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Steranes and hopanes are organic biomarkers used as indicators for the first appearance of eukaryotes and cyanobacteria on Earth. Oil-bearing fluid inclusions may provide a contamination-free source of Precambrian biomarkers, as the oil has been secluded from the environment since the formation of the inclusion. However, analysis of biomarkers in single oil-bearing fluid inclusions, which is often necessary due to the presence of different generations of inclusions, has not been possible due to the small size of most inclusions. Here, we have used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to monitor in real time the opening of individual inclusions trapped in hydrothermal veins of fluorite and calcite and containing oil from Ordovician source rocks. Opening of the inclusions was performed by using a focused C60+ ion beam and the in situ content was precisely analysed for C27–C29 steranes and C29–C32 hopanes using Bi3+ as primary ions. The capacity to unambiguously detect these biomarkers in the picoliter amount of crude oil from a single, normal-sized (15–30 μm in diameter) inclusion makes the approach promising in the search of organic biomarkers for life’s early evolution on Earth. [ABSTRACT FROM AUTHOR]

Abstract: Organic phases trapped inside natural mineral samples are of considerable interest in astrobiology, geochemistry and geobiology. Examples of such organic phases are microfossils, kerogen and oil. Information about these phases is usually retrieved through bulk crushing of the rock which means both a risk of contamination and that the composition and spatial distribution of the organics to its host mineral is lost. An attractive of way to retrieve information about the organics in the rock is depth profiling using a focused ion beam. Recently, it was shown that it is possible to obtain detailed mass spectrometric information from oil-bearing fluid inclusions, i.e. small amounts of oil trapped inside a mineral matrix, using ToF-SIMS. Using a 10keV C60 + sputter beam and a 25keV Bi3 + analysis beam, oil-bearing inclusions in different minerals were opened and analysed individually. However, sputtering with a C60 + beam also induced other changes to the mineral surface, such as formation of topographic features and carbon deposition. In this paper, the cause of these changes is explored and the consequences of the sputter-induced features on the analysis of organic phases in natural mineral samples (quartz, calcite and fluorite) in general and fluid inclusions in particular are discussed. The dominating topographical features that were observed when a several micrometers deep crater is sputtered with 10keV C60 + ions on a natural mineral surface are conical-shaped and ridge-like structures that may rise several micrometers, pointing in the direction of the incident C60 + ion beam, on an otherwise flat crater bottom. The sputter-induced structures were found to appear at places with different chemistry than the host mineral, including other minerals phases and fluid inclusions, while structural defects in the host material, such as polishing marks or scratches, did not necessarily result in sputter-induced structures. The ridge-like structures were often covered by a thick layer of deposited carbon. Despite the appearance of the sputter-induced structures and carbon deposition, most oil-bearing inclusions could successfully be opened and analysed. However, smaller inclusion (<15μm) could potentially become entirely covered by sputter-resistant structures and therefore difficult to open. Therefore, it might become necessary, to for example increase the ion energy and rotate the stage to successfully open smaller inclusions for analysis. SIMS, C60, carbon deposition, topography, mineral, fluid inclusions, geological samples, depth profiling. [Copyright &y& Elsevier]

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses., Competing Interests: Declaration of competing interest The authors have no conflict of interest. WRC is a co-founder and a member of the Board of Directors of Immunophotonics, Inc. LA, SSKL, and TH declare a conflict of interest as employees with minority ownership stakes of Immunophotonics, Inc., the manufacturer of the proprietary adjuvant GC., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Thermal ablation is reported to increase immunogenicity in tumor cells via expressing tumor antigens. IP-001, a synthesized molecule, is created by attaching galactose molecules to the free amino groups of partially deacetylated glucosamine polymers. As a member of a new class of polycationic immunoadjuvants that activate multiple immune response pathways, IP-001 can both sequester ablation-released tumor antigens in situ and independently recruit and stimulate antigen presenting cells (APCs) to induce a potent tumor-specific Th1 type T cell response., Methods: An orthotopic HCC rat model is established by implantation of 5 × 10 6 N1-S1 cells into the left lobe of liver. When tumor size reached 1.0-1.5 cm 3 , the animals were divided randomly into 4 groups, (1) MWA+IP-001; (2) MWA+saline; (3) sham MWA+IP-001 and (4) sham MWA+saline ( n  = 5 each group)., Results: IP001 + MWA treatment significantly suppressed tumor growth in comparison to the other 3 groups. Significantly increased infiltration of inflammatory/immune cells were found in the tumor adjacent tissues of MWA+IP-001 mice, compared to the other 3 groups. Flow cytometry results indicated that there were significant increases of cytotoxic T cells, macrophages, dendritic cells and NK cell in the combination of MWA and IP001 treated mice, compared to other 3 groups ( p  < 0.01). Significantly decreased number of Treg cells were found in all the treatment arms compared to untreated control ( p  < 0.01)., Conclusion: Combination of MWA and IP001 enhances tumor suppression in an orthotopic HCC rat model. The tumor suppression is associated to the enhanced immune responses in terms of recruiting the important cell subpopulations such as CD8 + T-cells and NK cells into tumor microenvironment and abolishing immune suppressor such as Treg cells.

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., Competing Interests: TH, CFW, SSKL declare a conflict of interest as employees with minority ownership stakes of Immunophotonics, Inc., the manufacturer of the proprietary immune stimulant GC. RML declares a conflict of interest as an advisor with minority ownership stake in Immunophotonics. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Ablation therapies have emerged as an effective tool for destroying cancerous tissue, but for advanced and disseminated tumors their application remains mainly a palliative measure. However, it is becoming increasingly clear that this limitation can be redressed by the use of intratumoral immune stimulating agents for amplifying potential antitumor immune responses that are induced by ablation therapies. A novel immune stimulating drug IP-001, a specific variant of the N-dihydrogalactochitosan (GC) family of molecules, has shown to be effective against metastatic tumors, when combined with different forms tumor ablation. It acts as a multi-function immune stimulant both by directly inhibiting cell membrane repair and recycling of ablation-damaged tumor cells, and indirectly by sequestering ablation-released tumor antigens, as well as recruiting and stimulating antigen presenting cells to induce a potent Th1 type T cell response against the cancer. In this review, we briefly discuss the current applications of local ablation for cancer treatment and the effects of GC in combination with other ablation therapies, a therapeutic approach that is pioneering the field of Interventional Immuno-Oncology (IIO).

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival., Competing Interests: Declaration of Competing Interest Tomas Hode and Samuel S.K. Lam declare a conflict of interest as employees of Immunophotonics Inc. which manufactures N-dihydrogalactochitosan. The other authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

It is becoming apparent that to obtain robust and prolonged antitumor responses in cancer immunotherapy, appropriate adjunct agents promoting both tumor antigen delivery and immune rejection enhancement are critically required. The semisynthetic biopolymer N-dihydrogalactochitosan (GC) is emerging as a promising such candidate. In the present study, the effects of GC were investigated when combined with cancer vaccines generated by photodynamic therapy (PDT) using mouse tumor model SCCVII (squamous cell carcinoma). The adjunct GC treatment was found to enhance therapeutic benefit obtained with PDT vaccine, while reducing the numbers of myeloid-derived suppressor cells. Another important property of GC is promoting directly the death of SCCVII cells sustaining injury from PDT mediated by various photosensitizers. This effect is extended to cells treated by cryoablation therapy (CAT) performed by exposure to -80 °C. A capacity of GC for preferential binding to PDT treated cells was demonstrated using fluorescence microscopy. In vitro testing with specific caspase-1 inhibitor revealed a pro-survival role of this enzyme in membrane lipid repair mechanisms following combined PDT plus GC treatment. In conclusion, GC represents a uniquely promising adjunct for various PDT protocols, photothermal and similar rapid tumor-ablating therapies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Manipulation of immune system toward the rejection of established cancers has become the standard of care in some patients. Here we propose the development of an in situ autologous cancer vaccine, inCVAX, for the treatment of hepatocellular cancer (HCC). inCVAX is based on the induction of local immunogenic cancer cell death combined with local dendritic cell stimulation by intratumoral injection of the immune-activator N -dihydro-galacto-chitosan (GC). In a first set of experiments, cellular and molecular studies were performed to investigate the effect of inCVAX on immune activation in a murine model of HCC that we previously developed. Once large tumors were formed in mice, the tumor is surgically exposed and a laser fiber was inserted into the center of an individual tumor mass. Using a 10 mm diffuser tip, laser irradiation of 1.5 W was applied to heat the tumor at different durations (6-10 min) to assess tolerability of photothermal application at different temperatures. The laser application was followed by immediate injection of GC, and each mouse received one laser treatment and one GC injection. ELISA was used to assess the level of cytokines; immunohistochemical staining was conducted to analyze the effect of inCVAX on immune cell tumor-filtration and expression of tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Results indicate that survival correlated to thermal exposure. At lower temperatures the photothermal effect was sufficient to induce tumor necrosis, but without obvious complication to the mice, although at these temperatures the treatment didn't alter the level of TSAs and TAAs, so further optimization is suggested. Nevertheless, in response to the inCVAX treatment, cytotoxic cytokine IFN-γ was significantly increased, but suppressive cytokine TGF-β was dramatically reduced. Furthermore, inCVAX prompted tumor infiltration of CD3 + , CD4 + , and CD8 + T cells; but modulated macrophage subsets differently. In conclusion, while the protocol needs further optimization, it would appear that inCVAX for the treatment of HCC activates an immune response in tumor-bearing mice, which in turn may have potential for the treatment of HCC.

A novel, promising potential cancer vaccine strategy was proposed to use a two-injection procedure for solid tumors to prompt the immune system to identify and systemically eliminate primary and metastatic cancers. The two-injection procedure consists of local photothermal application on a selected tumor intended to liberate whole cell tumor antigens, followed by a local injection of an immunoadjuvant that consists of a semi-synthetic functionalized glucosamine polymer, N-dihydro-galacto-chitosan (GC), which is intended to activate antigen presenting cells and facilitate an increased uptake of tumor antigens. This strategy is thus proposed as an in situ autologous cancer vaccine (inCVAX) that may activate antigen presenting cells and expose them to tumor antigens in situ, with the intention of inducing a systemic tumor specific T-cell response. Here, the development of inCVAX for the treatment of metastatic cancers in the past decades is systematically reviewed. The antitumor immune responses of local photothermal treatment and immunological stimulation with GC are also discussed. This treatment approach is also commonly referred to as laser immunotherapy (LIT)., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Since the invention of Coley's toxin by William Coley in early 1900s, the path for cancer immunotherapy has been a convoluted one. Although still not considered standard of care, with the FDA approval of trastuzumab, Provenge and ipilimumab, the medical and scientific community has started to embrace the possibility that immunotherapy could be a new hope for cancer patients with otherwise untreatable metastatic diseases. This review aims to summarize the development of some major strategies in cancer immunotherapy, from the earliest peptide vaccine and transfer of tumor specific antibodies/T cells to the more recent dendritic cell (DC) vaccines, whole cell tumor vaccines, and checkpoint blockade therapy. Discussion of some major milestones and obstacles in the shaping of the field and the future perspectives is included. Photoimmunotherapy is also reviewed as an example of emerging new therapies combining phototherapy and immunotherapy.

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.

Laser immunotherapy (LIT) uses a synergistic approach to treat cancer systemically through local laser irradiation and immunological stimulation. Currently, LIT utilizes dye-assisted noninvasive laser irradiation to achieve selective photothermal interaction. However, LIT faces difficulties treating deeper tumors or tumors with heavily pigmented overlying skin. To circumvent these barriers, we use interstitial laser irradiation to induce the desired photothermal effects. The purpose of this study is to analyze the thermal effects of interstitial irradiation using proton resonance frequency (PRF). An 805-nm near-infrared laser with an interstitial cylindrical diffuser was used to treat rat mammary tumors. Different power settings (1.0, 1.25, and 1.5 W) were applied with an irradiation duration of 10 min. The temperature distributions of the treated tumors were measured by a 7 T magnetic resonance imager using PRF. We found that temperature distributions in tissue depended on both laser power and time settings, and that variance in tissue composition has a major influence in temperature elevation. The temperature elevations measured during interstitial laser irradiation by PRF and thermocouple were consistent, with some variations due to tissue composition and the positioning of the thermocouple's needle probes. Our results indicated that, for a tissue irradiation of 10 min, the elevation of rat tumor temperature ranged from 8 to 11°C for 1 W and 8 to 15°C for 1.5 W. This is the first time a 7 T magnetic resonance imager has been used to monitor interstitial laser irradiation via PRF. Our work provides a basic understanding of the photothermal interaction needed to control the thermal damage inside a tumor using interstitial laser treatment. Our work may lead to an optimal protocol for future cancer treatment using interstitial phototherapy in conjunction with immunotherapy.

We report our preliminary results of a pilot clinical trial of late-stage breast cancer patients treated by laser immunotherapy (LIT), a local intervention using an 805 nm laser for non-invasive irradiation, indocyanine green for selective thermal effect, and immunoadjuvant (glycated chitosan) for immunological stimulation. Ten breast cancer patients were enrolled in this study; all patients were considered to be out of other available treatment options. Toxicity was individually evaluated through physical exams and laboratory tests. Adverse reactions only occurred in the area of treatment due to photothermal injury and local administration of immunoadjuvant. No grade 3 or 4 side effects were observed. Treatment efficacy of LIT was also evaluated by physical examination and tomography. In 8 patients available for evaluation, the objective response rate was 62.5% and the clinical beneficial response rate was 75%. While the study is still ongoing, the initial outcomes of this clinical trial show that LIT is well tolerated and is promising in the treatment of metastatic breast cancer.

The Siljan ring structure (368 +/- 1.1 Ma) is the largest known impact structure in Europe. It isa 65-km-wide, eroded, complex impact structure, displaying several structural units, including a central uplifted region surrounded by a ring-shaped depression. Associated with the impact crater are traces of a post-impact hydrothermal system indicated by precipitated and altered hydrothermal mineral assemblages. Precipitated hydrothermal minerals include quartz veins and breccia fillings associated with granitic rocks at the outer margin of the central uplift, and calcite, fluorite, galena, and sphalerite veins associated with Paleozoic carbonate rocks located outside the central uplift. Two-phase water/gas and oil/gas inclusions in calcite and fluorite display homogenization temperatures between 75 degrees C and 137 degrees C. With an estimated erosional unloading of approximately 1 km, the formation temperatures were probably not more than 10-15 degrees C higher. Fluid inclusion ice-melting temperatures indicate a very low salt content, reducing the probability that the mineralization was precipitated during the Caledonian Orogeny. Our findings suggest that large impacts induce low-temperature hydrothermal systems that may be habitats for thermophilic organisms. Large impact structures on Mars may therefore be suitable targets in the search for fossil thermophilic organisms.