Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Evaluating the relative status of health and safety programs for minority academic and research institutions

A variety of occupational hazards are indigenous to academic and research institutions, ranging from traditional life safety concerns, such as fire safety and fall protection, to specialized occupational hygiene issues such as exposure to carcinogenic chemicals, radiation sources, and infectious microorganisms. Institutional health and safety programs are constantly challenged to establish and maintain adequate protective measures for this wide array of hazards. A unique subset of academic and research institutions are classified as historically Black universities which provide educational opportunities primarily to minority populations. State funded minority schools receive less resources than their non-minority counterparts, resulting in a reduced ability to provide certain programs and services. Comprehensive health and safety services for these institutions may be one of the services compromised, resulting in uncontrolled exposures to various workplace hazards. Such a result would also be contrary to the national health status objectives to improve preventive health care measures for minority populations.^ To determine if differences exist, a cross-sectional survey was performed to evaluate the relative status of health and safety programs present within minority and non-minority state-funded academic and research institutions. Data were obtained from direct mail questionnaires, supplemented by data from publicly available sources. Parameters for comparison included reported numbers of full and part-time health and safety staff, reported OSHA 200 log (or equivalent) values, and reported workers compensation experience modifiers. The relative impact of institutional minority status, institution size, and OSHA regulatory environment, was also assessed. Additional health and safety program descriptors were solicited in an attempt to develop a preliminary profile of the hazards present in this unique work setting.^ Survey forms were distributed to 24 minority and 51 non-minority institutions. A total of 72% of the questionnaires were returned, with 58% of the minority and 78% of the non-minority institutions participating. The mean number of reported full-time health and safety staff for the responding minority institutions was determined to be 1.14, compared to 3.12 for the responding non-minority institutions. Data distribution variances were stabilized using log-normal transformations, and although subsequent analysis indicated statistically significant differences, the differences were found to be predicted by institution size only, and not by minority status or OSHA regulatory environment. Similar results were noted for estimated full-time equivalent health and safety staffing levels. Significant differences were not noted between reported OSHA 200 log (or equivalent) data, and a lack of information provided on workers compensation experience modifiers prevented comparisons on insurance premium expenditures. Other health and safety program descriptive information obtained served to validate the study's presupposition that the inclusion criteria would encompass those organizations with occupational risks from all four major hazard categories. Worker medical surveillance programs appeared to exist at most institutions, but the specific tests completed were not readily identifiable.^ The results of this study serve as a preliminary description of the health and safety programs for a unique set of workplaces have not been previously investigated. Numerous opportunities for further research are noted, including efforts to quantify the relative amount of each hazard present, the further definition of the programs reported to be in place, determination of other means to measure health outcomes on campuses, and comparisons among other culturally diverse workplaces. ^

High-Resolution Ice Cores from US ITASE (West Antarctica): Development and Validation of Chronologies and Determination of Precision and Accuracy

Shallow ice cores were obtained from widely distributed sites across the West Antarctic ice sheet, as part of the United States portion of the International Trans-Antarctic Scientific Expedition (US ITASE) program. The US ITASE cores have been dated by annual-layer counting, primarily through the identification of summer peaks in non-sea-salt sulfate (nssSO(4)(2-)) concentration. Absolute dating accuracy of better than 2 years and relative dating accuracy better than 1 year is demonstrated by the identification of multiple volcanic marker horizons in each of the cores, Tambora, Indonesia (1815), being the most prominent. Independent validation is provided by the tracing of isochronal layers from site to site using high-frequency ice-penetrating radar observations, and by the timing of mid-winter warming events in stable-isotope ratios, which demonstrate significantly better than 1 year accuracy in the last 20 years. Dating precision to 1 month is demonstrated by the occurrence of summer nitrate peaks and stable-isotope ratios in phase with nssSO(4)(2-), and winter-time sea-salt peaks out of phase, with phase variation of < 1 month. Dating precision and accuracy are uniform with depth, for at least the last 100 years.

POPULATION ASSESSMENTS OF INFECTION WITH SCHISTOSOMA MANSONI: EFFECTS OF BIAS CAUSED BY THE RELATIVE SENSITIVITY OF DIAGNOSTIC TECHNIQUES (PUERTO RICO)

This study establishes the extent and relevance of bias of population estimates of prevalence, incidence, and intensity of infection with Schistosoma mansoni caused by the relative sensitivity of stool examination techniques. The population studied was Parcelas de Boqueron in Las Piedras, Puerto Rico, where the Centers for Disease Control, had undertaken a prospective community-based study of infection with S. mansoni in 1972. During each January of the succeeding years stool specimens from this population were processed according to the modified Ritchie concentration (MRC) technique. During January 1979 additional stool specimens were collected from 30 individuals selected on the basis of their mean S. mansoni egg output during previous years. Each specimen was divided into ten 1-gm aliquots and three 42-mg aliquots. The relationship of egg counts obtained with the Kato-Katz (KK) thick smear technique as a function of the mean of ten counts obtained with the MRC technique was established by means of regression analysis. Additionally, the effect of fecal sample size and egg excretion level on technique sensitivity was evaluated during a blind assessment of single stool specimen samples, using both examination methods, from 125 residents with documented S. mansoni infections. The regression equation was: Ln KK = 2.3324 + 0.6319 Ln MRC, and the coefficient of determination (r('2)) was 0.73. The regression equation was then utilized to correct the term "m" for sample size in the expression P ((GREATERTHEQ) 1 egg) = 1 - e('-ms), which estimates the probability P of finding at least one egg as a function of the mean S. mansoni egg output "m" of the population and the effective stool sample size "s" utilized by the coprological technique. This algorithm closely approximated the observed sensitivity of the KK and MRC tests when these were utilized to blindly screen a population of known parasitologic status for infection with S. mansoni. In addition, the algorithm was utilized to adjust the apparent prevalence of infection for the degree of functional sensitivity exhibited by the diagnostic test. This permitted the estimation of true prevalence of infection and, hence, a means for correcting estimates of incidence of infection. ^

Stable oxygen isotope record of Globigerinoides sacculifer and age determination of sediment cores from teh Somali Basin

This study documents the biological signatures impressed upon the sedimentary record underlying both the 5°N upwelling system of the Somali Current and the equatorial area of the Somali Basin out of the upwelling influence. The evolution of these two distinct hydrographic systems is compared for the last 160 kyr. Correspondence and cluster analyses are performed on combined radiolarian and planktonic foraminiferal quantitative data in order to study the changes of the planktonic assemblages through time and space. The Upwelling Radiolarian Index (URI) is used as a productivity proxy. The water temperature and hydrographic structure of the upper water masses appear to be the major factors controlling the distribution patterns of the fauna. The relative abundances of three groups of foraminifera, cold water form (dextral N. pachyderma), mixed layer dwellers (G. trilobus, G. ruber, G. sacculifer, G. conglobatus, and G. glutinata), and thermocline dwellers (G. menardii, G. tumida, N. dutertrei, G. crassaformis, and P. obliquiloculata), follow distinct evolutionary patterns at the two sites during the last 160 kyr. At the equatorial site (core MD 85668), downcore fluctuations in the relative abundances of the three groups are closely related to the glacial/interglacial cyclicity and provide some insights into the interpretation of hydrographic changes. The dominance of the mixed layer foraminifera at the transition intervals between isotope stages 6/5 and 2/1, combined with weak URI values, is thought to reflect the reorganization of the oceanographic circulation. These short-term events (with a duration of < 5000 year) could be related to the rapid inflow of oxygen-depleted water through the Indonesian straits as a result of sea level rise during deglaciation. Underneath the 5°N gyre (core MD 85674), the response to global climatic changes is overprinted by the regional effect of the Somalian upwelling, which has been persistent over the last 160 kyr.

Mineralogy and age determination of sediment core D11957P

The Tore Seamount is a circular, volcano-like feature 100 km in diameter with its summit at 2200 m water depth and a small, 5000 m deep basin in its interior. It is situated approximately 300 km west of Lisbon and is surrounded by deep abyssal plains. This site with a standard pelagic stratigraphy is the southernmost point where the so-called Heinrich events have so far been recorded. A succession of alternating interglacial/glacial periods reveals a stratigraphic record back to the beginning of isotopic stage 7 (225 kyr). Climatic changes are identifiable by coherent variations in colour, carbonate content and distribution of ice-rafted detritus in the carbonate-free fraction. Inputs of ice-rafted quartz are well defined. Characteristics in common with other sites showing Heinrich layers include a high terrigenous to biogenic ratio, a dramatic decrease in the accumulation rate of foraminifera shells, an increase in dolomite abundance and the occurrence of polar foraminiferal species indicating southwards penetration of cold waters which lead us to consider a wider southeastern extent of the North Atlantic ice-rafted detritus belt than hitherto. If the presently accepted position of the Polar Front is maintained, icebergs must have been swept southwards from the southern boundary of the pack ice in a current merging into the ancestral Canary Current, bringing ice-rafted material to the Tore Seamount. The coincidence of reddish-feldspar, probably derived from the northern Appalachian Triassic red facies, with the transparent quartz suggests at least a partial Labrador source for all the Heinrich layers here, including HL 3. In comparison to other sites in the entire North Atlantic, two exceptions stand out: the absence of HL 5 and the low detritus to biogenics ratio for HL 3. The simultaneous occurrence of these two types of ice-rafted minerals is a new piece in the puzzle of the origin of Heinrich layers.

Determination of sulfite and manganese in sediments from the K/T boundary

The solid-state-physics technique of electron spin resonance (ESR) has been employed in an exploratory study of marine limestones and impact-related deposits from Cretaceous-Tertiary (KT) boundary sites including Spain (Sopelana and Caravaca), New Jersey (Bass River), the U.S. Atlantic continental margin (Blake Nose, ODP Leg 171B/1049/A), and several locations in Belize and southern Mexico within -600 km of the Chicxulub crater. The ESR spectra of SO3(1-) (a radiation-induced point defect involving a sulfite ion substitutional for CO3(2-) which has trapped a positive charge) and Mn(2+) in calcite were singled out for analysis because they are unambiguously interpretable and relatively easy to record. ESR signal strengths of calcite-related SO3(1-) and Mn(2+) have been studied as functions of stratigraphic position in whole-rock samples across the KT boundary at Sopelana, Caravaca, and Blake Nose. At all three of these sites, anomalies in SO3(1-) and/or Mn(2+) intensities are noted at the KT boundary relative to the corresponding background levels in the rocks above and below. At Caravaca, the SO3(1-) background itself is found to be lower by a factor of 2.7 in the first 30,000 years of the Tertiary relative to its steady-state value in the last 15,000 years of the Cretaceous, indicating either an abrupt and quasi-permanent change in ocean chemistry (or temperature) or extinction of the marine biota primarily responsible for fixing sulfite in the late Cretaceous limestones. An exponential decrease in the Mn(2+) concentration per unit mass calcite, [Mn(2+)], as the KT boundary at Caravaca is approached from below (1/e characteristic length =1.4 cm) is interpreted as a result of post-impact leaching of the seafloor. Absolute ESR quantitative analyses of proximal impact deposits from Belize and southern Mexico group naturally into three distinct fields in a twodimensional [SO3(1-)]-versus-[Mn(2+)] scatter plot. These fields contain (I) limestone ejecta clasts, (II) accretionary lapilli, and (III) a variety of SO3(1-) -depleted/Mn(2+) enriched impact deposits. Data for the investigated non-impact-related Cretaceous and Tertiary marine limestones (Spain and Blake Nose) fall outside of these three fields. With reference to thes enon-impact deposits, fields I, II, and III can be respectively characterized as Mn(2+) -depleted, SO3(1-) -enhanced, and SO3(1-) -depleted. It is proposed that (1) field I represents calcites from the Yucatin Platform, and that the Mn(2+) -depleted signature can be used as an indicator of primary Chicxulub ejecta in deep marine environments and (2) field II represents calcites that include a component formed in the vapor plume, either from condensation in the presence of CO2/SO3(1-) -rich vapors, or reactions between CaO and CO2/SO3 rich vapors, and that this SO3(1-) -enhanced signature can be used as an indicator of impact vapor plume deposits. Given these two propositions, the ESR data for the Blake Nose deposits are ascribed to the presence of basal coarse calcitic Chicxulub ejecta clasts, while the finer components that are increasingly represented toward the top are interpreted to contain high- SO3(1-) calcite from the vapor plume. The apparently-undisturbed Bass River deposit may contain even higher concentrations of vapor-plume calcite. None of the three components included in field III appear to be represented at distal, deep marine KT-boundary sites; this field may include several types of impact-related deposits of diverse origins and diagenetic histories.

Age determination and grwoth rates of stalagmites and stalactites from the Okshola cave, Fauske, northern Norway

The sensitivity of terrestrial environments to past changes in heat transport is expected to be manifested in Holocene climate proxy records on millennial to seasonal timescales. Stalagmite formation in the Okshola cave near Fauske (northern Norway) began at about 10.4 ka, soon after the valley was deglaciated. Past monitoring of the cave and surface has revealed stable modern conditions with uniform drip rates, relative humidity and temperature. Stable isotope records from two stalagmites provide time-series spanning from c. 10380 yr to AD 1997; a banded, multi-coloured stalagmite (Oks82) was formed between 10380 yr and 5050 yr, whereas a pristine, white stalagmite (FM3) covers the period from ~7500 yr to the present. The stable oxygen isotope (delta18Oc), stable carbon isotope (delta13Cc), and growth rate records are interpreted as showing i) a negative correlation between cave/surface temperature and delta18Oc, ii) a positive correlation between wetness and delta13Cc, and iii) a positive correlation between temperature and growth rate. Following this, the data from Okshola show that the Holocene was characterised by high-variability climate in the early part, low-variability climate in the middle part, and high-variability climate and shifts between two distinct modes in the late part. A total of nine Scandinavian stalagmite delta18Oc records of comparable dating precision are now available for parts or most of the Holocene. None of them show a clear Holocene thermal optimum, suggesting that they are influenced by annual mean temperature (cave temperature) rather than seasonal temperature. For the last 1000 years, delta18Oc values display a depletion-enrichment-depletion pattern commonly interpreted as reflecting the conventional view on climate development for the last millennium. Although the delta18Oc records show similar patterns and amplitudes of change, the main challenges for utilising high-latitude stalagmites as palaeoclimate archives are i) the accuracy of the age models, ii) the ambiguity of the proxy signals, and iii) calibration with monitoring data.

Determination of polybrominated diphenyl ethers in water samples from Mendoza River Basin by HS-SPME-GC-MS/MS

Polybrominated diphenyl ethers (PBDEs) are considered persistent organic pollutants because of their ubiquity, persistence and bioaccumulation. Its harmful effects on human health and the environment, has led to its inclusion of the Stockholm Convention. Little information is found about PBDEs in abiotic systems of the South America in open literature. This paper reports the presence and concentration level of four PBDEs congeners in Mendoza River, Argentina. The selected PBDEs were: 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE- 100) and 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153). The analytical methodology used was head space-solid phase micro extraction combined with gas chromatographymass spectrometry (HS-SPME-GC-MS/MS). Several variables, including pH, salting out, extraction technique type and extraction time were studied and optimized over the relative response the target analytes. The precision of HS-SPME-GC-MS/MS evaluated over five replicate, leading RSDs values <13%, detection limits (S/N=3) ranging from 0.03 pg ml-1 to 0.12 pg ml-1 and the calibration graph was linear with r2=0.9959. BDE-47 and BDE-100 were the predominant congeners found in the analyzed samples. Their concentrations ranged from not detected to 1.9 pg ml-1 and to 0.5 pg ml-1, respectively.

Age determination, foraminiferal abundance and stable isotope ratios of sediment core CH82-20

A core from the Mid-Atlantic Ridge at 43.5°N and ~3 km water depth shows distinct evidence of the deglacial events known as Heinrich event 1 (probably the marine equivalent of Oldest Dryas cooling in Europe) and the Younger Dryas. The Heinrich event, dated at three levels to between 14.3 and 15.0 ka, is marked by a minimum in foraminifera per gram, by maxima in rates of sedimentation, ice rafted debris per gram, and relative abundance of N. pachyderma (s.), and by a delta18O minimum in planktonic foraminifera. The Younger Dryas event is marked by peak abundance of N. pachyderma (s.) and a planktonic delta18O maximum. Benthic foraminiferal delta13C reaches minimum values during both the Heinrich event and the Younger Dryas. Our data indicate pronounced changes in surface water properties were coupled with reduced production of North Atlantic Deep Water at each of these times.

Age determination and n-Alkane concentration of Arctic Ocean sediment cores

We have reconstructed the surface water environment of the Arctic Ocean over the last ? 50,000 years using measurements of the organic nitrogen and carbon isotope ratios, carbonate and total organic carbon concentrations (TOC), and terrestrial biomarkers (lignin and long-chain n-alkanes) in four multicores. Variations in nitrogen isotope ratios that are concordant with TOC and carbonate concentrations (representing foraminifera and excluding ice-rafted-debris) reflect differences in relative nutrient utilization of phytoplankton in the surface waters. However, d15N variations also appear to be dependent on the stratification of the water column and therefore potentially track the exchange of nutrients between deep and surface waters. Low Last Glacial Maximum (LGM) d15N values and higher Holocene values are opposite to those recorded in the Southern Ocean. The Arctic Ocean with higher nutrient utilization today compared to the LGM therefore acts as a counterpart to the Southern Ocean, although the global impact on carbon dioxide variations compared to the Southern Ocean is probably low.

(Table 4) Mesozoolpankton abundance and biomass and relative percentage of key zooplankton groups along the Bulgarian coast, Black Sea

Results of studies during Project of an international expedition onboard R/V Vladimir Parshin in September-October 2005 are presented. Intensive development of Bacillariophyceae and Dynophyceae was recorded in coastal waters of Bulgaria, Turkey, and in the Danube River delta during period of investigations. Increase in algae population was accompanied by rising of chlorophyll a concentration up to 2.0-5.5 µg/l. In the deep water region it did not exceed 0.5 µg/l. Phytoplankton growth rate in the surface water layer varied from 0.1 to 1.0 1/day. This parameter and NO2+NO3 concentration, as well as the silicon concentration were correlative, as was described by the Michaelis-Menten equation. Phytoplankton growth was affected by basic nutrients. Zooplankton grazing varied from 0.10 to 0.69 1/day and average values in different regions varied by 1.5 times. Microalgae size range is one of major factors of grazing regulation. Rate of phytoplankton consumption was decreasing with increasing the largest diatom Pseudosolenia calcar-avis impact on total biomass of nano- and microphytoplankton.

Age determination and paleomagnetic investigations on a sediment profile from laguna Potrok Aike

Paleomagnetic inclination, declination and relative paleointensity were reconstructed from the sediments of Laguna Potrok Aike in the framework of the International Continental scientific Drilling Program (ICDP) Potrok Aike maar lake Sediment Archive Drilling prOject (PASADO). Here we present the u-channel-based full vector paleomagnetic field reconstruction since 51.2 ka cal BP. The relative paleointensity proxy (RPI) was built by normalising the natural remanent magnetisation with the anhysteretic remanent magnetisation using the average ratio at 4 demagnetisation steps part of the ChRM interval (NRM/ARM10e40 mT). A grain size influence on the RPI was removed using a correction based on the linear relationship between the RPI and the median destructive field of the natural remanent magnetisation (MDFNRM). The new record is compared with other lacustrine and marine records and stacks from the mid- to high-latitudes of the Southern Hemisphere, revealing consistent millennial-scale variability, the identification of the Laschamp and possibly the Mono Lake geomagnetic excursions, and a direction swing possibly associated to the Hilina Pali excursion at 20 ka cal BP. Nonetheless, a global-scale comparison with other high-resolution records located on the opposite side of the Earth and with various dipole field references hint at a different behaviour of the geomagnetic field around southern South America at 46 ka cal BP.

Age determination of granite from Himalaya

The Gangdese belt, Tibet, records the opening and closure of the Neo-Tethyan ocean and the resultant collision between the Indian and Eurasian plates. Mesozoic magmatic rocks generated through subduction of the Tethyan oceanic slab constitute the main component of the Gangdese belt, and play a crucial role in understanding the formation and evolution of the Neo-Tethyan tectonic realm. U-Pb and Lu-Hf isotopic data for tonalite and granodiorite from the Xietongmen-Nymo segment of the Gangdese belt indicate a significant pulse of Jurassic magmatism from 184 Ma to 168 Ma. The magmatic rocks belong to metaluminous medium-K calc-alkaline series, characterized by regular variation in major element compositions with SiO2 of 61.35%-73.59 wt%, low to moderate MgO (0.31%-2.59%) with Mg# of 37-45. These magmatic rocks are also characterized by LREE enrichment with concave upward trend in MREE on the chondrite-normalized REE patterns, and also LILE enrichment and depletion in Nb, Ta and Ti in the primitive mantle normalized spidergrams. These rocks have high zircon ?Hf(t) values of + 10.94 to + 15.91 and young two-stage depleted mantle model ages (TDM2) of 192 Ma to 670 Ma. The low MgO contents and relatively depleted Hf isotope compositions, suggest that the granitoid rocks were derived from the partial melting of the juvenile basaltic lower crust with minor mantle materials injected. In combined with the published data, it is suggested that northward subduction of the Neo-Tethyan slab beneath the Lhasa terrane began by the Late-Triassic, which formed a major belt of arc-related magmatism.

Age determination of Southeast Atlantic sediment cores

Angola Basin and Cape Basin (southeast Atlantic) surface sediments and sediment cores show that maxima in the abundance of taraxerol (relative to other land-derived lipids) covary with maxima in the relative abundance of pollen from the mangrove tree genus Rhizophora and that in the surface sediments offshore maxima in the relative abundance of taraxerol occur at latitudes with abundant coastal mangrove forests. Together with the observation that Rhizophora mangle and Rhizophora racemosa leaves are extraordinarily rich in taraxerol, this strongly indicates that taraxerol can be used as a lipid biomarker for mangrove input to the SE Atlantic. The proxy-environment relations for taraxerol and Rhizophora pollen down-core show that increased taraxerol and Rhizophora pollen abundances occur during transgressions and periods with a humid climate. These environmental changes modify the coastal erosion and sedimentation patterns, enhancing the extent of the mangrove ecosystem and/or the transport of mangrove organic matter offshore. Analyses of mid-Pleistocene sediments show that interruption of the pattern of taraxerol maxima during precession minima occurs almost only during periods of low obliquity. This demonstrates the complex environmental response of the interaction between precession-related humidity cycles and obliquity-related sea-level changes on mangrove input.