankton ecosystem at the onset of Antarctic glaciation.

The circum-Antarctic Southern Ocean is an important region for global marine food webs and carbon cycling because of sea-ice formation and its unique plankton ecosystem. However, the mechanisms underlying the installation of this distinct ecosystem and the geological timing of its development remain unknown. Here, we show, on the basis of fossil marine dinoflagellate cyst records, that a major restructuring of the Southern Ocean plankton ecosystem occurred abruptly and concomitant with the first major Antarctic glaciation in the earliest Oligocene (~33.6 million years ago). This turnover marks a regime shift in zooplankton-phytoplankton interactions and community structure, which indicates the appearance of eutrophic and seasonally productive environments on the Antarctic margin. We conclude that earliest Oligocene cooling, ice-sheet expansion, and subsequent sea-ice formation were important drivers of biotic evolution in the Southern Ocean.

Relationship between uterine morphology and peripheral concentrations of sex steroid hormone in wild Japanese black bears (Ursus thibetanus japonicus).

Developing a better understanding of the reproductive physiology and breeding condition peculiar to wild Japanese black bears (Ursus thibetanus japonicus) is crucial for estimation of their habitat distribution. The aim of this study was to clarify the changes in morphology of the genital organs, cellular proliferation in the endometrium and sex steroid hormone concentrations along with the reproductive cycle in Japanese black bears. Samples were collected from a total of 24 female Japanese black bears (1-15 presumptive years old) that were caught in the wild in Iwate prefecture during the period between August 1999 and September 2005. Twenty-two out of the 24 animals were hunted from May to October. The ovaries from the 24 animals and the uteri from 23 animals were observed macroscopically and histologically to examine the relationship between morphology of the genital organs and the month of the year the animal was caught. The staining pattern of proliferating cell nuclear antigen (PCNA) in the endometrium was characterised. Peripheral concentrations of oestradiol-17beta and progesterone were determined by radioimmunoassay. All the animals that had a corpus luteum (n=12) were captured from August to October. The thickness of the endometrium in the animals captured from August to October (n=16) was significantly greater than those in animals captured from May to July (n=5) (P<0.05). From August to October, the thickness of the endometrium and the ratio of the area of the uterine glands to the area of the endometrium in the animals with a corpus luteum (n=12) were significantly greater than those without a corpus luteum (n=4) (P<0.01). Positive PCNA staining was only observed in the uteri of two animals captured in May. There was a significantly positive correlation between plasma progesterone concentrations and the thickness of the endometrium (rho=0.589, P<0.05). There were also significantly positive correlations between the progesterone to oestradiol-17beta ratio (P4/E2 ratio) and the thickness of the endometrium (rho=0.710, P<0.05), and between the P4/E2 ratio and the ratio of the uterine gland area in the endometrium (rho=0.626, P<0.01). These data suggest that the corpus luteum is formed during or just after the breeding season and that the cells in the endometrium and the uterine glands, which proliferate in the early breeding season, grow and develop under the influence of progesterone and oestradiol-17beta during the period of delayed implantation in Japanese black bears.

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress.

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.

N-cadherin signals through Rac1 determine the localization of connexin 43 in cardiac myocytes.

It has been proposed that the formation of gap junction is influenced by adherens junction in cardiac myocytes. To examine whether signals through N-cadherin are involved in the distribution of connexin 43 (Cx43), the distribution of cell-cell adhesion molecules, N-cadherin and Cx43, was analyzed in aligned cardiac myocytes. To induce cell orientation running in parallel to tension direction, neonatal rat cardiac myocytes were plated for 3 hours and exposed to 20% cyclic stretch for 24 hours on silicone dishes. The aligned cells cultured for 0-5 days were immunostained with anti- N-cadherin or anti-Cx43 antibody. After cultivation for 3-5 days, following the accumulation of N-cadherin, Cx43 was localized at the longitudinal cell termini. Adenoviral gene transfer of dominant negative N-cadherin significantly attenuated the localization of Cx43 at the longitudinal cell termini, suggesting that Cx43 localization is regulated downstream of N-cadherin. In the process of Cx43 localization, Rho family proteins, RhoA and Rac1, were activated, but not Cdc42. RhoA and Rac1 activation was inhibited by the transfection of dominant negative N-cadherin, indicating that RhoA and Rac1 were activated by N-cadherin in the oriented cardiac myocytes. The inhibition of Rho family proteins by Rho GDI significantly attenuated the accumulation of Cx43, but not that of N-cadherin. Furthermore, the translocation of Cx43 to longitudinal cell termini was prevented by the inhibition of Rac1, but not RhoA. Collectively, these findings suggest that the localization of Cx43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes.

Platelet activating factor induces cytoskeletal reorganization through Rho family pathway in THP-1 macrophages.

In the process of atherosclerosis, platelet activating factor (PAF) promotes the infiltration of inflammatory cells into atherosclerotic plaque by modulating their cytoskeleton. Here, we examined whether Rho family proteins are involved in PAF-induced cytoskeletal reorganization in THP-1 macrophages. PAF stimulation rapidly induced cell elongation, accompanied by filopodia formation. The inhibition of Rho family proteins by the overexpression of Rho-GDI attenuated the PAF-mediated morphological changes. Both RhoA and Cdc42 were activated in response to PAF. Inhibition of RhoA or Cdc42 by dominant negative mutants abrogated morphological changes induced by PAF. Collectively, PAF regulates cytoarchitecture through Rho family proteins in macrophages.