ABSTRACT
Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity. For complete details on the use and execution of this protocol, please refer to Xu et al.1.
ABSTRACT
BACKGROUND: Candida albicans is an opportunistic pathogen commonly found in human mucous membranes. In light of the escalating challenge posed by antibiotic resistance of C. albicans strains worldwide, it is an urgently necessary to explore alternative therapeutic options. OBJECTIVE: This study aims to assess the efficacy of two Cinnamaldehyde derivatives, 2-Cl Cinnamaldehyde (2-Cl CA) and 4-Cl Cinnamaldehyde (4-Cl CA), against C. albicans through both in vitro experiments and in vivo murine models and to evaluate their potential as new drug candidates for treating C. albicans. METHODS AND RESULTS: The minimum inhibitory concentrations (MICs) of Cinnamaldehyde 2-Cl and 4-Cl benzene ring derivatives against C. albicans were 25 µg/mL. Time-killing experiments revealed that both Cinnamaldehyde derivatives exhibited fungicidal activity against C. albicans at concentrations of 5 MIC and 10 MIC. In the checkerboard experiment, 4-Cl CA did not show any antagonistic effect when combined with first-line antifungal drugs. Instead, it exhibited additive effects in combination with nystatin. Both 2-Cl and 4-Cl CA demonstrated inhibitory activity against C. albicans biofilm formation, especially at 8 MIC and 16 MIC concentrations. In C. albicans biofilm eradication experiments, although high drug concentrations of 2-Cl and 4-Cl CA were unable to eradicate the biofilm completely, they were still effective in killing C. albicans cells within the biofilm. Moreover, sub-inhibitory concentrations of 4-Cl CA (ranging from 5 to 20 µg/mL) significantly inhibited cell aggregation and hyphal formation. Furthermore, 4-Cl CA effectively inhibited intracellular C. albicans infection in macrophages. Lastly, the effectiveness of 4-Cl CA was evaluated in a mouse model of hematogenous disseminated candidiasis caused by C. albicans, which revealed that 4-Cl CA significantly reduced fungal burden and improved mouse survival compared to the untreated controls. CONCLUSION: The 4-Cl CA exhibited inhibitory effects against C. albicans through both in vivo and in vitro models, demonstrating its therapeutic potential as a promising new drug candidate for treating drug-resistant candidiasis albicans.
ABSTRACT
The eradication of tuberculosis remains a global challenge. Despite being the only licensed vaccine, Bacillus Calmette-Guérin (BCG) confers limited protective efficacy in adults and individuals with latent tuberculosis infections (LTBI). There is an urgent need to develop novel vaccines that can enhance the protective effect of BCG. Protein subunit vaccines have garnered significant research interest due to their safety and plasticity. Based on previous studies, we selected three antigens associated with LTBI (Rv2028c, Rv2029c, Rv3126c) and fused them with an immunodominant antigen Ag85A, resulting in the construction of a multistage protein subunit vaccine named A986. We evaluated the protective effect of recombinant protein A986 adjuvanted with MPL/QS21 as a booster vaccine for BCG against Mycobacterium tuberculosis (Mtb) infection in mice. The A986 + MPL/QS21 induced the secretion of antigen-specific Th1 (IL-2+, IFN-γ+ and TNF-α+) and Th17 (IL-17A+) cytokines in CD4+ and CD8+ T cells within the lung and spleen of mice, while also increased the frequency of central memory and effector memory T cells. Additionally, it also induced the enhanced production of IgG antibodies. Compared to BCG alone, A986 + MPL/QS21 boosting significantly augmented the proliferation of antigen-specific multifunctional T cells and effectively reduced bacterial load in infected mice. Taken together, A986 + MPL/QS21 formulation induced robust antigen-specific immune responses and provided enhanced protection against Mtb infection as a booster of BCG vaccine.
Subject(s)
Antigens, Bacterial , BCG Vaccine , Cytokines , Mycobacterium tuberculosis , Tuberculosis , Vaccines, Subunit , Animals , Vaccines, Subunit/immunology , Mycobacterium tuberculosis/immunology , BCG Vaccine/immunology , Antigens, Bacterial/immunology , Female , Tuberculosis/prevention & control , Tuberculosis/immunology , Mice , Cytokines/metabolism , Immunization, Secondary , Disease Models, Animal , Adjuvants, Immunologic/administration & dosage , Tuberculosis Vaccines/immunology , Tuberculosis Vaccines/administration & dosage , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Mice, Inbred C57BL , Mice, Inbred BALB C , Acyltransferases/immunology , Lung/immunology , Lung/microbiology , Lung/pathology , Bacterial Proteins/immunology , HumansABSTRACT
Trained immunity is one of the mechanisms by which BCG vaccination confers persistent nonspecific protection against diverse diseases. Genomic differences between the different BCG vaccine strains that are in global use could result in variable protection against tuberculosis and therapeutic effects on bladder cancer. In this study, we found that four representative BCG strains (BCG-Russia, BCG-Sweden, BCG-China, and BCG-Pasteur) covering all four genetic clusters differed in their ability to induce trained immunity and nonspecific protection. The trained immunity induced by BCG was associated with the Akt-mTOR-HIF1α axis, glycolysis, and NOD-like receptor signaling pathway. Multi-omics analysis (epigenomics, transcriptomics, and metabolomics) showed that linoleic acid metabolism was correlated with the trained immunity-inducing capacity of different BCG strains. Linoleic acid participated in the induction of trained immunity and could act as adjuvants to enhance BCG-induced trained immunity, revealing a trained immunity-inducing signaling pathway that could be used in the adjuvant development.
Subject(s)
BCG Vaccine , Tuberculosis , Humans , Linoleic Acid , Trained Immunity , Multiomics , Adjuvants, Immunologic/pharmacologyABSTRACT
BACKGROUND: Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. RESULTS: In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1ß) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed. CONCLUSIONS: We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.
Subject(s)
Monocytes , Mycobacterium bovis , Humans , BCG Vaccine , Trained Immunity , Proto-Oncogene Proteins c-akt/genetics , THP-1 Cells , Phosphatidylinositol 3-Kinases , CytokinesABSTRACT
Self-powered wearable thermoelectric (TE) devices significantly reduce the inconvenience caused to users, especially in daily use of portable devices and monitoring personal health. The textile-based TE devices (TETs) exhibit the excellent flexibility, deformability, and light weight, which fulfill demands of long-term wearing for the human body. In comparison to traditional TE devices with their longstanding research history, TETs are still in an initial stage of growth. In recent years, TETs to provide electricity for low-power wearable electronics have attracted increasing attention. This review summarizes the recent progress of TETs from the points of selecting TE materials, scalable fabrication methods of TE fibers/yarns and TETs, structure design of TETs and reported high-performance TETs. The key points to develop TETs with outstanding TE properties and mechanical performance and better than available optimization strategies are discussed. Furthermore, remaining challenges and perspectives of TETs are also proposed to suggest practical applications for heat harvesting from human body.
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One-quarter of the world's population is infected with Mycobacterium tuberculosis (Mtb). After initial exposure, more immune-competent persons develop asymptomatic latent tuberculosis infection (LTBI) but not active diseases, creates an extensive reservoir at risk of developing active tuberculosis. Previously, we constructed a novel recombinant Sendai virus (SeV)-vectored vaccine encoding two dominant antigens of Mtb, which elicited immune protection against acute Mtb infection. In this study, nine Mtb latency-associated antigens were screened as potential supplementary vaccine candidate antigens, and three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immune-therapeutic effect in mice, and their elevated immune responses in LTBI human populations. Then, a recombinant SeV-vectored vaccine, termed SeV986A, that expresses three latency-associated antigens and Ag85A was constructed. In murine models, the doses, titers, and inoculation sites of SeV986A were optimized, and its immunogenicity in BCG-primed and BCG-naive mice were determined. Enhanced immune protection against the Mtb challenge was shown in both acute-infection and latent-infection murine models. The expression levels of several T-cell exhaustion markers were significantly lower in the SeV986A-vaccinated group, suggesting that the expression of latency-associated antigens inhibited the T-cell exhaustion process in LTBI infection. Hence, the multistage quarter-antigenic SeV986A vaccine holds considerable promise as a novel post-exposure prophylaxis vaccine against tuberculosis.
Subject(s)
Latent Tuberculosis , Mycobacterium tuberculosis , Tuberculosis , Humans , Animals , Mice , Latent Tuberculosis/prevention & control , Sendai virus/genetics , BCG Vaccine , Antigens, Bacterial/genetics , Tuberculosis/microbiology , Mycobacterium tuberculosis/genetics , Vaccines, Synthetic/geneticsABSTRACT
Objectives: Lipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap. Methods: A proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples. Results: The limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV- patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively. Conclusion: This study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.
ABSTRACT
Tuberculosis (TB), also known as the "White Plague", is caused by Mycobacterium tuberculosis (Mtb). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of Mtb, the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.
Subject(s)
COVID-19 , Tuberculosis Vaccines , Tuberculosis , Animals , Protein Subunits , COVID-19/prevention & control , Vaccines, Subunit , Tuberculosis/prevention & control , BCG VaccineABSTRACT
Starch hydrogels with biodegradability and cytocompatibility are good alternatives to traditional dressings. Herein, oxidized starch hydrogel loaded with coagulation factor Ca2+ ions (CaOMS) is successfully constructed by green hot-extrusion 3D printing technology (HE-3DP). In vitro study demonstrated the good water absorbing capacity (845.15-1194.20%) and blood cell and platelet adhesion of CaOMS to assist hemostasis owing to the boosted network structure density, gel strength, and the release of activated Ca2+ ions. More importantly, in vivo experiments further demonstrated CaOMS could maintain the weight loss caused by blood loss from wounds and has excellent hemostatic (65 s) and wound healing properties by promoting the secretion of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) expression. The advantages of CaOMS, including rapid and effective hemostasis, effective wound healing, low cost, easy usage, and adaptability to fit various wound shapes, make it a promising biomaterial for achieving fast hemostasis and wound healing.
Subject(s)
Hydrogels , Starch , Hydrogels/pharmacology , Hydrogels/chemistry , Starch/pharmacology , Vascular Endothelial Growth Factor A , Hemostasis , Printing, Three-Dimensional , IonsABSTRACT
The effects of coal metakaolin on the mechanical properties of high-belite sulphoaluminate cement under compressive loading were investigated. The composition and microstructure of hydration products at different hydration times were analyzed by X-ray diffraction and scanning electronic microscopy. The hydration process of blended cement was studied via electrochemical impedance spectroscopy. In particular, replacing a part of cement with CMK (10%, 20%, and 30%) was found to promote the hydration process, to refine the pore size, and to improve the compressive strength of the composite. The best compressive strength of the cement was achieved at a CMK content of 30% after 28 days hydration, being improved by 20.13 MPa, or 1.44 times relative to that of undoped specimens. Furthermore, the compressive strength is shown to correlate with the impedance parameter RCCP, which allows the latter to be used for nondestructive assessment of the compressive strength of blended cement materials.
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Objective: To compare the diagnostic performance of laboratory assays on the ultrasound-guided core needle biopsy samples for diagnosis of extra-pulmonary tuberculosis (EPTB) in HIV-positive and HIV-negative patients. Methods: A total of 217 patients suspected to have EPTB underwent lesion biopsy from 2017 to 2020. Results of laboratory tests on the biopsy and non-biopsy samples were collected with clinical data for retrospective analysis of test utility. The calculated diagnostic accuracy of the tests was stratified according to the specimen types and HIV status. Results: The cohort contained 118 patients with a final positive diagnosis of extrapulmonary tuberculosis (EPTB group, 54.4%) and 99 finally diagnosed as without TB (non-EPTB group, 45.6%). The risk factor for EPTB was HIV co-infection (OR 2.22, 95% CI 1.17-4.28, p = 0.014). In biopsy samples, GeneXpert (Xpert) showed higher sensitivity (96.6% [91.6-98.7], p < 0.0001) than culture (56.1% [47.0-64.9]). Regardless of HIV status, Xpert had the highest sensitivity (>95%) and specificity (nearly 100%) of any methods. In non-biopsy samples, only T-SPOT.TB (T-SPOT) showed higher sensitivity than culture (90.9% [62.3-99.5] vs 35.3% [17.3-58.7], p = 0.0037). Furthermore, the sensitivities of Xpert were lower in non-biopsy samples (60.0% [23.1-92.9], p = 0.022) than in biopsy samples (100% [86.7-100]). Even in smear-negative biopsy samples, Xpert still had higher sensitivity than culture and retained high specificity (100% [95.7-100]). Conclusion: Superior performance of Xpert in diagnosing EPTB was observed regardless of HIV status and specimen types. Nevertheless, the biopsy samples still substantially facilitated the accurate diagnosis of extrapulmonary tuberculosis.
Subject(s)
HIV Infections , Mycobacterium tuberculosis , Tuberculosis, Extrapulmonary , Tuberculosis , Humans , Tuberculosis/diagnosis , Retrospective Studies , Sensitivity and Specificity , HIV Infections/complications , Ultrasonography, InterventionalABSTRACT
High-hydration hydrogels based on carbohydrate polymers and green preparation methods have attracted intensive research focus recently. Driven by the attractive functions of starch, oxidized maize starch (OMS) was chosen and the related hydrogel (3D-OMS) was constructed by hot-extrusion 3D printing (HE-3DP). Meanwhile, the effect of different OMS concentrations (11 %-19 %) on its printability, structure and hydration properties were systematically investigated. The results showed that the formation of porous structure during HE-3DP environment contributed to rapid water absorption and well water holding capacity of 3D-OMS. Interestingly, as the OMS concentration increased from 11 % to 19 %, the 3D-OMS presented great hydration properties, with its maximum water absorption capacity and water holding capacity reaching 3013.43 % (11-OMS) and 93.53 % (19-OMS), respectively. Among them, 13 % was the best concentration for HE-3DP. Besides, 3D-OMS also exhibited good biodegradability and cytocompatibility. These results demonstrated potential for developing new starch-based biomedical hydrogel with great hydration properties through HE-3DP technology.
Subject(s)
Hydrogels , Starch , Hydrogels/chemistry , Porosity , Printing, Three-Dimensional , Starch/chemistry , WaterABSTRACT
BACKGROUND: Recombinant protein subunit vaccination is considered to be a safe, fast and reliable technique when combating emerging and re-emerging diseases such as coronavirus disease 2019 (COVID-19). Typically, such subunit vaccines require the addition of adjuvants to attain adequate immunogenicity. AS01, which contains adjuvants MPL and saponin QS21, is a liposome-based vaccine adjuvant system that is one of the leading candidates. However, the adjuvant effect of AS01 in COVID-19 vaccines is not well described yet. METHODS: In this study, we utilized a mixture of AS01 as the adjuvant for an S1 protein-based COVID-19 vaccine. RESULTS: The adjuvanted vaccine induced robust immunoglobulin G (IgG) binding antibody and virus-neutralizing antibody responses. Importantly, two doses induced similar levels of IgG binding antibody and neutralizing antibody responses compared with three doses and the antibody responses weakened only slightly over time up to six weeks after immunization. CONCLUSION: These results suggested that two doses may be enough for a clinical vaccine strategy design using MPL & QS21 adjuvanted recombinant protein, especially in consideration of the limited production capacity of COVID-19 vaccine in a public health emergency.
Subject(s)
Antigens, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Lipid A/analogs & derivatives , SARS-CoV-2/immunology , Saponins/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Vaccine/administration & dosage , Animals , Antibodies, Neutralizing , Antibodies, Viral/metabolism , Antibody Formation , COVID-19/virology , Dose-Response Relationship, Immunologic , Drug Combinations , Female , HEK293 Cells , Humans , Immunization , Immunogenicity, Vaccine , Lipid A/administration & dosage , Lipid A/immunology , Mice, Inbred BALB C , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Saponins/administration & dosageABSTRACT
Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.
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Based on molecular simulations, the synthetic route of water-insoluble gardenia blue pigment was prepared by the reaction of genipin and L-Phenylalanine methyl ester hydrochloride. A highly purified pigment was obtained after extraction by chloroform and purification by silica gel column chromatography, and the value of color is up to 288. A study on the structural characteristics of the pigment was implemented with a scanning electron microscope, ultraviolet-visible spectrophotometer, Fourier transform infrared spectrometer, X-ray photoelectron spectrometer, and quatropde-time of flight mass spectrometer. The results showed that the surface of the pigment was largely smooth and spherical; The λmax was 607 nm, and the main functional groups include O-C=O, C=O, C-N, C=C, OH, and benzene ring; We detrained six different molecular weight and chemical structures of pigments and speculated the particular structures and formation mechanisms of three kinds of pigment, whose molecular weights are 690.1156, 720.1226, and 708.1246 Da, respectively. The pigment was only able to be dissolved in ethanol, methanol, acetone, ethyl acetate, and other strong polar organic solvents, but was not able to be dissolved in water, ethyl ether, petroleum ether, and other weak polar organic solvents. In terms of light and thermal stabilities, water-insoluble gardenia blue pigment is significantly better than water-soluble gardenia blue pigment (p < 0.05). When it is under direct light for 7 days or incubated at 80-120 °C for 24 h, the pigment residual rates were 74.90, 95.26, 88.27, and 87.72%, respectively.
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A raw starch digesting α-amylase from Nile tilapia (Oreochromis niloticus) intestine was identified. The α-amylase, AMY-T, had an estimated molecular weight of 60 kDa and purified to near homogeneity. AMY-T showed an apparent KM 4.78 mg/mL and Vmax 0.44 mg/mL/min) towards soluble starch. It was highly stable for 24 h in the pH range 3.0-10.0, and to solvents like methanol, isopropanol, butanol, dimethylformamide, DMSO and ethyl-ether. AMY-T was able to digest different carbohydrates, mainly showing endo-activity. Importantly, AMY-T was catalytically efficient and adsorbing towards raw potato starch at temperature documented for other raw starch digesting α-amylases. Thin layer and anion exchange chromatography characterization showed that the end products of raw starch hydrolysis were glucose, maltose and maltodextrins, with degree of polymerisation ranging 1-8. Scanning electron microscopy analysis of the AMY-T treated starch granules documented both granular exo- and endo-attack by AMY-T. These catalytic capabilities suggest high potential for AMY-T for industrial use.
Subject(s)
Cichlids/metabolism , Intestines/enzymology , Starch/metabolism , alpha-Amylases/metabolism , Animals , Chromatography, Ion Exchange , Chromatography, Thin Layer , Hydrogen-Ion Concentration , Hydrolysis , Maltose/analysis , Polysaccharides/analysis , Starch/analysis , TemperatureABSTRACT
Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems.