Locally Sustainable Biodiesel Production from Waste Cooking Oil and Grease Using a Deep Eutectic Solvent: Characterization, Thermal Properties, and Blend Performance.

As part of local sustainability efforts, biodiesel was synthesized via transesterification using a deep eutectic solvent (DES) without further washing from on-campus, dining facility waste cooking oil and grease. Before moving forward with repurposing used DES as a solvent in chemistry teaching labs, we determined the suitability of the biodiesel as an alternative fuel blended with diesel to power campus utility vehicles. Biodiesel components were characterized by gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (1H NMR), viscometer, differential scanning calorimetry (DSC), and evolved gas analysis during pyrolysis with a thermogravimetric analyzer coupled with FTIR (TGA-FTIR). The four major components of fatty acid methyl esters (FAMEs) in the biodiesel were methyl oleate, methyl linoleate, methyl palmitate, and methyl stearate. Kinematic viscosity over typical temperature ranges was within optimal values recommended by the American Biodiesel Standard (ASTM D6751), with a 30:70 biodiesel/diesel blend experimental viscosity of 3.43 cSt at 40 °C and a calculated viscosity of 10.13 cSt at 0 °C. The pure biodiesel's cold-temperature onset of crystal formation is -10.1 °C versus -16.4 °C for a 30:70 biodiesel/diesel blend. Pyrolysis indicates good thermal stability, however, with an increased CO2 evolution in the blended fuel at higher temperatures as compared to that in the pure biodiesel and the pure diesel. Combustion gas analysis indicates virtually complete combustion of the blended fuel to CO2 and H2O with only trace amounts of CO. Overall results indicate that the biodiesel synthesized using DES is a suitable fuel for campus utility vehicles in the local moderate temperature climate and affords increased local sustainability by using used DES repurposed in our chemistry teaching labs.

Desensitized chimeric antigen receptor T cells selectively recognize target cells with enhanced antigen expression.

Chimeric antigen receptor (CAR) T cell therapy is an effective method for treating specific cancers. CARs are normally designed to recognize antigens, which are highly expressed on malignant cells but not on T cells. However, when T cells are engineered with CARs that recognize antigens expressed on the T cell surface, CAR T cells exhibit effector function on other T cells, which results in fratricide, or killing of neighboring T cells. Here, using human leukocyte antigen-DR (HLA-DR)-targeted CAR T cells, we show that weak affinity between CAR and HLA-DR reduces fratricide and induces sustained CAR downregulation, which consequently tunes the avidity of CAR T cells, leading to desensitization. We further demonstrate that desensitized CAR T cells selectively kill Epstein-Barr virus-transformed B cells with enhanced HLA-DR expression, while sparing normal B cells. Our study supports an avidity-tuning strategy that permits sensing of antigen levels by CAR T cells.

Relationship of cytokine levels and clinical effect on platelet-rich plasma-treated lateral epicondylitis.

Lateral epicondylitis (LE) is difficult to manage and can result in significant patient morbidity. Currently, the clinical use of platelet-rich plasma (PRP) for painful tendons has received attention, but its efficacy remains controversial. This study aimed to investigate the clinical effects of PRP and its biological components. A total of 156 patients with LE were randomly divided into group 1, treated with a single injection of 2-ml autologous PRP, and group 2, treated with a control received only physical therapy without injection. Both groups used a tennis elbow strap and performed stretching and strengthening exercises during 24 weeks' follow-up. Pain and functional improvements were assessed using the visual analog scale (VAS), Modified Mayo Clinic Performance Index for the elbow, and magnetic resonance imaging (MRI). White blood cell count, platelet count, and levels of platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor, epithelial growth factor, and interleukin-1 ß in PRP were measured and investigated for statistical correlation with the clinical score. At 24 weeks, all pain and functional variables, including VAS score, Mayo Clinic performance scores, and MRI grade, improved significantly in group 1 (p < 0.05). PDGF-AB, PDGF-BB, and TGF-ß levels were more significantly increased in PRP than in whole blood. TGF-ß level significantly correlated with Mayo Clinic performance score and MRI grade improvement. Thus, TGF-ß level in PRP is considered to play a pivotal role in tendon healing. These results may contribute to identifying the best protocol for PRP application in tendinopathies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:913-920, 2018.

The repopulating cancer cells in melanoma are characterized by increased mitochondrial membrane potential.

Although considerable effort has been expended in identifying definitive markers for cancer stem cells (CSCs) or cancer-initiating cells (CICs), the phenotypic plasticity of these cells obviates simple characterization using cell surface markers. We hypothesized that these cells could be characterized by their metabolic properties because they are in a quiescent state with low energy needs. We examined whether cancer cells differ in mitochondrial membrane potential (Δψm) when they are under stress. The Δψm of B16-F10 melanoma cells increased when they were exposed in vitro to serum starvation and chemotherapeutic agents, but not when exposed to hypoxia. Such TMREhigh cells were also present in tumor tissue. They primarily used glucose and/or lactate, and were superior to TMRElow B16-F10 cells in their ability to drive tumor growth. These findings suggest that CSCs or CICs could be identified in heterogeneous melanoma populations by measuring Δψm.

Observing Social Exclusion Leads to Dehumanizing the Victim.

We hypothesized that observing social exclusion would influence observers' judgments of the humanness of its victims and perpetrators. Specifically, we speculated that people would attribute victims and perpetrators to lower and higher mental capacities, respectively. Participants observed a simulated computer-based ball tossing game in which one of the players was either ostracized or not. They then rated the game players on traits associated with two dimensions of humanness, namely Human Nature (HN) and Human Uniqueness (HU). Overall, participants who witnessed an exclusion game judged the victim as less human on both domains compared to one of the perpetrators as well as to a player in the control condition. The perpetrator was attributed higher HN, but not significantly higher HU, compared to the control player. In addition, the less HN attributes a target was assigned, the more she was expected to be vulnerable to exploitation. On most of the other measures of target impression, however, the victim was rated more favorably than the perpetrator. The findings imply that social exclusion victims are often subtly derogated compared to the perpetrators, even while they are also more positively evaluated otherwise.

Authentic GITR Signaling Fails To Induce Tumor Regression unless Foxp3+ Regulatory T Cells Are Depleted.

The glucocorticoid-induced TNFR family-related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell-mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3(+)CD4(+) Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4(+) cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

4-1BB Signaling Enhances Primary and Secondary Population Expansion of CD8+ T Cells by Maximizing Autocrine IL-2/IL-2 Receptor Signaling.

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is primarily expressed on activated T cells and is known to enhance proliferation of T cells, prevent activation-induced cell death, and promote memory formation of CD8+ T cells. In particular, it is well acknowledged that 4-1BB triggering preferentially enhances the expansion of CD8+ T cells rather than CD4+ T cells, but the underlying mechanism remains unclear. Here we found that 4-1BB triggering markedly increased IL-2Rα (CD25) and IL-2 expressions of CD8+ T cells but minimally for CD4+ T cells. Proliferation of CD8+ T cells was moderately enhanced by direct 4-1BB triggering in the absence of signaling through IL-2Rα/IL-2 interactions, but further promoted in the presence of IL-2Rα/IL-2 interactions. Among the TNFRSF members including OX40, GITR, CD30, and CD27, 4-1BB was superior in the ability to induce IL-2Rα expression on CD8+ T cells. When the primary and secondary expansions of CD8+ T cells in vivo were examined by adoptively transferring OVA-specific CD8+ T cells along with the treatment with agonistic anti-4-1BB and/or antagonistic anti-CD25 F(ab')2 mAb, 4-1BB triggering enhanced both primary and secondary expansion of CD8+ T cells in vivo, and the 4-1BB effects were moderately suppressed in primary expansion while completely abolished in secondary expansion of OVA-specific CD8+ T cells by blocking IL-2Rα. These results suggest that 4-1BB-mediated increases of IL-2Rα and IL-2 prolong the effects of transient TCR- and 4-1BB-mediated signaling in CD8+ T cells, and that 4-1BB triggering preferentially enhances the expansion of CD8+ T cells through the amplification of autocrine IL-2/IL-2R signaling loop.

In vivo 4-1BB deficiency in myeloid cells enhances peripheral T cell proliferation by increasing IL-15.

4-1BB signals are considered positive regulators of T cell responses against viruses and tumors, but recent studies suggest that they have more complex roles in modulating T cell responses. Although dual roles of 4-1BB signaling in T cell responses have been suggested, the underlying mechanisms are still not fully understood. In this study, we tested whether 4-1BB expression affected T cell responses differently when expressed in myeloid versus lymphoid cells in vivo. By assessing the proliferation of 4-1BB(+/+) and 4-1BB(-/-) T cells in lymphocyte-deficient RAG2(-/-) and RAG2(-/-)4-1BB(-/-) mice, we were able to compare the effects on T cell responses of 4-1BB expression on myeloid versus T cells. Surprisingly, adoptively transferred T cells were more responsive in tumor-bearing RAG2(-/-)4-1BB(-/-) mice than in RAG2(-/-) mice, and this enhanced T cell proliferation was further enhanced if the T cells were 4-1BB deficient. Dendritic cells (DCs) rather than NK or tissue cells were the myeloid lineage cells primarily responsible for the enhanced T cell proliferation. However, individual 4-1BB(-/-) DCs were less effective in T cell priming in vivo than 4-1BB(+/+) DCs; instead, more DCs in the secondary lymphoid organs of RAG2(-/-)4-1BB(-/-) mice appeared to induce the enhanced T cell proliferation by producing and transpresenting more IL-15. Therefore, we conclude that in vivo 4-1BB signaling of myeloid cells negatively regulates peripheral T cell responses by limiting the differentiation of DCs and their accumulation in secondary lymphoid organs.

Adult stem cells from the hyaluronic acid-rich node and duct system differentiate into neuronal cells and repair brain injury.

The existence of a hyaluronic acid-rich node and duct system (HAR-NDS) within the lymphatic and blood vessels was demonstrated previously. The HAR-NDS was enriched with small (3.0-5.0 µm in diameter), adult stem cells with properties similar to those of the very small embryonic-like stem cells (VSELs). Sca-1(+)Lin(-)CD45(-) cells were enriched approximately 100-fold in the intravascular HAR-NDS compared with the bone marrow. We named these adult stem cells "node and duct stem cells (NDSCs)." NDSCs formed colonies on C2C12 feeder layers, were positive for fetal alkaline phosphatase, and could be subcultured on the feeder layers. NDSCs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(+), while VSELs were Oct4(+)Nanog(+)SSEA-1(+)Sox2(-). NDSCs had higher sphere-forming efficiency and proliferative potential than VSELs, and they were found to differentiate into neuronal cells in vitro. Injection of NDSCs into mice partially repaired ischemic brain damage. Thus, we report the discovery of potential adult stem cells that may be involved in tissue regeneration. The intravascular HAR-NDS may serve as a route that delivers these stem cells to their target tissues.

Nonmarrow hematopoiesis occurs in a hyaluronic-acid-rich node and duct system in mice.

A hyaluronic-acid-rich node and duct system (HAR-NDS) was found on the surface of internal organs of mice, and inside their blood and lymph vessels. The nodes (HAR-Ns) were filled with immune cells of the innate system and were especially enriched with mast cells and histiocytes. They also contained hematopoietic progenitor cells (HPCs), such as granulocyte-macrophage, erythroid, multipotential progenitors, and mast cell progenitors (MCPs). MCPs were the most abundant among the HPCs in HAR-Ns. Their frequency was fivefold higher than that of the MCPs in bone marrow. In addition, the system contained pluripotent stem cells (PSCs) capable of producing CD45(-)Flk1(+) hemangioblast-like cells, which subsequently generated various types of HPCs and differentiated blood cells. Although HAR-Ns did not appear to harbor enough number of cells capable of long-term reconstitution or short-term radioprotection of lethally irradiated recipients, bone marrow cells were able to engraft in the HAR-NDS and reconstitute hematopoietic potentials of the system. PSCs and HPCs were consistently found in intravenous, intralymphatic, and intestinal HAR-ND. We infer that PSCs and HPCs reside in the HAR-ND and that this novel system may serve as an alternative means to traffic immature and mature blood cells throughout the body.

Activation-free printed carbon nanotube field emitters.

When a carbon nanotube paste is formulated based on highly functional hyperbranched polymers such as dipentaerythritol hexaacrylate, the volume shrinkage during thermal curing builds up internal stress that generates microcrack patterns on the printed surface. The nanotubes exposed in the cracks emit electrons successfully at such an extremely low electric field as 0.5 V µm( - 1), and reach 25.5 mA cm( - 2) of current density at 2 Vµm( - 1) from an optimized paste concerning mainly the size and spatial uniformity of the crack. In addition to the superior field emission properties with low manufacturing cost, this activation-free technology can provide a minimized nanohazard in the device fabrication process, compared to those conventional activation technologies developing serious nanoflakes by using destructive methods.

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells.

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

"Same day" ex-vivo regional gene therapy: a novel strategy to enhance bone repair.

Ex-vivo regional gene therapy with bone marrow cells (BMCs) overexpressing bone morphogenetic protein-2 (BMP-2) has demonstrated efficacy in healing critical sized bone defects in preclinical studies. The purpose of this preclinical study was to compare the osteoinductive potential of a novel "same day" ex-vivo regional gene therapy versus a traditional two-step approach, which involves culture expansion of the donor cells before implantation. In the "same day" strategy buffy coat cells were harvested from the rat bone marrow, transduced with a lentiviral vector-expressing BMP-2 for 1 hour and implanted into a rat femoral defect in the same sitting. There was no significant difference (P = 0.22) with respect to the radiographic healing rates between the femoral defects treated with the "same day" strategy (13/13; 100%) versus the traditional two-step approach (11/14; 78%). However, the femoral defects treated with the "same day" strategy induced earlier radiographic bone healing (P = 0.004) and higher bone volume (BV) [micro-computed tomography (micro-CT); P < 0.001]. The "same day" strategy represents a significant advance in the field of ex-vivo regional gene therapy because it offers a solution to limitations associated with the culture expansion process required in the traditional ex vivo approach. This strategy should be cost-effective when adapted for human use.

Design of a Pep-1 peptide-modified liposomal nanocarrier system for intracellular drug delivery: Conformational characterization and cellular uptake evaluation.

In order to facilitate the intracellular delivery of macromolecules, Pep-1 peptide-modified liposomal (Pep1-Lipo) nanocarriers were designed and examined for their in vitro cell translocation capability. Pep-1 peptides were coupled via thiol-maleimide linkage to small unilamellar vesicles composed of phosphatidylcholine, Tween 80, and N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine (MPB-PE). The amount of Pep-1 peptide conjugated to the vesicle was effectively controlled by the amounts of maleimide groups on the vesicular surface, ranging from 70 to 700 molecules per vesicle. Systems were evaluated for cell uptake capacity by monitoring entrapped fluorescence-labeled bevacizumab, a model protein for poorly permeable macromolecule, using confocal microscopy. The novel carriers rapidly bound to the cell membrane and migrated into the cells within 1 h, exhibiting better translocation of macromolecules compared to that of conventional liposomes. Cellular uptake of Pep1-Lipo was proportional to the amount of Pep-1 peptide on the liposomal surface. In conclusion, we found that the Pep1-Lipo formulation was a promising nanocarrier system for intracellular delivery of macromolecules.

Intramitochondrial crystalline inclusions in nonalcoholic steatohepatitis.

UNLABELLED: Mitochondrial dysfunction is an important element in the pathogenesis of nonalcoholic steatohepatitis (NASH). Intramitochondrial crystals (IMCs) are a well-documented morphological abnormality seen on transmission electron microscopy in this disease. It has been suggested that IMCs consist of phospholipids, but their exact composition remain uncertain many years after their discovery. Micellar phase transitions of phospholipid bilayers is a well-known but little-studied phenomenon in living systems. Its presence in the mitochondria of NASH would offer significant insight into the disease with possible therapeutic implications. We postulated that intramitochondrial disturbances in NASH are sufficient to produce such transitions and that their detection in fresh biopsies would therefore be a dynamic process. To test this, we performed a blinded, prospective analysis of fresh liver biopsy samples immediately fixed under different conditions. Quantitative transmission electron microscopy morphometry, performed by systematically counting total mitochondria and IMCs within areas of uniform dimension, showed a stepwise decline in IMCs with cooler fixation temperature in each subject studied. Randomization testing (Monte Carlo resampling) confirmed that the detection of IMCs was strongly dependent on fixation temperature (P < 0.0001). CONCLUSION: These results indicate that the intramitochondrial crystals characteristic of NASH are highly dynamic and unstable structures. The findings offer the strongest support yet for their origin in micellar phase transitions. We speculate that such transitions result from microenvironmental changes within the mitochondria and carry therapeutic implications, especially in regard to dietary manipulations of mitochondrial lipid composition.

The epidemiology of hepatocellular cancer: from the perspectives of public health problem to tumor biology.

The epidemiology of hepatocellular cancer (HCC) can be viewed from several important perspectives. The conventional perspective includes the overall public health impact of HCC, which is increasing in incidence in many regions of the world. The epidemiology of HCC can also be viewed from the perspective of variation in underlying disease associations such as viral hepatitis or the recently recognized link to nonalcoholic fatty liver disease (NAFLD). Of perhaps increasing importance with recent advances in therapy of HCC, the epidemiology of HCC can also be viewed from the perspective of variation in HCC biology. This lesser known perspective may depend in part on the underlying liver disease and the cell origin of the cancer, whether of hepatocyte or stem cell origin. This aspect is likely to become central to diagnosis and management of HCC with the further development of targeted therapeutics. The relative efficacy of these agents will likely depend on the biochemical pathways active in a given hepatocellular malignancy. This, in turn, is likely to be related to the epidemiological associations of HCC.

The effects of 48 weeks of rosiglitazone on hepatocyte mitochondria in human nonalcoholic steatohepatitis.

UNLABELLED: Rosiglitazone, a thiazolidinedione peroxisome proliferator-activated receptor gamma ligand, reduces disease activity in nonalcoholic steatohepatitis (NASH), a disease associated with hepatocyte mitochondrial crystalline inclusions that are not seen in animal models of NASH. In human and animal studies of adipose tissue, thiazolidinediones may induce mitochondrial biogenesis and associated morphological changes. To determine if rosiglitazone alters the hepatocyte mitochondrial morphology in human NASH, we prospectively and systematically examined liver biopsies from human subjects with NASH before and after 48 weeks of rosiglitazone by transmission electron microscopy. Twenty patients (body mass index = 34 +/- 7) were studied. Four coded sections from each of 20 pretherapy biopsies and each of 20 posttherapy biopsies were examined by transmission electron microscopy. The total hepatocyte mitochondria and crystal-containing mitochondria were counted, and semiquantitative scoring was performed for macrosteatosis, microsteatosis, dilated endoplasmic reticulum, apoptosis, Mallory bodies, and hepatocyte enlargement. The total mitochondria count was unchanged after therapy, but there was a significant increase in crystal-containing mitochondria from 4.0% (95% confidence interval = 1.8-8.8) to 7.2% (95% confidence interval = 3.9-12.6; odds ratio = 1.80; P = 0.04) after the treatment with rosiglitazone. Macrosteatosis (P < 0.001) and Mallory bodies (P = 0.05) significantly decreased, but no change was evident in microsteatosis, cellular enlargement, dilated endoplasmic reticulum, or apoptosis. CONCLUSION: Rosiglitazone therapy of NASH is associated with increased crystalline inclusions in hepatocyte mitochondria. Whether these are adaptive or pathological remains unknown, and further studies are warranted to assess hepatic mitochondrial function during thiazolidinedione therapy for NASH.