Chemical and palaeoentomological evidence of a relationship between early Eocene Belgian and Oise (France) ambers.

Of the early Eocene amber deposits known across the world, Belgian amber has been mostly absent from the relevant literature. We reinvestigated amber held in the palaeobotanical collection of the Royal Belgian Institute of Natural Sciences, Brussels, which derived from three localities in Belgium that originated from two geographical areas (Leval-Trahegnies and Orp-le-Grand). Using Fourier transform infrared (FTIR) spectroscopy we show the close chemical relationship of Belgian amber to the early Eocene Oise amber from the Paris Basin, and highlight the potential effect of weathering on the amber chemistry. The amber derives from a very similar botanical source as the Oise amber (Combretaceae or Leguminosae-Caesalpinioideae), but from different coeval basins. The two Leval-Trahegnies localities provided amber that exhibit different stages of weathering (heavily fissured and crazed, darkened) and lacking any inclusions. The Orp-le-Grand locality provided the least weathered amber, with one amber piece containing two inclusions: a mite and a new genus and species of hemipteran (Cativolcus uebruum gen. et sp. nov.), and a second one that preserved the impression of insect wings pressed into the surface.

Mimes of the past: Eocene midges of the tribe Pseudochironomini (Chironomidae, Diptera) reveal their peculiarities.

This is the first study focused on Eocene dipterans of the tribe Pseudochironomini (subfamily Chironominae, family Chironomidae), based on unique materials from Baltic amber. Two new genera and three new species: Eomicromimus gen. nov. with Eomicromimus polliciformis sp. nov. and Eomicromimus serpens sp. nov., and Eoriethia gen. nov. with Eoriethia ursipes sp. nov. are presented. The systematic position of the new taxa is discussed, and an amended key to the identification of adult males of extinct and extant Pseudochironomini genera is provided. The presented analysis of the morphology of the tribe's fossil members allowed us to verify the concepts regarding the origin/homology of male diagnostic structures crucial in defining new taxa, their phylogeny, and to consolidate the terminology used in chironomid research. A new habitual name for Chironomidae, "mime midges", is also proposed.

Morphological and organic spectroscopic studies of a 44-million-year-old leaf beetle (Coleoptera: Chrysomelidae) in amber with endogenous remains of chitin.

This study details the quality of preservation of amber deposits in the Eocene. Through Baltic amber crack-out studies using Synchrotron Micro-Computed Tomography and Scanning Electron Microscopy it was found that the cuticle of a specimen of leaf beetle (Crepidodera tertiotertiaria (Alticini: Galerucinae: Chrysomelidae)) is exceptionally well preserved. Spectroscopic analysis using Synchrotron Fourier Transform Infrared Spectroscopy suggests presence of degraded [Formula: see text]-chitin in multiple areas of the cuticle, and Energy Dispersive Spectroscopy supports the presence of organic preservation. This remarkable preservation is likely the result of several factors such as the favourable antimicrobial and physical shielding properties of Baltic amber as compared to other depositional media, coupled to rapid dehydration of the beetle early in its taphonomic process. We provide evidence that crack-out studies of amber inclusions, although inherently destructive of fossils, are an underutilised method for probing exceptional preservation in deep time.

Sulfurized diterpenoids in amber as diagenetic indicators of sulfate-reducing processes in past depositional environments.

Two novel compounds isolated from an amber sample from the Santonian of Piolenc (Vaucluse, SE France) were identified using nuclear magnetic resonance and high-resolution mass spectrometry as sulfurized analogues of diterpenic acids from the isopimaric series originating from ancient conifers possibly related to the Cupressaceae family. These two compounds are members of a diterpenoid series corresponding to early diagenetic transformation products of resin diterpenoids. They were likely formed once plant resin comes into contact with reduced sulfur species originating from bacterial sulfate reduction occurring in anaerobic settings such as mangroves or marshes. They represent the first evidence of sulfurization processes affecting plant resin prior to diagenetic transformation into amber. Given their mode of formation, these compounds may be used as indicators of sulfate-reducing processes in past depositional environments.

Identification of treated Baltic amber by FTIR and FT-Raman - A feasibility study.

Heat-treated succinite modified under air, oxygen or inert gas atmosphere to improve its colour and clarity, was studied with Fourier-Transform Infrared and Raman spectroscopy, and referenced to variously altered untreated samples. For treated amber, the macroscopic observations revealed diagnostic features such as (1) the presence of discoidal " sun sparkles" inside the crumbs; (2) unusual colours (e.g. dark red) for transparent specimens; (3) mottled colour patches in "beeswax" and "dragon's blood" amber; (4) luminescence quenching; and (5) significant reduction of pine-tree resinous smell. On FTIR spectra the heat treatment of succinite is mainly marked by: (1) the intensity decrease of 2932 cm-1 band (>CH2 and -CH3), followed by the intensity increase of line at 1732 cm-1; (2) the shifts of the band from ν(C = O) towards lower wavenumbers (from 1735 cm-1 to 1714 cm-1); (3) the decrease of intensity of the band at 888 cm-1 ((=CH2 or -CH = CH-); (4) some slight distortion of "Baltic shoulder" in the region 1100-1300 cm-1. The Principal Component Analysis applied to FTIR spectra enabled to distinguish not only untreated amber from treated one, but also recognize the modifications made under oxygen conditions from the ones in an inert gas. On RS spectra of the treated Baltic amber, the heat modifications are indicated by: (1) slight value increase of the relative intensity ratio I1650/1445 compared to untreated succinite; (2) the shift of bands near 1650 (C = C) and 1445 cm-1 (CH2, CH3) towards higher wavenumbers.

Non-destructive comparative evaluation of fossil amber using terahertz time-domain spectroscopy.

Fossilized plant resins, or ambers, offer a unique paleontological window into the history of life. A natural polymer, amber can preserve aspects of ancient environments, including whole organisms, for tens or even hundreds of millions of years. While most amber research involves imaging with visual light, other spectra are increasingly used to characterize both organismal inclusions as well as amber matrix. Terahertz (THz) radiation, which occupies the electromagnetic band between microwave and infrared light wavelengths, is non-ionizing and frequently used in polymer spectroscopy. Here, we evaluate the utility of amber terahertz spectroscopy in a comparative setting for the first time by analyzing the terahertz optical properties of samples from 10 fossil deposits ranging in age from the Miocene to the Early Cretaceous. We recover no clear relationships between amber age or botanical source and terahertz permittivity; however, we do find apparent deposit-specific permittivity among transparent amber samples. By comparing the suitability of multiple permittivity models across sample data we find that models with a distribution of dielectric relaxation times best describe the spectral permittivity of amber. We also demonstrate a process for imaging amber inclusions using terahertz transmission and find that terahertz spectroscopy can be used to identify some synthetic amber forgeries.

Widespread mineralization of soft-bodied insects in Cretaceous amber.

Fossilized tree resin, or amber, commonly contains fossils of animals, plants and microorganisms. These inclusions have generally been interpreted as hollow moulds or mummified remains coated or filled with carbonaceous material. Here, we provide the first report of calcified and silicified insects in amber from the mid-Cretaceous Kachin (Burmese) amber. Data from light microscopy, scanning electron microscopy (SEM), energy-dispersive and wavelength-dispersive X-ray spectroscopy (EDX and WDX), X-ray micro-computed tomography (Micro-CT) and Raman spectroscopy show that these Kachin fossils owe their preservation to multiple diagenetic mineralization processes. The labile tissues (e.g. eyes, wings and trachea) mainly consist of calcite, chalcedony and quartz with minor amounts of carbonaceous material, pyrite, iron oxide and phyllosilicate minerals. Calcite, quartz and chalcedony also occur in cracks as void-filling cements, indicating that the minerals formed from chemical species that entered the fossil inclusions through cracks in the resin. The results demonstrate that resin and amber are not always closed systems. Fluids (e.g. sediment pore water, diagenetic fluid and ground water) at different burial stages have chances to interact with amber throughout its geological history and affect the preservational quality and morphological fidelity of its fossil inclusions.

Amber Extract Reduces Lipid Content in Mature 3T3-L1 Adipocytes by Activating the Lipolysis Pathway.

Amber-the fossilized resin of trees-is rich in terpenoids and rosin acids. The physiological effects, such as antipyretic, sedative, and anti-inflammatory, were used in traditional medicine. This study aims to clarify the physiological effects of amber extract on lipid metabolism in mouse 3T3-L1 cells. Mature adipocytes are used to evaluate the effect of amber extract on lipolysis by measuring the triglyceride content, glucose uptake, glycerol release, and lipolysis-related gene expression. Our results show that the amount of triacylglycerol, which is stored in lipid droplets in mature adipocytes, decreases following 96 h of treatment with different concentrations of amber extract. Amber extract treatment also decreases glucose uptake and increases the release of glycerol from the cells. Moreover, amber extract increases the expression of lipolysis-related genes encoding perilipin and hormone-sensitive lipase (HSL) and promotes the activity of HSL (by increasing HSL phosphorylation). Amber extract treatment also regulates the expression of other adipocytokines in mature adipocytes, such as adiponectin and leptin. Overall, our results indicate that amber extract increases the expression of lipolysis-related genes to induce lipolysis in 3T3-L1 cells, highlighting its potential for treating various obesity-related diseases.

Anti-inflammatory activities of amber extract in lipopolysaccharide-induced RAW 264.7 macrophages.

Amber is a type of fossil tree resin with several bioactive properties and has been traced in traditional medicines used in Russia and China. However, its anti-inflammatory activities are poorly characterized. Here, the anti-inflammatory effects of the extract of amber mined from Kaliningrad, Russia was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The effect of the amber extract on cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Further, its effects on the production of intracellular reactive oxygen species (ROS), NO, and inflammatory cytokines were assessed by 2',7'-dichlorodihydrofluorescein diacetate staining, Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. Western blotting and real-time reverse transcription-polymerase chain reaction analysis were performed to assess the mRNA and protein expression levels of the inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The translocation of the nuclear factor-kappa B (NF-κB) p65 subunit was observed by immunofluorescent staining. Amber extract negatively regulated the LPS-induced differentiation of RAW 264.7 cells to dendritic-like cells and reduced the LPS-induced increase in ROS and NO levels. It also reduced the level of mRNA and protein expressions of TNF-α, IL-6, COX-2, and iNOS in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, amber extract suppressed the nuclear translocation of the NF-κB p65 subunit. These findings suggest that the potent anti-inflammatory effect of the amber extract is mediated by the inhibition of the NF-κB p65 signaling pathway. Collectively, this study renders amber extract as a potential pharmacological alternative to treat inflammation-related diseases.

Successful extraction of insect DNA from recent copal inclusions: limits and perspectives.

Insects entombed in copal, the sub-fossilized resin precursor of amber, represent a potential source of genetic data for extinct and extant, but endangered or elusive, species. Despite several studies demonstrated that it is not possible to recover endogenous DNA from insect inclusions, the preservation of biomolecules in fossilized resins samples is still under debate. In this study, we tested the possibility of obtaining endogenous ancient DNA (aDNA) molecules from insects preserved in copal, applying experimental protocols specifically designed for aDNA recovery. We were able to extract endogenous DNA molecules from one of the two samples analyzed, and to identify the taxonomic status of the specimen. Even if the sample was found well protected from external contaminants, the recovered DNA was low concentrated and extremely degraded, compared to the sample age. We conclude that it is possible to obtain genomic data from resin-entombed organisms, although we discourage aDNA analysis because of the destructive method of extraction protocols and the non-reproducibility of the results.

Recent developments in using the molecular decay dating method: a review.

The dating of organic findings is a fundamental task for many scientific fields. Radiocarbon dating is currently the most commonly used method. For wood, dendrochronology is another state-of-the-art method. Both methods suffer from systematic restrictions, leading to samples that have not yet been able to be dated. Molecular changes over time are reported for many materials under different preservation conditions. Many of them are intrinsically monotonous. These monotonous molecular decay (MD) patterns can be understood as clocks that start at the time when a given molecule was formed. Factors that influence these clocks include input material composition and preservation conditions. Different wood species, degrees of pyrolysis, and pretreatments lead to different prediction models. Preservation conditions might change the speed of a given clock and lead to different prediction models. Currently published models for predicting the age of wood, paper, and parchment depend on infrared spectroscopy. In contrast to radiocarbon dating, dating via MD does not comprise a single methodology. Some clocks may deliver less precise results than the others. Ultimately, developing a completely different, new dating strategy-such as MD dating-will help to bring to light a treasure trove of information hidden in the darkness of organic findings.

DNA from resin-embedded organisms: Past, present and future.

Past claims have been made for fossil DNA recovery from various organisms (bacteria, plants, insects and mammals, including humans) dating back in time from thousands to several million years BP. However, many of these recoveries, especially those described from million-year-old amber (fossil resin), have faced criticism as being the result of modern environmental contamination and for lack of reproducibility. Using modern genomic techniques, DNA can be obtained with confidence from a variety of substrates (e.g. bones, teeth, gum, museum specimens and fossil insects) of different ages, albeit always less than one million years BP, and results can also be obtained from much older materials using palaeoproteomics. Nevertheless, new attempts to determine if ancient DNA (aDNA) is present in insects preserved in 40 000-year old sub-fossilised resin, the precursor of amber, have been unsuccessful or not well documented. Resin-embedded specimens are therefore regarded as unsuitable for genetic studies. However, we demonstrate here, for the first time, that although a labile molecule, DNA is still present in platypodine beetles (Coleoptera: Curculionidae) embedded in six-year-old and two-year-old resin pieces from Hymenaea verrucosa (Angiospermae: Fabaceae) collected in Madagascar. We describe an optimised method which meets all the requirements and precautions for aDNA experiments for our purpose: to explore the DNA preservation limits in resin. Our objective is far from starting an uncontrolled search for aDNA in amber as it was in the past, but to start resolving basic aspects from the DNA preservation in resin and search from the most modern samples to the ancient ones, step by step. We conclude that it is therefore possible to study genomics from resin-embedded organisms, although the time limits remain to be determined.

Phloem sap in Cretaceous ambers as abundant double emulsions preserving organic and inorganic residues.

Fossilized remains preserved in amber provide abundant data on the paleobiota surrounding the resin-producing plants, but relatively scarcer information about the resinous sources themselves. Here, dark pseudoinclusions in kidney-shaped amber pieces from the Early Cretaceous (Albian) amber from Spain are studied. This type of fossilized remain, abundant in Cretaceous ambers, was first interpreted as fossilized vacuole-bearing microorganisms, but later regarded as artifactual and probably secreted by the resinous trees, although their origin remained unclear. Using complementary microscopy (light, electron, confocal), spectroscopy (infrared, micro-Raman), mass spectrometry and elemental analysis techniques, we demonstrate that the pseudoinclusions correspond to droplets of phloem sap containing amber spheroids and preserving both organic and inorganic residues consistent with degraded components from the original sap. The amber pieces containing pseudoinclusions are fossilized, resin-in-sap-in-resin double emulsions, showing banding patterns with differential content of resin-in-sap emulsion droplets. Our findings represent the first time fossilized phloem sap, 105 million years old, has been recognized and characterized, and open new lines of paleontological research with taxonomic, taphonomic, physiological and ecological implications.

Unravelling the mystery of "Madagascar copal": Age, origin and preservation of a Recent resin.

The loss of biodiversity during the Anthropocene is a constant topic of discussion, especially in the top biodiversity hotspots, such as Madagascar. In this regard, the study of preserved organisms through time, like those included in "Madagascar copal", is of relevance. "Madagascar copal" originated from the leguminous tree Hymenaea verrucosa, which produced and produces resin abundantly. In the last 20 years, interest has focused on the scientific study of its biological inclusions, mainly arthropods, described in dozens of publications. The age and origin of the deposits of "Madagascar copal" have not yet been resolved. Our objectives are to determine its age and geographical origin, and thus increase its scientific value as a source of biological/palaeobiological information. Although Hymenaea was established in Madagascar during the Miocene, we did not find geological deposits of copal or amber in the island. It is plausible that the evolution of those deposits was negatively conditioned by the type of soil, by the climate, and by the development of soil/litter microorganisms, which inhibit preservation of the resin pieces in the litter and subsoil over 300 years. Our results indicate that "Madagascar copal" is a Recent resin, up to a few hundred years old, that originated from Hymenaea trees growing in the lowland coastal forests, one of the most endangered ecosystems in the world. The included and preserved biota is representative of that ecosystem today and during historical times. Inclusions in this Recent resin do not have the palaeontological significance that has been mistakenly attributed to them, but they do have relevant implications for studies regarding Anthropocene biodiversity loss in this hottest hotspot.

Predicting Reactive Cysteines with Implicit-Solvent-Based Continuous Constant pH Molecular Dynamics in Amber.

Cysteines existing in the deprotonated thiolate form or having a tendency to become deprotonated are important players in enzymatic and cellular redox functions and frequently exploited in covalent drug design; however, most computational studies assume cysteines as protonated. Thus, developing an efficient tool that can make accurate and reliable predictions of cysteine protonation states is timely needed. We recently implemented a generalized Born (GB) based continuous constant pH molecular dynamics (CpHMD) method in Amber for protein pKa calculations on CPUs and GPUs. Here we benchmark the performance of GB-CpHMD for predictions of cysteine pKa's and reactivities using a data set of 24 proteins with both down- and upshifted cysteine pKa's. We found that 10 ns single-pH or 4 ns replica-exchange CpHMD titrations gave root-mean-square errors of 1.2-1.3 and correlation coefficients of 0.8-0.9 with respect to experiment. The accuracy of predicting thiolates or reactive cysteines at physiological pH with single-pH titrations is 86 or 81% with a precision of 100 or 90%, respectively. This performance well surpasses the traditional structure-based methods, particularly a widely used empirical pKa tool that gives an accuracy less than 50%. We discuss simulation convergence, dependence on starting structures, common determinants of the pKa downshifts and upshifts, and the origin of the discrepancies from the structure-based calculations. Our work suggests that CpHMD titrations can be performed on a desktop computer equipped with a single GPU card to predict cysteine protonation states for a variety of applications, from understanding biological functions to covalent drug design.

Inhibition of melanin production and promotion of collagen production by the extract of Kuji amber.

Kuji amber is fossilized tree resin of the Late Cretaceous in Japan. In this study, new biological activities of ethanol extract of Kuji amber (EtOH ext.) and supercritical carbon dioxide fluid extract of Kuji amber (scCO2 ext.) were examined. Both EtOH ext. and scCO2 ext. inhibited melanin production in B16 mouse melanoma cells and promoted collagen production in human skin fibroblast SF-TY cells. The scCO2 ext. had more potent activity than that of EtOH ext. and may depend on the efficiency of the extraction. The main new biologically active compound in Kuji amber, kujigamberol had no activities against melanin production, however, it promoted collagen production at low concentrations. A biologically active compound having a different structure, spirolactone norditerpenoid, showed both the inhibition activity against melanin production and the promotion activity of collagen production in a dose dependent manner. EtOH ext. and scCO2 ext., which include both kujigamberol and spirolactone norditerpenoid, have not only anti-allergy activity, but also inhibit melanin production and promote collagen production.

A direct association between amber and dinosaur remains provides paleoecological insights.

Hadrosaurian dinosaurs were abundant in the Late Cretaceous of North America, but their habitats remain poorly understood. Cretaceous amber is also relatively abundant, yet it is seldom found in direct stratigraphic association with dinosaur remains. Here we describe an unusually large amber specimen attached to a Prosaurolophus jaw, which reveals details of the contemporaneous paleoforest and entomofauna. Fourier-transform Infrared spectroscopy and stable isotope composition (H and C) suggest the amber formed from resins exuded by cupressaceous conifers occupying a coastal plain. An aphid within the amber belongs to Cretamyzidae, a Cretaceous family suggested to bark-feed on conifers. Distinct tooth row impressions on the amber match the hadrosaur's alveolar bone ridges, providing some insight into the taphonomic processes that brought these remains together.

Carbon speciation in organic fossils using 2D to 3D x-ray Raman multispectral imaging.

The in situ two-dimensional (2D) and 3D imaging of the chemical speciation of organic fossils is an unsolved problem in paleontology and cultural heritage. Here, we use x-ray Raman scattering (XRS)-based imaging at the carbon K-edge to form 2D and 3D images of the carbon chemistry in two exceptionally preserved specimens, a fossil plant dating back from the Carboniferous and an ancient insect entrapped in 53-million-year-old amber. The 2D XRS imaging of the plant fossil reveals a homogeneous chemical composition with micrometric "pockets" of preservation, likely inherited from its geological history. The 3D XRS imaging of the insect cuticle displays an exceptionally well preserved remaining chemical signature typical of polysaccharides such as chitin around a largely hollowed-out inclusion. Our results open up new perspectives for in situ chemical speciation imaging of fossilized organic materials, with the potential to enhance our understanding of organic specimens and their paleobiology.

Amber.

David Grimaldi introduces amber and the fossils contained therein.

Baltic amber teething necklaces: could succinic acid leaching from beads provide anti-inflammatory effects?

BACKGROUND: Baltic amber teething necklaces have been popularized as a safe and natural alternative to conventional or pharmacological medicines for the management of teething pain. However, claims made by retailers regarding the efficacy and mechanism of action of these necklaces lack scientific or clinical basis. The claim most closely resembling science is the assertion that succinic acid will leach out of the beads and through the skin of the wearer and carry out anti-inflammatory and analgesic effects. The objective of the current research is to scientifically assess this claim. METHODS: Beads from necklaces were powdered for identification by infrared spectroscopy, and dissolved in sulfuric acid for quantification of succinic acid using HPLC. Succinic acid release from beads was assessed by long-term submersion of amber beads (separated according to light, medium and dark brown colour) in solvents relevant to human skin conditions. The potential for succinic acid to have anti-inflammatory effects was assessed by measuring the release of inflammatory cytokines IL-1α, IL-1ß, IL-8 and TNFα, and the inflammatory messenger PGE2, from THP-1 human macrophages after treatment with succinic acid and LPS. RESULTS: Amber teething necklaces were positively identified as Baltic amber, by comparison of the beads' infrared spectrum to the literature, and by their succinic acid content (1.5 mg per bead; 1.44% w/w). However, whole amber beads submerged in octanol or pH 5.5 phosphate buffered saline did not release any measurable succinic acid, except for the light-coloured beads in octanol which broke into tiny fragments. Additionally, treatment of macrophages with succinic acid did not reduce the release of any inflammatory cytokines measured, and displayed toxicity to the cells at high concentrations. CONCLUSIONS: While amber teething necklaces are genuine Baltic amber, we have found no evidence to suggest that the purported active ingredient succinic acid could be released from the beads into human skin. Additionally, we found no evidence to suggest that succinic acid has anti-inflammatory properties.