The Search for Life on Mars - Preparation for Sample Return

The purpose of the thesis work has been to develop methods and concepts to aid in the search, detection and assessment of ancient microfossils here on Earth as a guide to the search for ancient life on Mars. The intention has been to identify and characterize environments on Earth that may be considered analogous to Martian environments and in which fossil preservation is expected to be good, and to develop and apply methods to characterize the isotopic and chemical composition of possible traces of life in order to assess their biogenicity and biological affinities. An investigation of the Siljan impact structure, Sweden, demonstrated that niches for thermophilic organisms were created in the associated hydrothermal system. The temperature regimes were favorable for thermophilic life in the outer parts of the structure during the early and main stages of the hydrothermal system, but that these niches moved toward the center of the crater during the final cooling stages. It was demonstrated that the hydrothermal system contains traces of a thermophilic microbial community, represented by fossilized extracellular polymeric substances (EPS). Given the presence of water on Mars, similar impact-induced hydrothermal systems were probably generated on Mars as well. These regions, like those at Siljan, may have supported hyperthermophilic microbial communities on the red planet, emphasizing the relevance of searching for impact-induced hydrothermal deposits for evidence of microbial life on Mars. A method for the determination of stable carbon isotopes with high lateral resolution of TEM (transmission electron microscopy) samples has been developed. The method is based on alpha-particle Rutherford backscattering (RBS), it is non-destructive, and therefore suitable for analysis of extraterrestrial and other rare or irreplaceable material. Also, a novel concept to extract fluid inclusions without ablating the sample has been proposed, and a proof-of-concept has been demonstrated. The purpose is to analyze organic biomarkers trapped in fluid inclusions without risking contamination, and also to extract and analyze single fluid inclusions. The minimized contamination risk and the potential to extract single fluid inclusions could make the method a useful tool in the search for organic biomarkers in early-Earth material, and eventually, in samples returned from Mars

Development of a non-destructive micro-analytical method for stable carbon isotope analysis of transmission electron microscope (TEM) samples

The biogenicity of ancient morphological microfossil-like objects can be established by linking morphological (e.g. cell remnants and extracellular polymeric matrix) and chemical (e.g. isotopes, biomarkers and biominerals) evidence indicative of microorganisms or microbial activity. We have developed a non-destructive micro-analytical ion beam system capable of measuring with high spatial resolution the stable carbon isotope ratios of thin samples used for transmission electron microscopy. The technique is based on elastic scattering of alpha particles with an energy of 2.751 MeV. At this energy the C-13 cross section is enhanced relative to the pure Rutherford cross section for C-13, whereas the C-12 cross section is reduced relative to its pure Rutherford cross section. Here we report the initial results of this experimental approach used to characterize ultramicrotomed sections of sulfur-embedded graphite and microbial cells. (C) 2009 Elsevier B.V. All rights reserved

Analysis of organic biomarkers in single Precambrian oil-bearing fluid inclusions using ToF-SIMS

Organic biomarkers are valuable sources of information on the biodiversity and environment of early Earth. However, with organic biomarkers, especially in old samples, there are often problems of syngeneity and many of the most ancient biomarkers are suspected of being younger contamination. A type of sample where biomarkers are better constrained in the rock is oil-bearing fluid inclusions, especially if single inclusions can be analysed. However, most inclusions, including Precambrian oil-bearing fluid inclusions are so small (less than 10 mu m) that analyzing single ones with conventional techniques is not possible. Therefore, we have developed an approach employing time-of-flight secondary ion mass spectrometry (ToF-SIMS) to selectively open individual oil-bearing inclusions by C (sub 60) (super +) ion etching, and to subsequently analyse their content. Using this approach steranes and hopanes could be detected in single Ordovician oil-bearing inclusions (15-30 mu m) from the Siljan impact structure in Sweden. Now the developed approach has been applied on Precambrian samples. Four different oil-bearing fluid inclusions trapped in a 1.43 Ga sandstone from the Roper Superbasin in Australia were opened and analysed with ToF-SIMS. The ToF-SIMS spectra of the oil in the different inclusions were similar to each other indicating that the same oil was trapped in all inclusions. In addition, the ToF-SIMS spectra contained peaks that could be assigned to alkanes, cycloalkanes, aromatic moities, steranes and hopanes. With further development and if applied on other Precambrain samples this approach could help answering questions regarding early evolution of life on Earth.1 page(s

Analysis of single oil-bearing fluid inclusions in mid-Proterozoic sandstones (Roper Group, Australia)

Hydrocarbons and organic biomarkers extracted from black shales and other carbonaceous sedimentary rocks are valuable sources of information on the biodiversity and environment of early Earth. However, many Precambrian hydrocarbons including biomarkers are suspected of being younger contamination. An alternative approach is to study biomarkers trapped in oil-bearing fluid inclusions by bulk crushing samples and subsequently analysing the extracted hydrocarbons with gas chromatography-mass spectrometry. However, this method does not constrain the hydrocarbons to one particular oil inclusion, which means that if several different generations of oil inclusions are present in the sample, a mix of the content from these oil inclusions will be analysed. In addition, samples with few and/or small inclusions are often below the detection limit. Recently, we showed that it is possible to detect organic biomarkers in single oil-bearing fluid inclusions using time-of-flight secondary ion mass spectrometry (ToF-SIMS). In the present study, single fluid inclusion analysis has been performed on Proterozoic samples for the first time. Four individual oil-bearing fluid inclusions, found in 1430. Ma sandstone from the Roper Superbasin in Northern Australia, were analysed with ToF-SIMS. The ToF-SIMS spectra of the oil in the different inclusions are very similar to each other and are consistent with the presence of n-alkanes/branched alkanes, monocyclic alkanes, bicyclic alkanes, aromatic hydrocarbons, and tetracyclic and pentacyclic hydrocarbons. These results are in agreement with those obtained from bulk crushing of inclusions trapped in the same samples. The capability to analyse the hydrocarbon and biomarker composition of single oil-bearing fluid inclusions is a major breakthrough, as it opens up a way of obtaining molecular compositional data on ancient oils without the ambiguity of the origin of these hydrocarbons. Additionally, this finding suggests that it will be possible to analyse minute oil samples beyond the capability of established techniques. This may allow the biomarker record of the biosphere, as preserved in fluid inclusions, to be extended further back in time, and hence makes it possible to more accurately trace the early evolution of life on Earth, and search for life on other planets or moons.16 page(s

Chitin, Chitosan, and Glycated Chitosan Regulate Immune Responses: The Novel Adjuvants for Cancer Vaccine

With the development of cancer immunotherapy, cancer vaccine has become a novel modality for cancer treatment, and the important role of adjuvant has been realized recently. Chitin, chitosan, and their derivatives have shown their advantages as adjuvants for cancer vaccine. In this paper, the adjuvant properties of chitin and chitosan were discussed, and some detailed information about glycated chitosan and chitosan nanoparticles was also presented to illustrate the trend for future development

N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2.

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of β-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse

Single-cell RNA sequencing reveals localized tumourablation and intratumoural immunostimulant deliverypotentiate T cell mediated tumour killing

Background: Metastatic breast cancer poses great challenge in cancer treatment. N-dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV-PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated.Methods: Using depletion antibodies, we studied the contributions of CD8+ and CD4+ T cells to the therapeutic effect of LAIT. Using single-cell RNA-sequencing (scRNAseq), we analysed tumour-infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC).Results: Loss of CD8+ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8+ and CD4+ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co-stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8+ and CD4+ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8+and CD4+T cells that positively correlated with extended survival of breast cancer patients.Conclusions: Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity.Financial support was provided by the University of Oklahoma Libraries’ Open Access Fund. National Cancer Institute, Grant/AwardNumbers: R01CA205348, R01CA269897;Oklahoma Center for the Advancement ofScience and Technology, Grant/AwardNumbers: HR16-085, HF20-019.Ye