Preservation of proteins in the geosphere.

Deep-time protein preservation has attracted increasing interest and rapid research activity within the palaeobiological community in recent years, but there are several different viewpoints without a cohesive framework for the interpretation of these proteins. Therefore, despite this activity, crucial gaps exist in the understanding of how proteins are preserved in the geological record and we believe it is vital to arrive at a synthesis of the various taphonomic pathways in order to proceed forward with their elucidation. Here we take a critical look at the state of knowledge regarding deep-time protein preservation and argue for the necessity of a more nuanced approach to understanding the molecular taphonomy of proteins through the lens of diagenetic pathways. We also propound an initial framework with which to comprehend the chemical changes undergone by proteins via the concept of 'proteagen'.

The diagenetic fate of collagen as revealed by analytical pyrolysis of fossil fish scales from deep time.

The mechanism of protein degradation has remained a topic of debate (specifically concerning their preservation in deep time), which has recently been invigorated due to multiple published reports of preservation ranging from Miocene to the Triassic that potentially challenge the convention that protein preservation beyond the Cenozoic is extremely uncommon or is expected to be absent altogether, and thus have attracted skepticism. In this paper, we analyze fossil fish scales from the Cretaceous, Jurassic, and Triassic using comprehensive pyrolysis gas chromatography coupled with time-of-flight mass spectrometry and compare the pyrolytic products so obtained with a well-preserved fish scale from Late Pliocene, in an attempt to better understand the effects of diagenesis on protein degradation at the molecular level through deep time. We find that the Pliocene fish scale displays a large number of N-bearing pyrolytic products, including abundant substituted cyclic 2,5-diketopiperazines (2,5-DKPs) which are diagnostic products of peptide and amino acid pyrolysis. We identify N-bearing compounds in the Mesozoic fish scales-however, among the 2,5-DKPs that were identified in the Pliocene scale, only diketodipyrrole (or cyclo (Pyr-Pyr)) is present in the Mesozoic scales. We discuss the implications of N-bearing pyrolytic products with emphasis on 2,5-DKPs in geological samples and conclude that the discrepancy in abundance and variety of N-bearing products between Pliocene and Mesozoic scales indicates that the protein component in the latter has been extensively diagenetically altered, while a suite of DKPs such as in the former would imply stronger evidence to indicate preservation of protein. We conclude that analytical pyrolysis is an effective tool for detecting preservation of intact proteins, as well as for providing insights into their degradation mechanisms, and can potentially be utilized to assign proteinaceous origin to a fossil sample of unknown affinity.

Slurry phase biodegradation of heavy oily sludge and evidence of asphaltene biotransformation.

Oily sludge management is a global environmental concern due to its hazardous nature. Oily sludge obtained from a refinery in India had 19-21% oil content. The oil was highly enriched in the asphaltene fraction. Slurry phase biodegradation of this oily sludge in presence of a 3-membered bacterial consortium was optimized in presence of Triton X-100 to increase the bioavailability of hydrocarbons. Triton X-100 at 4 times the critical micelle concentration (CMC) showed the highest degradation where oil removal of 53.1% was achieved from a 10% sludge slurry over 90 days. GCxGC analysis of n-alkanes present in the oily sludge after the biodegradation study showed an increase in the lower n-alkanes, i.e., dodecane and tridecane over the first 30 days, whereas the higher n-alkanes were removed to a much higher extent. Heptadecane showed the maximum extent of degradation with 94.9% removal in 90 days and an initial degradation rate of 0.079 day-1. The, maximum rate of degradation was observed for pentacosane (0.083 day-1) with 93.7% removal in 90 days. The increase in the lower n-alkanes may be attributed to biotic transformation of the asphaltene fraction which was also confirmed through FTIR and pyrolysis GCxGC analysis. Biodegradation was found to cause changes in the pyrolysis product of asphaltenes where four and three-ring pyrolysis products decreased while the one and two-ring pyrolysis products increased. In presence of the consortium asphaltene removal over 90 days was 12% whereas only 0.4% removal was obtained in the abiotic controls.

Characterization and biodegradability assessment of water-soluble fraction of oily sludge using stir bar sorptive extraction and GCxGC-TOF MS.

Percolation of water through oily sludge during storage and handling of the sludge can cause soil and groundwater contamination. In this study, oily sludge from a refinery was equilibrated with water to obtain the water-soluble fraction (WSF) of oily sludge. The WSF had dissolved organic carbon (DOC) of 166 mg/L. Human cell line-based toxicity assay revealed IC50 of 41 mg/L indicating its toxic nature. The predominant compounds in WSF of oily sludge included isomers of methyl, dimethyl and trimethyl quinolines and naphthalenes along with phenol derivatives and other polynuclear aromatic hydrocarbons (PAHs). Biodegradation of WSF of oily sludge was studied using a consortium of Rhodococcus ruber, Bacillus sp. and Bacillus cereus isolated from the refinery sludge. The consortium of the three strains resulted in 70% degradation over 15 days with a first-order degradation rate of 0.161 day-1. Further analysis of the WSF was performed using the stir-bar sorptive extraction (SBSE) followed by GCxGC-TOF MS employing a PDMS Twister. The GCxGC analysis showed that Bacillus cereus was capable of degrading the quinoline, phenol and naphthalene derivatives in WSF of oily sludge at a faster rate compared to pyridine and benzoquinoline derivatives. Quinoline, phenol, biphenyl, naphthalene, pyridine and benzoquinolines derivatives in the WSF of oily sludge were reduced by 87%, 92%, 88%, 77%, 40% and 62%, respectively with respect to the controls. The WSF of oily sludge contained, n-alkanes, ranging from n-C12 to n-C18 which were removed within 2 days of biodegradation.

Identification of Chitin in Pliocene Fungi Using Py-GC × GC-TOFMS: Potential Implications for the Study of the Evolution of the Fungal Clade in Deep Time.

Molecular dating estimates the origin of the fungal clade to the Pre-Cambrian. Yet, the oldest unambiguous fungal fossils date to the Ordovician and show remarkable diversity and organizational development. Recent studies have suggested that the dates for the emergence of fungi in the fossil record may be pushed back to the Proterozoic. However, the nonspecificity of the methods used in those studies necessitates the employment of a wider variety of analytical techniques that can independently verify the presence of chitin, a crucial prerequisite in the assignment of fungal affinity, particularly of putative fossils from the Pre-Cambrian. In this paper, we propose Py-GC × GC-TOFMS as an example of one such technique. We analyze fungal fossils from the Pliocene. We find that a suite of N-bearing compounds are present in the pyrolysis products of these fossils, from which we suggest that 3-acetamidopyrones and their methylated homologues can serve as specific pyrolytic markers for chitin. We discuss both how this technique can potentially be used to differentiate between biopolymers, including those similar to chitin such as peptidoglycan, and the potential implications of identifying such markers in fossils from deep time. We conclude that Py-GC × GC-TOFMS is a promising technique that can potentially be used alongside, or independent of, staining methods to detect the presence of chitin in fossils.

Southeast Asian Dipterocarp origin and diversification driven by Africa-India floristic interchange.

The evolution and diversification of ancient megathermal angiosperm lineages with Africa-India origins in Asian tropical forests is poorly understood because of the lack of reliable fossils. Our palaeobiogeographical analysis of pollen fossils from Africa and India combined with molecular data and fossil amber records suggest a tropical-African origin of Dipterocarpaceae during the mid-Cretaceous and its dispersal to India during the Late Maastrichtian and Paleocene, leading to range expansion of aseasonal dipterocarps on the Indian Plate. The India-Asia collision further facilitated the dispersal of dipterocarps from India to similar climatic zones in Southeast Asia, which supports their out-of-India migration. The dispersal pathway suggested for Dipterocarpaceae may provide a framework for an alternative biogeographic hypothesis for several megathermal angiosperm families that are presently widely distributed in Southeast Asia.

A Late Cretaceous amber biota from central Myanmar.

Insect faunas are extremely rare near the latest Cretaceous with a 24-million-year gap spanning from the early Campanian to the early Eocene. Here, we report a unique amber biota from the Upper Cretaceous (uppermost Campanian ~72.1 Ma) of Tilin, central Myanmar. The chemical composition of Tilin amber suggests a tree source among conifers, indicating that gymnosperms were still abundant in the latest Campanian equatorial forests. Eight orders and 12 families of insects have been found in Tilin amber so far, making it the latest known diverse insect assemblage in the Mesozoic. The presence of ants of the extant subfamilies Dolichoderinae and Ponerinae supports that tropical forests were the cradle for the diversification of crown-group ants, and suggests that the turnover from stem groups to crown groups had already begun at ~72.1 Ma. Tilin amber biota fills a critical insect faunal gap and provides a rare insight into the latest Campanian forest ecosystem.

Remarkable preservation of terpenoids and record of volatile signalling in plant-animal interactions from Miocene amber.

Plants produce and release a large array of volatile organic compounds that play many ecological functions. These volatile plant metabolites serve as pollinator attractants, herbivore and pathogen repellents and protect plants from abiotic stresses. To date, the geological evolution of these organic compounds remains unknown. The preservation potential of these metabolites in the fossil record is very poor due to their low boiling points. Here we report a series of volatile sesquiterpenoids, including δ-elemene, α-copaene, ß-elemene, ß-caryophyllene, α-humulene, germacrene D, δ-cadiene and spathunenol, from early Miocene (~17 million year) amber from eastern India. The survival of these unaltered bioterpenoids can be attributed to the existence of extraordinary taphonomic conditions conducive to the preservation of volatile biomolecules through deep time. Furthermore, the occurrence of these volatiles in the early Miocene amber suggests that the plants from this period had evolved metabolic pathways to synthesize these organic molecules to play an active role in forest ecology, especially in plant-animal interactions.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Terpenoid compositions and botanical origins of Late Cretaceous and Miocene amber from China.

The terpenoid compositions of the Late Cretaceous Xixia amber from Central China and the middle Miocene Zhangpu amber from Southeast China were analyzed by gas chromatography-mass spectrometry (GC-MS) to elucidate their botanical origins. The Xixia amber is characterized by sesquiterpenoids, abietane and phyllocladane type diterpenoids, but lacks phenolic abietanes and labdane derivatives. The molecular compositions indicate that the Xixia amber is most likely contributed by the conifer family Araucariaceae, which is today distributed primarily in the Southern Hemisphere, but widely occurred in the Northern Hemisphere during the Mesozoic according to paleobotanical evidence. The middle Miocene Zhangpu amber is characterized by amyrin and amyrone-based triterpenoids and cadalene-based sesquiterpenoids. It is considered derived from the tropical angiosperm family Dipterocarpaceae based on these compounds and the co-occurring fossil winged fruits of the family in Zhangpu. This provides new evidence for the occurrence of a dipterocarp forest in the middle Miocene of Southeast China. It is the first detailed biomarker study for amber from East Asia.

A diverse paleobiota in early eocene Fushun amber from China.

Paleogene arthropod biotas have proved important for tracing the faunal turnover and intercontinental faunal interchange driven by climatic warming and geodynamic events [1-5]. Despite the large number of Paleogene fossil arthropods in Europe and North America [5-8], little is known about the typical Asian (Laurasia-originated) arthropod biota. Here, we report a unique amber biota (50-53 million years ago) from the Lower Eocene of Fushun in northeastern China, which fills a large biogeographic gap in Eurasia. Fushun amber is derived from cupressaceous trees, as determined by gas chromatography-mass spectrometry, infrared spectroscopy, and paleobotanical observations. Twenty-two orders and more than 80 families of arthropods have been reported so far, making it among the most diverse amber biotas. Our results reveal that an apparent radiation of ecological keystone insects, including eusocial, phytophagous, and parasitoid lineages, occurred at least during the Early Eocene Climatic Optimum. Some insect taxa have close phylogenetic affinities to those from coeval European ambers, showing a biotic interchange between the eastern and western margins of the Eurasian landmass during the Early Paleogene.

Direct chemical evidence for eumelanin pigment from the Jurassic period.

Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms.