TAMpepK Suppresses Metastasis through the Elimination of M2-Like Tumor-Associated Macrophages in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) accounts for approximately 10-15% of all breast cancer cases and is characterized by high invasiveness, high metastatic potential, relapse proneness, and poor prognosis. M2-like tumor-associated macrophages (TAMs) contribute to tumorigenesis and are promising targets for inhibiting breast cancer metastasis. Therefore, we investigated whether melittin-conjugated pro-apoptotic peptide (TAMpepK) exerts therapeutic effects on breast cancer metastasis by targeting M2-like TAMs. TAMpepK is composed of M2-like TAM binding peptide (TAMpep) and pro-apoptotic peptide d(KLAKLAK)2 (dKLA). A metastatic mouse model was constructed by injecting 4T1-luc2 cells either orthotopically or via tail vein injection, and tumor burden was quantified using a bioluminescence in vivo imaging system. We found that TAMpepK suppressed lung and lymph node metastases of breast cancer by eliminating M2-like TAMs without affecting the viability of M1-like macrophages and resident macrophages in the orthotopic model. Furthermore, TAMpepK reduced pulmonary seeding and the colonization of tumor cells in the tail vein injection model. The number of CD8+ T cells in contact with TAMs was significantly decreased in tumor nodules treated with TAMpepK, resulting in the functional activation of cytotoxic CD8+ T cells. Taken together, our findings suggest that TAMpepK could be a novel therapeutic agent for the inhibition of breast cancer metastasis by targeting M2-like TAMs.

Targeting of adipose tissue macrophages by bee venom phospholipase A2 attenuates high-fat diet-induced obesity.

BACKGROUND/OBJECTIVES: Adipose tissue macrophages (ATMs) exist in either the M1 or M2 form. The anti-inflammatory M2 ATMs accumulate in lean individuals, whereas the pro-inflammatory M1 ATMs accumulate in obese individuals. Bee venom phospholipase A2 (bvPLA2), a major component in honeybee (Apis mellifera) venom, exerts potent anti-inflammatory effects via interactions with regulatory T cells (Treg) and macrophages. This study investigated the effects of bvPLA2 on a high-fat diet (HFD)-induced obesity in mice. SUBJECTS/METHODS: For in vivo experiments, male C57BL/6, CD206-deficient, and Treg-depleted mice models were fed either a normal diet 41.86 kJ (ND, 10 kcal% fat) or high-fat diet 251.16 kJ (HFD, 60 kcal% fat). Each group was i.p. injected with PBS or bvPLA2 (0.5 mg/kg) every 3 days for 11 weeks. Body weight and food intake were measured weekly. Histological changes in the white adipose tissue (WAT), liver, and kidney as well as the immune phenotypes of the WAT were examined. Immune cells, cytokines, and lipid profiles were also evaluated. The direct effects of bvPLA2 on 3T3-L1 pre-adipocytes and bone marrow-derived macrophages were measured in vitro. RESULTS: bvPLA2 markedly decreased bodyweight in HFD-fed mice. bvPLA2 treatment also decreased lipid accumulation in the liver and reduced kidney inflammation in the mice. It was confirmed that bvPLA2 exerted immunomodulatory effects through the CD206 receptor. In addition, bvPLA2 decreased M1 ATM and alleviated the M1/M2 imbalance in vivo. However, bvPLA2 did not directly inhibit adipogenesis in the 3T3-L1 adipose cells in vitro. CONCLUSIONS: bvPLA2 is a potential therapeutic strategy for the management of obesity by regulating adipose tissue macrophage homeostasis.

Hippocampal-striatal functional connectivity supports processing of temporal expectations from associative memory.

The hippocampus and dorsal striatum are both associated with temporal processing, but they are thought to play distinct roles. The hippocampus has been reported to contribute to storing temporal structure of events in memory, whereas the striatum contributes to temporal motor preparation and reward anticipation. Here, we asked whether the striatum cooperates with the hippocampus in processing the temporal context of memorized visual associations. In our task, participants were trained to implicitly form temporal expectations for one of two possible time intervals associated to specific cue-target associations, and subsequently were scanned using ultra-high-field 7T functional magnetic resonance imaging. During scanning, learned temporal expectations could be violated when the pairs were presented at either the associated or not-associated time intervals. When temporal expectations were met during testing trials, activity in left and right hippocampal subfields and right putamen decreased, compared to when temporal expectations were not met. Further, psycho-physiological interactions showed that functional connectivity between left hippocampal subfields and caudate decreased when temporal expectations were not met. Our results indicate that the hippocampus and striatum cooperate to process implicit temporal expectation from mnemonic associations. Our findings provide further support for a hippocampal-striatal network in temporal associative processing.

IbMPK3/IbMPK6-mediated IbSPF1 phosphorylation promotes tolerance to bacterial pathogen in sweetpotato.

KEY MESSAGE: IbSPF1, a novel target of IbMPK3/IbMPK6, regulates biotic stress response in sweetpotato. Environmental stresses due to biotic and abiotic factors negatively affect crop quality and productivity. To minimize the damage caused by these factors, numerous stress signaling pathways are activated in plants. Among these, the mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in diverse plant stress responses. MPK3 and MPK6 function in several cellular signaling pathways by phosphorylating downstream partner proteins in response to environmental stresses. However, little is known about the MPK3/MPK6 signaling pathway in sweetpotato [Ipomoea batatas (L.) Lam]. We recently confirmed that IbMPK3 and IbMPK6, two pathogen-responsive MAPKs, play essential roles in defense gene activation in sweetpotato. In this study, we show that sweetpotato SP8-binding factor (IbSPF1), a substrate of IbMPK3/IbMPK6, functions as a transcriptional regulator of biotic stress signaling in sweetpotato. IbSPF1 specifically interacts with IbMPK3 and IbMPK6, which phosphorylate Ser75 and Ser110 residues of IbSPF1. This increases the affinity of IbSPF1 for the W-box element in target gene promoters. Additionally, the expression of IbSPF1 was up-regulated under various stress conditions and different hormone treatments involved in plant defense responses. Interestingly, the phospho-mimicking mutant of IbSPF1 showed enhanced resistance to Pseudomonas syringae pv. tabaci, and transient expression of mutant IbSPF1 induced the expression of pathogenesis-related genes. These results indicate that the phosphorylation of IbSPF1 by IbMPK3/IbMPK6 plays a critical role in plant immunity by up-regulating the expression of downstream genes.

A single amino acid change at position 96 (Arg to His) of the sweetpotato Orange protein leads to carotenoid overaccumulation.

KEY MESSAGE: IbOr-R96H resulted in carotenoid overaccumulation and enhanced abiotic stress tolerance in transgenic sweetpotato calli. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and tolerance to various environmental stresses. Sweetpotato IbOr, with strong holdase chaperone activity, protects a key enzyme, phytoene synthase (PSY), in the carotenoid biosynthetic pathway and stabilizes a photosynthetic component, oxygen-evolving enhancer protein 2-1 (PsbP), under heat and oxidative stresses in plants. Previous studies of various plant species demonstrated that a single-nucleotide polymorphism (SNP) from Arg to His in Or protein promote a high level of carotenoid accumulation. Here, we showed that the substitution of a single amino acid at position 96 (Arg to His) of wild-type IbOr (referred to as IbOr-R96H) dramatically increases carotenoid accumulation. Sweetpotato calli overexpressing IbOr-WT or IbOr-Ins exhibited 1.8- or 4.3-fold higher carotenoid contents than those of the white-fleshed sweetpotato Yulmi (Ym) calli, and IbOr-R96H overexpression substantially increased carotenoid accumulation by up to 23-fold in sweetpotato calli. In particular, IbOr-R96H transgenic calli contained 88.4-fold higher levels of ß-carotene than those in Ym calli. Expression levels of carotenogenesis-related genes were significantly increased in IbOr-R96H transgenic calli. Interestingly, transgenic calli overexpressing IbOr-R96H showed increased tolerance to salt and heat stresses, with similar levels of malondialdehyde to those in calli expressing IbOr-WT or IbOr-Ins. These results suggested that IbOr-R96H is a useful target for the generation of efficient industrial plants, including sweetpotato, to cope with growing food demand and climate change by enabling sustainable agriculture on marginal lands.

Orange: a target gene for regulating carotenoid homeostasis and increasing plant tolerance to environmental stress in marginal lands.

Carotenoids play essential roles in various light-harvesting processes in plants and help protect the photosynthetic machinery from photo-oxidative damage. Orange genes, which play a role in carotenoid accumulation, have recently been isolated from several plant species, and their functions have been intensively investigated. The Orange gene (IbOr) of sweet potato [Ipomoea batatas (L.) Lam] helps maintain carotenoid homeostasis to improve plant tolerance to environmental stress. IbOr, a protein with strong holdase chaperone activity, directly interacts with phytoene synthase, a key enzyme involved in carotenoid biosynthesis, in plants under stress conditions, resulting in increased carotenoid accumulation and abiotic stress tolerance. In addition, IbOr interacts with the oxygen-evolving enhancer protein 2-1, a member of a protein complex in photosystem II that is denatured under heat stress. Transgenic sweet potato plants overexpressing IbOr showed enhanced tolerance to high temperatures (47 °C). These findings indicate that IbOr protects plants from environmental stress not only by controlling carotenoid biosynthesis, but also by directly stabilizing photosystem II. In this review, we discuss the functions of IbOr and Or proteins in other plant species and their possible biotechnological applications for molecular breeding for sustainable development on marginal lands.

Overexpression of Arabidopsis P3B increases heat and low temperature stress tolerance in transgenic sweetpotato.

BACKGROUND: Sweetpotato (Ipomoea batatas [L.] Lam) is suitable for growth on marginal lands due to its abiotic stress tolerance. However, severe environmental conditions including low temperature pose a serious threat to the productivity and expanded cultivation of this crop. In this study, we aimed to develop sweetpotato plants with enhanced tolerance to temperature stress. RESULTS: P3 proteins are plant-specific ribosomal P-proteins that act as both protein and RNA chaperones to increase heat and cold stress tolerance in Arabidopsis. Here, we generated transgenic sweetpotato plants expressing the Arabidopsis ribosomal P3 (AtP3B) gene under the control of the CaMV 35S promoter (referred to as OP plants). Three OP lines (OP1, OP30, and OP32) were selected based on AtP3B transcript levels. The OP plants displayed greater heat tolerance and higher photosynthesis efficiency than wild type (WT) plants. The OP plants also exhibited enhanced low temperature tolerance, with higher photosynthesis efficiency and less membrane permeability than WT plants. In addition, OP plants had lower levels of hydrogen peroxide and higher activities of antioxidant enzymes such as peroxidase and catalase than WT plants under low temperature stress. The yields of tuberous roots and aerial parts of plants did not significantly differ between OP and WT plants under field cultivation. However, the tuberous roots of OP transgenic sweetpotato showed improved storage ability under low temperature conditions. CONCLUSIONS: The OP plants developed in this study exhibited increased tolerance to temperature stress and enhanced storage ability under low temperature compared to WT plants, suggesting that they could be used to enhance sustainable agriculture on marginal lands.

Bee Venom Phospholipase A2, a Novel Foxp3+ Regulatory T Cell Inducer, Protects Dopaminergic Neurons by Modulating Neuroinflammatory Responses in a Mouse Model of Parkinson's Disease.

Foxp3-expressing CD4(+) regulatory T cells (Tregs) are vital for maintaining immune tolerance in animal models of various immune diseases. In the present study, we demonstrated that bee venom phospholipase A2 (bvPLA2) is the major BV compound capable of inducing Treg expansion and promotes the survival of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. We associated this neuroprotective effect of bvPLA2 with microglial deactivation and reduction of CD4(+) T cell infiltration. Interestingly, bvPLA2 had no effect on mice depleted of Tregs by injecting anti-CD25 Ab. This finding indicated that Treg-mediated modulation of peripheral immune tolerance is strongly involved in the neuroprotective effects of bvPLA2. Furthermore, our results showed that bvPLA2 directly bound to CD206 on dendritic cells and consequently promoted the secretion of PGE2, which resulted in Treg differentiation via PGE2 (EP2) receptor signaling in Foxp3(-)CD4(+) T cells. These observations suggest that bvPLA2-CD206-PGE2-EP2 signaling promotes immune tolerance through Treg differentiation and contributes to the prevention of various neurodegenerative diseases, including Parkinson's disease.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8.

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

Neuroprotective effects of bee venom phospholipase A2 in the 3xTg AD mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a severe neuroinflammatory disease. CD4(+)Foxp3(+) regulatory T cells (Tregs) modulate various inflammatory diseases via suppressing Th cell activation. There are increasing evidences that Tregs have beneficial roles in neurodegenerative diseases. Previously, we found the population of Treg cells was significantly increased by bee venom phospholipase A2 (bvPLA2) treatment in vivo and in vitro. METHODS: To examine the effects of bvPLA2 on AD, bvPLA2 was administered to 3xTg-AD mice, mouse model of Alzheimer's disease. The levels of amyloid beta (Aß) deposits in the hippocampus, glucose metabolism in the brain, microglia activation, and CD4(+) T cell infiltration were analyzed to evaluate the neuroprotective effect of bvPLA2. RESULTS: bvPLA2 treatment significantly enhanced the cognitive function of the 3xTg-AD mice and increased glucose metabolism, as assessed with 18F-2 fluoro-2-deoxy-D-glucose ([F-18] FDG) positron emission tomography (PET). The levels of Aß deposits in the hippocampus were dramatically decreased by bvPLA2 treatment. This neuroprotective effect of bvPLA2 was associated with microglial deactivation and reduction in CD4(+) T cell infiltration. Interestingly, the neuroprotective effects of bvPLA2 were abolished in Treg-depleted mice. CONCLUSIONS: The present studies strongly suggest that the increase of Treg population by bvPLA2 treatment might inhibit progression of AD in the 3xTg AD mice.

Immunotherapy with methyl gallate, an inhibitor of Treg cell migration, enhances the anti-cancer effect of cisplatin therapy.

Foxp3(+) CD25(+)CD4(+) regulatory T (Treg) cells are crucial for the maintenance of immunological self-tolerance and are abundant in tumors. Most of these cells are chemo-attracted to tumor tissues and suppress anti-tumor responses inside the tumor. Currently, several cancer immunotherapies targeting Treg cells are being clinically tested. Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. While cisplatin is a powerful drug for the treatment of multiple cancers, there are obstacles that limit its use, such as renal dysfunction and the development of cisplatin-resistant cancer cells after its use. To minimize these barriers, combinatorial therapies of cisplatin with other drugs have been developed and have proven to be more effective to treat cancer. In the present study, we evaluated the eff ect of the combination therapy using methyl gallate with cisplatin in EL4 murine lymphoma bearing C57BL/6 mice. The combinatorial therapy of methyl gallate and cisplatin showed stronger anti-cancer eff ects than methyl gallate or cisplatin as single treatments. In Treg cell-depleted mice, however, the eff ect of methyl gallate vanished. It was found that methyl gallate treatment inhibited Treg cell migration into the tumor regardless of cisplatin treatment. Additionally, in both the normal and cisplatin-treated tumor-bearing mice, there was no renal toxicity attributed to methyl gallate treatment. These findings suggest that methyl gallate treatment could be useful as an adjuvant method accompanied with cisplatin therapy.

Immune Checkpoint-Blocking Nanocages Cross the Blood-Brain Barrier and Impede Brain Tumor Growth.

Glioblastoma (GBM) is the deadliest tumor of the central nervous system, with a median survival of less than 15 months. Despite many trials, immune checkpoint-blocking (ICB) therapies using monoclonal antibodies against the PD-1/PD-L1 axis have demonstrated only limited benefits for GBM patients. Currently, the main hurdles in brain tumor therapy include limited drug delivery across the blood-brain barrier (BBB) and the profoundly immune-suppressive microenvironment of GBM. Thus, there is an urgent need for new therapeutics that can cross the BBB and target brain tumors to modulate the immune microenvironment. To this end, we developed an ICB strategy based on the BBB-permeable, 24-subunit human ferritin heavy chain, modifying the ferritin surface with 24 copies of PD-L1-blocking peptides to create ferritin-based ICB nanocages. The PD-L1pep ferritin nanocages first demonstrated their tumor-targeting and antitumor activities in an allograft colon cancer model. Next, we found that these PD-L1pep ferritin nanocages efficiently penetrated the BBB and targeted brain tumors through specific interactions with PD-L1, significantly inhibiting tumor growth in an orthotopic intracranial tumor model. The addition of PD-L1pep ferritin nanocages to triple in vitro cocultures of T cells, GBM cells, and glial cells significantly inhibited PD-1/PD-L1 interactions and restored T-cell activity. Collectively, these findings indicate that ferritin nanocages displaying PD-L1-blocking peptides can overcome the primary hurdle of brain tumor therapy and are, therefore, promising candidates for treating GBM.

Comparative transcriptome profiling of sweetpotato storage roots during curing-mediated wound healing.

Sweetpotato (Ipomoea batatas L. Lam) is an economically important crop that is cultivated for its storage roots. Storage roots provide a source of valuable nutrients, processed foods, animal feeds, and pigments. Sweetpotato storage roots spoil during post-harvest handling because of wounding, which makes them more susceptible to disease-causing microorganisms. Curing to promote wound healing is a common method to control microbial spoilage during post-harvest storage. However, molecular mechanisms underlying the process of curing in sweetpotato storage roots are unknown. To better understand the biology behind curing, the transcriptome of the sweetpotato cultivar, Pungwonmi, was studied using RNA-seq. Storage roots of sweetpotato were treated at 33 °C (Curing) and 13 °C (Control) for 3 days. RNA-seq data identified 78,781 unigenes and 3,366 differentially expressed genes by over log2 fold change (FC) > 2 and <-2. During curing, DEGs encoded genes related to drought/salt stress responses, phyto-hormones (e.g., auxin, ethylene and jasmonic acid), and proteolysis, were up-regulated, whereas those related to redox state, phyto-hormones (e.g., salicylic acid and brassinosteroids), and lignin and flavonoid biosynthesis were down-regulated. Additionally, among the candidate genes, DEGs encoded genes related to proteolysis and pathogen defense, such as protease inhibitors and lipid transfer proteins, were highly up-regulated during curing and storage. This study provides a valuable resource to further understand the molecular basis of curing-mediated wound healing in sweetpotato storage roots. Moreover, genes revealed in this work could present targets for the development of sweetpotato varieties with improved post-harvest storage characteristics.

Flooding Tolerance in Sweet Potato (Ipomoea batatas (L.) Lam) Is Mediated by Reactive Oxygen Species and Nitric Oxide.

Flooding is harmful to almost all higher plants, including crop species. Most cultivars of the root crop sweet potato are able to tolerate environmental stresses such as drought, high temperature, and high salinity. They are, however, relatively sensitive to flooding stress, which greatly reduces yield and commercial value. Previous transcriptomic analysis of flood-sensitive and flood-resistant sweet potato cultivars identified genes that were likely to contribute to protection against flooding stress, including genes related to ethylene (ET), reactive oxygen species (ROS), and nitric oxide (NO) metabolism. Although each sweet potato cultivar can be classified as either tolerant or sensitive to flooding stress, the molecular mechanisms of flooding resistance in ET, ROS, and NO regulation-mediated responses have not yet been reported. Therefore, this study characterized the regulation of ET, ROS, and NO metabolism in two sweet potato cultivars-one flood-tolerant cultivar and one flood-sensitive cultivar-under early flooding treatment conditions. The expression of ERFVII genes, which are involved in low oxygen signaling, was upregulated in leaves during flooding stress treatments. In addition, levels of respiratory burst oxidase homologs and metallothionein-mediated ROS scavenging were greatly increased in the early stage of flooding in the flood-tolerant sweet potato cultivar compared with the flood-sensitive cultivar. The expression of genes involved in NO biosynthesis and scavenging was also upregulated in the tolerant cultivar. Finally, NO scavenging-related MDHAR expressions and enzymatic activity were higher in the flood-tolerant cultivar than in the flood-sensitive cultivar. These results indicate that, in sweet potato, genes involved in ET, ROS, and NO regulation play an important part in response mechanisms against flooding stress.

Adoptive therapy with amyloid-ß specific regulatory T cells alleviates Alzheimer's disease.

Rationale: Neuroinflammation is a primary feature of Alzheimer's disease (AD), for which an increasing number of drugs have been specifically developed. The present study aimed to define the therapeutic impact of a specific subpopulation of T cells that can suppress excessive inflammation in various immune and inflammatory disorders, namely, CD4+CD25+Foxp3+ regulatory T cells (Tregs). Methods: To generate Aß antigen-specific Tregs (Aß+ Tregs), Aß 1-42 peptide was applied in vivo and subsequent in vitro splenocyte culture. After isolating Tregs by magnetic bead based purification method, Aß+ Tregs were adoptively transferred into 3xTg-AD mice via tail vein injection. Therapeutic efficacy was confirmed with behavior test, Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining (IHC). In vitro suppression assay was performed to evaluate the suppressive activity of Aß+ Tregs using flow cytometry. Thy1.1+ Treg trafficking and distribution was analyzed to explore the infused Tregs migration into specific organs in an antigen-driven manner in AD mice. We further assessed cerebral glucose metabolism using 18F-FDG-PET, an imaging approach for AD biological definition. Subsequently, we evaluated the migration of Aß+ Tregs toward Aß activated microglia using live cell imaging, chemotaxis, antibody blocking and migration assay. Results: We showed that Aß-stimulated Tregs inhibited microglial proinflammatory activity and modulated the microglial phenotype via bystander suppression. Single adoptive transfer of Aß+ Tregs was enough to induce amelioration of cognitive impairments, Aß accumulation, hyper-phosphorylation of tau, and neuroinflammation during AD pathology. Moreover, Aß-specific Tregs effectively inhibited inflammation in primary microglia induced by Aß exposure. It may indicate bystander suppression in which Aß-specific Tregs promote immune tolerance by secreting cytokines to modulate immune responses during neurodegeneration. Conclusions: The administration of Aß antigen-specific regulatory T cells may represent a new cellular therapeutic strategy for AD that acts by modulating the inflammatory status in AD.

Thermopneumatic Soft Micro Bellows Actuator for Standalone Operation.

Typical pneumatic soft micro actuators can be manufactured without using heavy driving components such as pumps and power supplies by adopting an independent battery-powered mechanism. In this study, a thermopneumatically operated soft micro bellows actuator was manufactured, and the standalone operation of the actuator was experimentally validated. Thermopneumatic actuation is based on heating a sealed cavity inside the elastomer of the actuator to raise the pressure, leading to deflection of the elastomer. The bellows actuator was fabricated by casting polydimethylsiloxane (PDMS) using the 3D-printed soluble mold technique to prevent leakage, which is inherent in conventional soft lithography due to the bonding of individual layers. The heater, manufactured separately using winding copper wire, was inserted into the cavity of the bellows actuator, which together formed the thermopneumatic actuator. The 3D coil heater and bellows allowed immediate heat transfer and free movement in the intended direction, which is unachievable for conventional microfabrication. The fabricated actuator produced a stroke of 2184 µm, equivalent to 62% of the body, and exerted a force of 90.2 mN at a voltage of 0.55 V. A system in which the thermopneumatic actuator was driven by alkaline batteries and a control circuit also demonstrated a repetitive standalone operation.

The Effects of Human Bone Marrow-Derived Mesenchymal Stem Cell Conditioned Media Produced with Fetal Bovine Serum or Human Platelet Lysate on Skin Rejuvenation Characteristics.

BACKGROUND AND OBJECTIVES: Human mesenchymal stem cell-conditioned medium (MSC-CM) is produced using mesenchymal stem cell culture technology and has various benefits for the skin, including wrinkle removal, skin regeneration, and increased antioxidant activity. Its popularity is thus increasing in the field of functional cosmetics. METHODS AND RESULTS: In this study, we analyzed the effects of fetal bovine serum-supplemented MSC-CM (FBSMSC-CM) and human platelet lysate-supplemented MSC-CM (hPL-MSC-CM) on skin rejuvenation characteristics. We found that the concentrations of important growth factors (VEGF, TGF-ß1, and HGF) and secretory proteins for skin regeneration were significantly higher in hPL-MSC-CM than in FBS-MSC-CM. Furthermore, the capacity for inducing proliferation of human dermal fibroblast (HDF) and keratinocytes, the migration ability of HDF, extracellular matrix (ECM) production such as collagen and elastin was higher in hPL-MSC-CM than that in FBSMSC-CM. CONCLUSIONS: These results support the usefulness and high economic value of hPL-MSC-CM as an alternative source of FBS-MSC-CM in the cosmetic industry for skin rejuvenation.

Culturing at Low Cell Density Delays Cellular Senescence of Human Bone Marrow-Derived Mesenchymal Stem Cells in Long-Term Cultures.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) have immense therapeutic potential for treating intractable and immune diseases. They also have applications in regenerative medicine in which distinct treatments do not exist. Thus, MSCs are gaining attention as important raw materials in the field of cell therapy. Importantly, the number of MSCs in the bone marrow is limited and they are present only in small quantities. Therefore, mass production of MSCs through long-term culture is necessary to use them in cell therapy. However, MSCs undergo cellular senescence through repeated passages during mass production. In this study, we explored methods to prolong the limited lifetime of MSCs by culturing them with different seeding densities. METHODS AND RESULTS: We observed that in long-term cultures, low-density (LD, 50 cells/cm2) MSCs showed higher population doubling level, leading to greater fold increase, than high-density (HD, 4,000 cells/cm2) MSCs. LD-MSCs suppressed the expression of aging-related genes. We also showed that reactive oxygen species (ROS) were decreased in LD-MSCs compared to that in HD-MSCs. Further, proliferation potential increased when ROS were inhibited in HD-MSCs. CONCLUSIONS: The results in this study suggest that MSC senescence can be delayed and that life span can be extended by controlling cell density in vitro. These results can be used as important data for the mass production of stem cell therapeutic products.

Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs.

The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to co-cultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors.

Overexpression of the Golden SNP-Carrying Orange Gene Enhances Carotenoid Accumulation and Heat Stress Tolerance in Sweetpotato Plants.

Carotenoids function as photosynthetic accessory pigments, antioxidants, and vitamin A precursors. We recently showed that transgenic sweetpotato calli overexpressing the mutant sweetpotato (Ipomoea batatas [L.] Lam) Orange gene (IbOr-R96H), which carries a single nucleotide polymorphism responsible for Arg to His substitution at amino acid position 96, exhibited dramatically higher carotenoid content and abiotic stress tolerance than calli overexpressing the wild-type IbOr gene (IbOr-WT). In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H under the control of the cauliflower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediated transformation. The total carotenoid contents of IbOr-R96H storage roots (light-orange flesh) and IbOr-WT storage roots (light-yellow flesh) were 5.4-19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The ß-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, IbOr-R96H plants showed greater tolerance to heat stress (47 °C) than NT and IbOr-WT plants, possibly because of higher DPPH radical scavenging activity and ABA contents. These results indicate that IbOr-R96H is a promising strategy for developing new sweetpotato cultivars with improved carotenoid contents and heat stress tolerance.