Exogenous Loading Research Articles

Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses

Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8+ T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4–HCV paradox.

Human perturbation on phosphorus cycles in one of China’s most eutrophicated lakes

Lake Dianchi is one of the most phosphorus-polluted lakes in China. Understanding its phosphorus budget and the corresponding limitations for research are fundamental to lake management. We have therefore systematically analyzed publications and publicly available datasets regarding Dianchi P pollution since the year 1960 to explore the anthropogenic perturbation on phosphorus budget dynamics. Results show that current exogenous loading (> 600 tons ⋅ a −1 ) remains significant; inflow rivers and drainage pipes around the lakeshore are the main pathways (accounting for around 80% of loading). Besides shifts in the spatial–temporal pattern of exogenous loading, artificial treatments have also reshaped P removal patterns; consequently, only a small amount of P can be removed through lake outflows (<20%, around 140 tons ⋅ a −1 ). Although a reduction has been observed in exogenous P loading over the past decade, endogenous P loading has significantly increased, of which P flux via sediment release alone approximates more than 180 tons ⋅ a −1 in 2017. Generally, P budget improvements have been due to artificial treatments such as the Niulan River water diversion project, shifting directions on tail water emissions, and sediment dredging projects. However, these treatments also introduce negative consequences and therefore require further caution. The reuse of ”waste P” via treatments has especially become a challenge. To date, exogenous loading via atmospheric dry deposition, wintering birds, water diversion, degradation in geological phosphorus-rich regions and endogenous P cycling fluxes, etc., need further study. Here, suggestions for corresponding research are given. It is suggested that the restoration of spontaneous ”P balance” in the Dianchi system, and the modification of the regional urban development strategy should present issues facing plateau freshwater lakes in SW China which need to be addressed. Moreover, synthesized analyses and machine learning processes are new attempts worth promoting to other lakes where monitoring data is incomplete. • P budget dynamics of Lake Dianchi were calculated based on 254 publications. • Human perturbation has reshaped the spatial–temporal pattern of exogenous loading. • Current P ”balance” can be maintained only with the help of artificial treatments. • Artificial treatments introduce unintended consequences and require further caution. • P mitigation issue in Lake Dianchi epitomize eutrophication concerns in SW China.

A call for the standardised reporting of factors affecting the exogenous loading of extracellular vesicles with therapeutic cargos.

Extracellular vesicles (EVs) are complex nanoparticles required for the intercellular transfer of diverse biological cargoes. Unlike synthetic nanoparticles, EVs may provide a natural platform for the enhanced targeting and functional transfer of therapeutics across complex and often impenetrable biological boundaries (e.g. the blood-brain barrier or the matrix of densely organised tumours). Consequently, there is considerable interest in utilising EVs as advanced drug delivery systems for the treatment of a range of challenging pathologies. Within the past decade, efforts have focused on providing standard minimal requirements for conducting basic EV research. However, no standard reporting framework has been established governing the therapeutic loading of EVs for drug delivery applications. The purpose of this review is to critically evaluate progress in the field, providing an initial set of guidelines that can be applied as a benchmark to enhance reproducibility and increase the likelihood of translational outcomes.

Micromechanical properties and functional activity of granulocytes when simulating exogenous loading with ATP in vitro

The micromechanical properties of leukocytes make a certain contribution to the blood flow velocity in the microcirculatory bed, while the micromechanical properties themselves change under the influence of a complex network of purinergic signals.The aim of the work was to study the micromechanical properties and functional activity of granulocytes in normal conditions and in patients with acute lymphoblastic leukemia when simulating exogenous loading with ATP in vitro.Materials and methods. Leukocytes were isolated from the blood of patients with acute lymphoblastic leukemia and healthy people. Each sample was divided into a test sample and a control sample. In the test samples, the loading with ATP in vitro was simulated. Leukocytes of the control samples were incubated in the culture medium without the addition of ATP. Young’s modulus and adhesion force were measured using an atomic force microscope in the force spectroscopy mode. The cell surface potential was measured in an atomic force microscope in the Kelvin mode. To assess the functional activity of granulocytes, hypoosmotic tests in vitro and determination of migration activity were used.Results. In tests with exogenous ATP, both in samples from healthy people and from patients with acute lymphoblastic leukemia, a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties of leukocytes and migration activity were found. At the same time, the responses of granulocytes to the osmotic loading were different: for example, in the group of healthy people, the loading with ATP caused cell contraction and a decrease in the use of the membrane reserve by the cell in a hypotonic environment, and in patients with acute lymphoblastic leukemia, it caused an increase in the volume and more intensive use of the membrane reserve in volume regulation.Conclusion. The revealed effects indicate the leading role of the ATP molecule in the signal transduction mechanisms between blood cells in the microvasculature. The increase in the adhesive properties of the cell surface of granulocytes revealed in the study, in parallel with the increase in their migration activity under the influence of the ATP molecule, can contribute to the development of inflammation in the vessel wall.

БИОФИЗИЧЕСКИЕ СВОЙСТВА КЛЕТОЧНОЙ ПОВЕРХНОСТИ И ФУНКЦИОНАЛЬНАЯ АКТИВНОСТЬ ГРАНУЛОЦИТОВ БОЛЬНЫХ ОСТРЫМ ЛИМФОБЛАСТНЫМ ЛЕЙКОЗОМ ПРИ МОДЕЛИРОВАНИИ НАГРУЗКИ С АТФ IN VITRO

The current study is devoted to the investigation of the biophysical properties of the cell surface and the functional activity of granulocytes in patients with acute lymphoblastic leukemia when simulating the ATP load in vitro. The experiment was performed on the peripheral blood of patients with acute lymphoblastic leukemia (ALL) who underwent a standard course of chemotherapy. In experimental tests, exogenous loading with ATP was simulated in vitro by adding 100.0 μM adenosine-5-triphosphate disodium salt trihydrate to the granulocyte suspension. Incubation with the drug was carried out for 15 min at 370 C. As a control, a suspension of granulocytes in RPMI 1640 medium from the same patient, but without the addition of the drug, was used, this was incubated for 15 min at a temperature of 370 C. After the incubation time, the biophysical properties (rigidity, charge of the cell surface, the strength of intercellular adhesion) of granulocytes in experimental and control samples were studied using atomic force microscopy, and the migration activity of cells was also assessed in a direct capillary test under agarose. A model with exogenous ATP in ALL patients showed a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties and migration activity of granulocytes. The revealed effects point to the key role of the ATP molecule in the mechanisms of intercellular signaling in the microvasculature.

Extracellular Vesicles Derived from a Human Brain Endothelial Cell Line Increase Cellular ATP Levels.

Engineered cell-derived extracellular vesicles (EVs) such as exosomes and microvesicles hold immense potential as safe and efficient drug carriers due to their lower immunogenicity and inherent homing capabilities to target cells. In addition to innate vesicular cargo such as lipids, proteins, and nucleic acids, EVs are also known to contain functional mitochondria/mitochondrial DNA that can be transferred to recipient cells to increase cellular bioenergetics. In this proof-of-concept study, we isolated naïve EVs and engineered EVs loaded with an exogenous plasmid DNA encoding for brain-derived neurotrophic factor (BDNF-EVs) from hCMEC/D3, a human brain endothelial cell line, and RAW 264.7 macrophages. We tested whether mitochondrial components in naïve or engineered EVs can increase ATP levels in the recipient brain endothelial cells. EVs (e.g., exosomes and microvesicles; EXOs and MVs) were isolated from the conditioned medium of either untreated (naïve) or pDNA-transfected (Luc-DNA or BDNF-DNA) cells using a differential centrifugation method. RAW 264.7 cell line-derived EVs showed a significantly higher DNA loading and increased luciferase expression in the recipient hCMEC/D3 cells at 72h compared with hCMEC/D3 cell line-derived EVs. Naïve EVs from hCMEC/D3 cells and BDNF-EVs from RAW 264.7 cells showed a small, but a significantly greater increase in the ATP levels of recipient hCMEC/D3 cells at 24 and 48h post-exposure. In summary, we have demonstrated (1) differences in exogenous pDNA loading into EVs as a function of cell type using brain endothelial and macrophage cell lines and (2) EV-mediated increases in the intracellular ATP levels in the recipient hCMEC/D3 monolayers.

Exosomes and Extracellular Vesicles as Emerging Theranostic Platforms in Cancer Research.

Exosomes are endosome-derived nanovesicles produced by healthy as well as diseased cells. Their proteic, lipidic and nucleic acid composition is related to the cell of origin, and by vehiculating bioactive molecules they are involved in cell-to-cell signaling, both in healthy and pathologic conditions. Being nano-sized, non-toxic, biocompatible, scarcely immunogenic, and possessing targeting ability and organotropism, exosomes have been proposed as nanocarriers for their potential application in diagnosis and therapy. Among the different techniques exploited for exosome isolation, the sequential ultracentrifugation/ultrafiltration method seems to be the gold standard; alternatively, commercially available kits for exosome selective precipitation from cell culture media are frequently employed. To load a drug or a detectable agent into exosomes, endogenous or exogenous loading approaches have been developed, while surface engineering procedures, such as click chemistry, hydrophobic insertion and exosome display technology, allow for obtaining actively targeted exosomes. This review reports on diagnostic or theranostic platforms based on exosomes or exosome-mimetic vesicles, highlighting the diverse preparation, loading and surface modification methods applied, and the results achieved so far.

Gene Editing by Extracellular Vesicles.

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.

Exploiting synergistic effect of externally loaded bFGF and endogenous growth factors for accelerated wound healing using heparin functionalized PCL/gelatin co-spun nanofibrous patches

Growth factors (GFs) are signaling molecules that are principle mediators in tissue regeneration. Biomaterial scaffolds employed as wound dressings are often hampered by their limitations to deliver GFs exogenously due to their instability and low half-life. The key to overcome this challenge lies in the better organization and use of endogenous pro-regenerative GFs released at regenerative site, with an aim to minimize the sole dependency on exogenous factors. Considering such challenges, this research utilizes the exogenous and endogenous GFs sequestering capability of heparin functionalized PCL/gelatin co-spun nanofabrics to mediate synergistically driven tissue regeneration by utilizing combined therapeutic effect of exogenous and endogenous GFs, and thereby minimizing the sole dependency on exogenous GFs for tissue regeneration. Basic fibroblast growth factor (bFGF) was chosen as GF for exogenous loading whereas vascular endothelial growth factor (VEGF) was chosen as a representative example to demonstrate the endogenous pro-regenerative GF sequestration capability of fabricated nanofabrics. From our results, the fabricated nanofabrics showed loading efficiency of 80% for exogenous bFGF and can sequester 15-fold more amount of endogenous VEGF compared to non-heparin functionalized nanofibrous dressings. When applied as wound dressings, heparin functionalized nanofibers showed better therapeutic capability compared to control groups that were treated using patches without heparin functionalization, indicating endogenously driven tissue regeneration. This was indicated by significant higher number of newly formed skin appendages, lesser scarring and lower inflammatory levels in newly formed granulation. Additionally, further improvements in therapeutic effect was observed when exogenous bFGF was employed indicating effectiveness of synergistically mediated tissue regeneration.

Cancer Nanomedicine Special Issue Review Anticancer Drug Delivery with Nanoparticles: Extracellular Vesicles or Synthetic Nanobeads as Therapeutic Tools for Conventional Treatment or Immunotherapy

Both natural and synthetic nanoparticles have been proposed as drug carriers in cancer treatment, since they can increase drug accumulation in target tissues, optimizing the therapeutic effect. As an example, extracellular vesicles (EV), including exosomes (Exo), can become drug vehicles through endogenous or exogenous loading, amplifying the anticancer effects at the tumor site. In turn, synthetic nanoparticles (NP) can carry therapeutic molecules inside their core, improving solubility and stability, preventing degradation, and controlling their release. In this review, we summarize the recent advances in nanotechnology applied for theranostic use, distinguishing between passive and active targeting of these vehicles. In addition, examples of these models are reported: EV as transporters of conventional anticancer drugs; Exo or NP as carriers of small molecules that induce an anti-tumor immune response. Finally, we focus on two types of nanoparticles used to stimulate an anticancer immune response: Exo carried with A Disintegrin And Metalloprotease-10 inhibitors and NP loaded with aminobisphosphonates. The former would reduce the release of decoy ligands that impair tumor cell recognition, while the latter would activate the peculiar anti-tumor response exerted by γδ T cells, creating a bridge between innate and adaptive immunity.

The Ins and Outs of TAPBPR.

Peptide presentation on MHC class I molecules (MHC-I) is central to mounting effective antiviral and antitumoral immune responses. The tapasin-related protein TAPBPR is an MHC-I peptide editor which shapes the final peptide repertoire displayed on the cell surface. Here, we review recent findings which further elucidate the mechanisms by which TAPBPR performs peptide editing on a molecular level, and how glycosylation on MHC-I influences the interaction with TAPBPR and the peptide loading complex. We also explore how the function of TAPBPR can be utilized to promote exogenous peptide loading directly onto plasma-membrane expressed MHC-I. This has led to the development of new assays to investigate TAPBPR-mediated peptide editing and uncovered translational opportunities of utilizing TAPBPR to treat human disease.

Human Activities Inducing High CH4 Diffusive Fluxes in an Agricultural River Catchment in Subtropical China

Methane (CH4) is one of the key greenhouse gases (GHGs) in the atmosphere with current concentration of 1859 ppb in 2017 due to climate change and anthropogenic activities. Rivers are of increasing concern due to sources of atmospheric CH4. However, knowledge and data limitations exist for field studies of subtropical agricultural river catchments, particularly in southern China. The headspace balance method and the diffusion model method were employed to assess spatiotemporal variations of CH4 diffusive fluxes from April 2015 to January 2016 in four order reaches (S1, S2, S3, and S4) of the Tuojia River, Hunan, China. Results indicated that both the dissolved concentrations and diffusive fluxes of CH4 showed obvious spatiotemporal variations. The observed mean concentration and diffusive flux of CH4 were 0.40 ± 0.02 μmol L−1 and 41.19 ± 2.50 µg m−2 h−1, respectively, showing the river to be a strong source of atmospheric CH4. The CH4 diffusive fluxes during the rice-growing seasons were significantly greater than the winter fallow season (an increase of 80.26%). The spatial distribution of CH4 diffusive fluxes increased gradually from (17.58 ± 1.42) to (55.56 ± 4.32) µg m−2 h−1 due to the organic and nutrient loading into the river waterbodies, with the maximum value at location S2 and the minimum value at location S1. Correlation analysis showed that the CH4 diffusive fluxes exhibited a positive relationship with the dissolved organic carbon (DOC), salinity, and water temperature (WT), while a negative correlation occurred between CH4 diffusive fluxes and the dissolved oxygen (DO) concentration, as well as the pH value. Our findings highlighted that a good understanding of exogenous nutrient loading in agricultural catchments will clarify the influence of human activities on river water quality and then constrain the global CH4 budget.

P729The functional relevance of bile acids in the improvement of HDL-mediated endothelial protection after bariatric surgery

Abstract Introduction Roux-en-Y gastric bypass (RYGB) reduces cardiovascular mortality. We showed that high density lipoproteins (HDL)-mediated vasoprotection is improved early after RYGB. Circulating BAs increase upon RYGB and contribute to the weight-loss independent metabolic improvements after surgery. Bile acids (BA) are signaling molecules increasingly recognized as regulators of cardiometabolic homeostasis. BAs circulate in the blood either free or bound to albumin and HDL. The signaling role of HDL-bound BAs (HDL-BAs) is unknown. Indeed, HDL may facilitate BA delivery directly to endothelial cells where BA may synergize with HDL to promote vasoprotection. Purpose We studied whether RYGB changes the composition of HDL-BA and whether HDL functional properties may be modulated by specific BA bound to HDL. Methods HDL were isolated by ultracentrifugation from 29 morbidly obese patients before and 1 year after RYGB. The HDL-BA composition was determined by liquid chromatography-mass spectrometry (LC/MS-MS) and HDL vasoprotective properties were evaluated ex-vivo in human aortic endothelial cells (HAEC). The size and abundance of HDL particles were determined by NMR spectroscopy in plasma. Results The increase in total BA concentrations observed in plasma 1 year after RYGB also translated into higher concentrations (up to 25%) of BA bound to HDL. Moreover, obesity-induced HDL dysfunction was reversed after surgery, as shown by improved HDL-mediated endothelial NO production, anti-apoptotic effects and cholesterol efflux capacity. The size function analyses showed a post-operative shift towards larger HDL. After RYGB there was a remodeling of BA bound to HDL, which are either agonists of the endothelial nuclear farnesoid X receptor (FXR), e.g. chenodeoxy-CA (CDCA), cholic acid (CA) or for the membrane TGR5 receptor, e.g. deoxy-CA (DCA). The composition-function analysis revealed that among all BA subclasses, the specific enrichment in CA and in CDCA bound to HDL correlated with an improved endothelial anti-apoptotic capacity of HDL (R −0.52, p=0.006 for CA-HDL and R −0.35, p=0.07 for CDCA-HDL). Further, the exogenous loading of CA onto healthy native HDL isolated from human serum significantly enhanced their endothelial anti-apoptotic function. In the case of obese, dysfunctional, pro-apoptotic HDL, exogenous CA loading was able to restore HDL anti-apoptotic function. Conclusion Exogenous loading of CA restored HDL anti-apoptotic function of HDL from obese patients mimicking the beneficial remodeling of BA bound to HDL observed after RYGB. These results suggest a crucial interaction between endothelial cells and BA in the improvement of HDL's vasoprotective properties. Acknowledgement/Funding Swiss national Science Foundation Ambizione and PRIMA grant to EO

Sensitization of Tumors for Attack by Virus-Specific CD8+ T-Cells Through Antibody-Mediated Delivery of Immunogenic T-Cell Epitopes.

Anti-tumor immunity is limited by a number of factors including the lack of fully activated T-cells, insufficient antigenic stimulation and the immune-suppressive tumor microenvironment. We addressed these hurdles by developing a novel class of immunoconjugates, Antibody-Targeted Pathogen-derived Peptides (ATPPs), which were designed to efficiently deliver viral T-cell epitopes to tumors with the aim of redirecting virus-specific memory T-cells against the tumor. ATPPs were generated through covalent binding of mature MHC class I peptides to antibodies specific for cell surface-expressed tumor antigens that mediate immunoconjugate internalization. By means of a cleavable linker, the peptides are released in the endosomal compartment, from which they are loaded into MHC class I without the need for further processing. Pulsing of tumor cells with ATPPs was found to sensitize these for recognition by virus-specific CD8+ T-cells with much greater efficiency than exogenous loading with free peptides. Systemic injection of ATPPs into tumor-bearing mice enhanced the recruitment of virus-specific T-cells into the tumor and, when combined with immune checkpoint blockade, suppressed tumor growth. Our data thereby demonstrate the potential of ATPPs as a means of kick-starting the immune response against “cold” tumors and increasing the efficacy of checkpoint inhibitors.

Abstract 2303: In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models

Abstract Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures. Citation Format: Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2303.

An Invertible Mathematical Model of Cortical Bone\u2019s Adaptation to Mechanical Loading

Determination of mechanical loading regimen that would induce a prescribed new bone formation rate and its site-specific distribution, may be desirable to treat some orthopaedic conditions such as bone loss due to muscle disuse, e.g. because of space flight, bed-rest, osteopenia etc. Site-specific new bone formation has been determined earlier experimentally and numerically for a given loading regimen; however these models are mostly non-invertible, which means that they cannot be easily inverted to predict loading parameters for a desired new bone formation. The present work proposes an invertible model of bone remodeling, which can predict loading parameters such as peak strain, or magnitude and direction of periodic forces for a desired or prescribed site-specific mineral apposition rate (MAR), and vice versa. This fast, mathematical model has a potential to be developed into an important aid for orthopaedic surgeons for prescribing exercise or exogenous loading of bone to treat bone-loss due to muscle disuse.

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Glucose and lipid metabolic adaptations during postprandial starvation of Japanese flounder Paralichthys olivaceus previously fed different levels of dietary carbohydrates

Exogenous glucose loading is generally used to assess the capacity of glucose utilization in carnivorous fish. Examining the use of endogenous glucose during fasting provides a new perspective on assessing glucose metabolism in fish. Japanese flounder Paralichthys olivaceus, a marine carnivorous fish species, were starved for 3, 9, 24, 48 h, and 3, 7, 14, 21, 28 days, respectively, after a 10-week feeding trial with 12% (C12: 49.49% crude protein, 9.65% crude lipid and gross energy of 18.98 kJ g−1) and 20% (C20: 49.99% crude protein, 9.75% crude lipid and gross energy of 19.11 kJ g−1) of dietary carbohydrates (alpha-starch and corn starch). After feeding trial, fish fed different diets showed similar specific growth rate (3.62 vs 3.61) (P > .05). During starvation period, compared with 3 h, plasma glucose and insulin concentration increased at 9 h in both groups. A similar trend was found for the glycogen content and the mRNA levels of genes involved in glycolysis and pentose phosphate pathway in liver. And the gluconeogenic genes mRNA levels significantly increased since 9 h in C12 and 24 h in C20 (P < .05). Subsequently, liver glycogen in C12 fell significantly on the 3 d compared with 48 h and was depleted on the 7 d (P < .05). It significantly decreased on the 7 d compared with 3 d and was minimized on the 14 d in C20 (P < .05). Plasma glucose peaked at 48 h and 7 d in both groups. As for lipid metabolism, high levels of plasma triglyceride, cholesterol concentration and lipogenetic genes mRNA levels were observed within 9 h. Compared with 3 h, liver lipid significantly decreased on the 14 d in both groups (P < .05). The mRNA levels of genes related to beta-oxidation and ketogenesis was enhanced in later stages in both groups. In addition, in C20, greater changes of mRNA levels of genes involved in gluconeogenesis and lipolysis in liver were observed. It was concluded that primary substrate of energy sources switched from glycogen to lipid occurring on the 3 d-7 d in C12 and 7 d-14 d in C20. Besides, high dietary carbohydrate (C20) feeding activated greater gluconeogenesis and lipolysis during starvation. These observations demonstrated glucose and lipid metabolic responses of Japanese flounder during starvation were influenced by previous feeding with different levels of dietary carbohydrates.

Static Preload Inhibits Loading-Induced Bone Formation.

Nearly all exogenous loading models of bone adaptation apply dynamic loading superimposed upon a time invariant static preload (SPL) in order to ensure stable, reproducible loading of bone. Given that SPL may alter aspects of bone mechanotransduction (eg, interstitial fluid flow), we hypothesized that SPL inhibits bone formation induced by dynamic loading. As a first test of this hypothesis, we utilized a newly developed device that enables stable dynamic loading of the murine tibia with SPLs ≥ -0.01 N. We subjected the right tibias of BALB/c mice (4-month-old females) to dynamic loading (-3.8 N, 1 Hz, 50 cycles/day, 10 s rest) superimposed upon one of three SPLs: -1.5 N, -0.5 N, or -0.03 N. Mice underwent exogenous loading 3 days/week for 3 weeks. Metaphyseal trabecular bone adaptation (μCT) and midshaft cortical bone formation (dynamic histomorphometry) were assessed following euthanasia (day 22). Ipsilateral tibias of mice loaded with a -1.5-N SPL demonstrated significantly less trabecular bone volume/total volume (BV/TV) than contralateral tibias (-12.9%). In contrast, the same dynamic loading superimposed on a -0.03-N SPL significantly elevated BV/TV versus contralateral tibias (12.3%) and versus the ipsilateral tibias of the other SPL groups (-0.5 N: 46.3%, -1.5 N: 37.2%). At the midshaft, the periosteal bone formation rate (p.BFR) induced when dynamic loading was superimposed on -1.5-N and -0.5-N SPLs was significantly amplified in the -0.03-N SPL group (>200%). These data demonstrate that bone anabolism induced by dynamic loading is markedly inhibited by SPL magnitudes commonly implemented in the literature (ie, -0.5 N, -1.5 N). The inhibitory impact of SPL has not been recognized in bone adaptation models and, as such, SPLs have been neither universally reported nor standardized. Our study therefore identifies a previously unrecognized, potent inhibitor of mechanoresponsiveness that has potentially confounded studies of bone adaptation and translation of insights from our field. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.