Cryptotanshinone (CT), isolated from the plant Salvia miltiorrhiza Bunge, has been reported to have potential anticancer effects on human prostate and breast cancer cells. However, the mechanisms of action of CT on gastric cancer (GC) cells are not well understood. Here we investigated the antitumor effects of CT on GC cells and its possible molecular mechanism. We found CT suppressed viability of twelve GC cell lines in a dose-dependent manner. CT induced cell cycle arrest at the G2/M phase and mitochondrial apoptosis accompanying the accumulation of reactive oxygen species (ROS). Pretreatment with ROS inhibitor N-acetyl-L-cysteine (NAC) blocked CT-induced apoptosis. CT increased p-JNK and p-p38, and decreased p-ERK and p-STAT3 protein expression, these effects were prevented by NAC. Furthermore, a xenograft assay showed that CT significantly inhibited MKN-45 cell-induced tumor growth in vivo by increasing expression of pro-apoptotic proteins (p-JNK, p-38 and cleaved-caspase-3) and reducing expression of anti-apoptotic proteins (p-ERK and p-STAT3) without adverse effects on nude mice weight. In conclusion, CT induced apoptosis and cell cycle arrest in GC cells via ROS-mediated MAPK and AKT signaling pathways, and this CT may be a useful compound for the developing anticancer agents for GC.

Utilizing the permeability enhancement and irreversible biomolecule denaturation caused by hyperthermia, photothermal-chemo synergistic therapy has shown great potential in clinical cancer treatment.

It is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies.

Partial substitution of Ce in Nd-Fe-B magnets is a feasible way to cope with the crisis of Nd and Dy in Nd-Fe-B production and reduce the cost of Nd-Fe-B magnets. In the present paper, the Nd-Ce-Fe-B films with high performance have been successfully fabricated by using an ultra-high vacuum (UHV) magnetron sputtering system. High magnetic performance with a ceorcivity of 13.3 kOe, a remanence of 11.4 kGs and a maximum energy product of 29.4 GMOe is obtained with the Ce substitution for more than 50 wt.% Nd without Dy addition. The high coercivity and (BH)max achieved in this work are much larger than those of previously reported Nd-Ce-Fe-B magnets with the same Ce concentration. The phase structure, microstructure and coercivity mechanism are analyzed. The coercivity mechanism is determined to be mainly dominated by nucleation. Based on the microstructure observation and coercivity mechanism analysis, the fine and well separated grains, smooth grain surface, small and less inhomogeneities should be responsible for the high coercivity. Our results encourage the further improvement of magnetic properties in Ce magnets including the bulk material with high Ce concentration.

Anti-CTLA-4 monoclonal antibodies (mAbs) confer a cancer immunotherapeutic effect (CITE) but cause severe immunotherapy-related adverse events (irAE). Targeting CTLA-4 has shown remarkable long-term benefit and thus remains a valuable tool for cancer immunotherapy if the irAE can be brought under control. An animal model, which recapitulates clinical irAE and CITE, would be valuable for developing safer CTLA-4-targeting reagents. Here, we report such a model using mice harboring the humanized Ctla4 gene. In this model, the clinically used drug, Ipilimumab, induced severe irAE especially when combined with an anti-PD-1 antibody; whereas another mAb, L3D10, induced comparable CITE with very mild irAE under the same conditions. The irAE corresponded to systemic T cell activation and resulted in reduced ratios of regulatory to effector T cells (Treg/Teff) among autoreactive T cells. Using mice that were either homozygous or heterozygous for the human allele, we found that the irAE required bi-allelic engagement, while CITE only required monoallelic engagement. As with the immunological distinction for monoallelic vs bi-allelic engagement, we found that bi-allelic engagement of the Ctla4 gene was necessary for preventing conversion of autoreactive T cells into Treg cells. Humanization of L3D10, which led to loss of blocking activity, further increased safety without affecting the therapeutic effect. Taken together, our data demonstrate that complete CTLA-4 occupation, systemic T cell activation and preferential expansion of self-reactive T cells are dispensable for tumor rejection but correlate with irAE, while blocking B7-CTLA-4 interaction impacts neither safety nor efficacy of anti-CTLA-4 antibodies. These data provide important insights for the clinical development of safer and potentially more effective CTLA-4-targeting immunotherapy.

Niemann-Pick proteins type C2 (NPC2) are carriers of cholesterol in vertebrates, with a single member in each species. The high sequence conservation between mammals and across vertebrates is related to their common function. In contrast, NPC2 proteins in arthropods have undergone extensive duplication and differentiation, probably under environmental pressure, and are likely to have different functions. Recent studies have suggested that in arthropods these proteins might act as carriers for semiochemicals and other hydrophobic compounds. In this study we focused on the function of a specific NPC2 gene in the moth Helicoverpa armigera (HarmNPC2-1). This protein binds several flavonoids with micromolar dissociation constants. The best ligand was gossypol, present in cotton, one of the main host plants for H. armigera. Western blot revealed the presence of HarmNPC2-1 in different parts of the body, including the antennae, proboscis, and abdomen. In the antennae, in situ hybridization experiments produced strong staining in auxiliary cells at the base of sensilla trichodea, basiconica, coeloconica, and chaetica. Immunocytochemistry confirmed the expression of the protein in sensilla chaetica. Our results support a role of semiochemical carriers for NPC2 proteins in insects and indicate such proteins as new targets for insecticide-free pest population control.

Acute gouty arthritis (AGA) is one of the most common forms of auto-inflammatory arthritis. IL-17 is a key proinflammatory cytokine which has been implicated in several autoimmune diseases. However, to date little is known about the role of IL-17 in AGA. In the present study, we show that serum IL-17 levels are significantly elevated in AGA patients early in the onset of symptoms of gout, and decrease gradually as symptoms diminish. Correlation analysis indicated that IL-17 expression is not only positively correlated with disease activity, but is also correlated with serum levels of IL-1β which plays a critical role in the differentiation of IL-17- γδT cells into IL-17+γδT cells. Flow cytometry analysis indicated that γδ T cells are a major source of IL-17 production during the early onset of AGA. We therefore identify IL-17 as a potential novel biomarker for AGA and suggest that targeting the γδ T cell/IL-17 immune axis is a potential strategy for treatment of acute flares of AGA.

Multiple nuclear markers provide genetic polymorphism data for molecular systematics and population genetic studies. They are especially required for the coalescent-based analyses that can be used to accurately estimate species trees and infer population demographic histories. However, in avian evolutionary studies, these powerful coalescent-based methods are hindered by the lack of a sufficient number of markers. In this study, we designed PCR primers to amplify 136 nuclear protein-coding loci (NPCLs) by scanning the published Red Junglefowl (Gallus gallus) and Zebra Finch (Taeniopygia guttata) genomes. To test their utility, we amplified these loci in 41 bird species representing 23 Aves orders. The sixty-three best-performing NPCLs, based on high PCR success rates, were selected which had various mutation rates and were evenly distributed across 17 avian autosomal chromosomes and the Z chromosome. To test phylogenetic resolving power of these markers, we conducted a Neoavian phylogenies analysis using 63 concatenated NPCL markers derived from 48 whole genomes of birds. The resulting phylogenetic topology, to a large extent, is congruence with results resolved by previous whole genome data. To test the level of intraspecific polymorphism in these makers, we examined the genetic diversity in four populations of the Kentish Plover (Charadrius alexandrinus) at 17 of NPCL markers chosen at random. Our results showed that these NPCL markers exhibited a level of polymorphism comparable with mitochondrial loci. Therefore, this set of pan-avian nuclear protein-coding loci has great potential to facilitate studies in avian phylogenetics and population genetics.

Epitopes serve an important role in influenza infection. It may be useful to screen universal influenza virus vaccines, analyzing the epitopes of multiple subtypes of the hemagglutinin (HA) protein. A total of 40 monoclonal antibodies (mAbs) previously obtained from flu virus HA antigens (development and characterization of 40 mAbs generated using H1N1 influenza virus split vaccines were previously published) were used to detect and classify mAbs into distinct flu virus sub-categories using the ELISA method. Following this, the common continuous amino acid sequences were identified by multiple sequence alignment analysis with the GenBank database and DNAMAN software, for use in predicting the epitopes of the HA protein. Synthesized peptides of these common sequences were prepared, and used to verify and determine the predicted linear epitopes through localization and distribution analyses. With these methods, nine HA linear epitopes distributed among different strains of influenza virus were identified, which included three from influenza A, four from 2009 H1N1 and seasonal influenza, and two from H1. The present study showed that considering a combination of the antigen-antibody reaction specificity, variation in the influenza virus HA protein and linear epitopes may present a useful approach for designing effective multi-epitope vaccines. Furthermore, the study aimed to clarify the cause and pathogenic mechanism of influenza virus HA-induced flu, and presents a novel idea for identifying the epitopes of other pathogenic microorganisms.

Coxsackievirus 16 (CA16) causes hand, foot, and mouth disease (HFMD) in young children and infants, and it can lead to fatal neurological complications. This study investigated antiviral effects of Siji Antiviral Mixture (SAM) on CA16 in neonatal mice and the protective effects of SAM on CA16 induced brain injuries. Neonatal BALB/c mice and SH-SY5Y cells were used and injected with CA16 stains to study the efficacy. ELISA and Western blotting were used to measure the cytokines levels and proteins expression. Genes transduction was also used to verify interaction mechanism. As the results shown, SAM could reduce the clinical scores at the beginning and delay disease development in vivo. Treatment with SAM decreased the levels of LDH, CK-MB, caspase 3 and Bax, ER stress, and inflammatory reaction induced by CA16 infection. Further siRNA transfection results showed that CA16 induced ER stress and inflammatory reaction through PERK/STAT3/NF-κB signaling and the protective effects of SAM might be through inhibiting PERK/STAT3/NF-κB signaling. HPLC analysis showed fingerprint profiles of SAM had 42 chromatographic peaks. Collectively, our study highlighted distinct roles of SAM in inhibiting CA16 infection and brain injury. The molecular mechanism of SAM might be through inhibiting PERK/STAT3/NF-κB signaling.

Osteoradionecrosis of the jaws (ORNJ) is a complication of oral and maxillofacial malignancy that arises following radiotherapy; progressive jaw necrosis severely decreases the quality of life of patients. Human bone marrow mesenchymal stem cells (hBMMSCs) are a cell type with self‑renewal and pluripotent differentiation potential in the bone marrow stroma. These cells are associated with bone tissue regeneration and are one of the primary cell types affected by bone tissue radiation injury. α‑2‑macroglobulin (α2M) is a glycoprotein‑rich macromolecule that interacts with cytokines, growth factors and hormones to serve a variety of biological roles. In addition, α2M possesses radio‑protective effects. The aim of the present study was to investigate whether α2M has protective effects against radiation injury of hBMMSCs. Cell counting kit‑8 and colony formation assays were used to monitor cell proliferation. Western blot analysis and reverse transcription‑quantitative polymerase chain reaction were used to detect Beclin1, microtubule‑associated protein 1A/1B, sex determining region Y, Nanog, runt‑related transcription factor 2, osteoglycin and manganese superoxide dismutase expression. The formation of calcium nodules was evaluated by Alizarin red staining after osteogenic induction. Flow cytometric analysis of Annexin‑V and propidium iodide double staining was used to detect changes in apoptosis rate. Alkaline phosphatase and superoxide dismutase activity were determined using colorimetric assays. Reactive oxygen species levels were detected using 2',7'‑dichlorodihydrofluorescein diacetate. The results of the present study revealed that α2M increased the rate of proliferation, reduced autophagy, alleviated pluripotent differentiation injury, increased the osteogenic differentiation ability and decreased the rate of apoptosis in hBMMSCs following irradiation via an antioxidative pathway. In conclusion, α2M exhibited protective effects against radiation injury in hBMMSCs and may be considered a potential therapeutic agent for the prevention and treatment of ORNJ.

Cyclocarya paliurus has been widely used as an ingredient in functional foods in China. However, the antioxidant properties of phenolic compounds and the effect of the plant origin remain unclear. The present study evaluated the geographical variation of this plant in term of its phenolic composition and antioxidant activities based on leaf materials collected from five regions. high-performance liquid chromatography (HPLC) analysis showed that there are three major components, quercetin-3-O-glucuronide, kaempferol-3-O-glucuronide, and kaempferol-3-O-rhamnoside, and their contents varied significantly among sampling locations. The investigated phenolic compounds showed substantial antioxidant activities, both in vitro and in vivo, with the highest capacity observed from Wufeng and Jinzhongshan. Correlation analysis revealed that quercetin and kaempferol glycosides might be responsible for the antioxidant activities. Our results indicate the importance of geographic origin, with sunny hours and temperature as the main drivers affecting the accumulation of C. paliurus phenolics and their antioxidant properties.

Hadal biosphere represents the deepest part of the ocean with water depth >6,000 m. Accumulating evidence suggests the existence of unique microbial communities dominated by heterotrophic processes in this environment. However, investigations of the microbial diversity and their metabolic potentials are limited because of technical constraints for sample collection. Here, we provide a detailed metagenomic analysis of three seawater samples at water depths 5,000-6,000 m below sea level (mbsl) and three surface sediment samples at water depths 4,435-6,578 mbsl at the Yap Trench of the western Pacific. Distinct microbial community compositions were observed with the dominance of Gammaproteobacteria in seawater and Thaumarchaeota in surface sediment. Comparative analysis of the genes involved in carbon, nitrogen and sulfur metabolisms revealed that heterotrophic processes (i.e., degradation of carbohydrates, hydrocarbons, and aromatics) are the most common microbial metabolisms in the seawater, while chemolithoautotrophic metabolisms such as ammonia oxidation with the HP/HB cycle for CO2 fixation probably dominated the surface sediment communities of the Yap Trench. Furthermore, abundant genes involved in stress response and metal resistance were both detected in the seawater and sediments, thus the enrichment of metal resistance genes is further hypothesized to be characteristic of the hadal microbial communities. Overall, this study sheds light on the metabolic versatility of microorganisms in the Yap Trench, their roles in carbon, nitrogen, and sulfur biogeochemical cycles, and how they have adapted to this unique hadal environment.

The pace-of-life hypothesis predicts no impact of urbanization on stress responses. Accordingly, several studies have been inconsistent in showing differences in breath rate (BR), a proxy of acute stress responses to handling in passerines, between rural and urban areas. However, this evidence is limited to a single bird species and a limited geographic region (SW Europe). No study addressed whether this pattern is also apparent in other species or regions, such as in tropical environments, or whether it is dependent on the level of diet specialization, given that diet restriction and change influence stress responses. Here, we tested whether there were differences in BR between habitats and diet groups using eight highly diverse passerine assemblages experiencing different levels of anthropogenic disturbance (i.e., natural, rural, and urban locations) in SW China. We predicted that insectivores and herbivores (frugivores, nectarivores, and seed-eating species) would show higher BR than omnivores. We also predicted no differences in BR among habitat types. BR was a moderately repeatable trait, which showed a negative relationship with body mass and a positive relationship with the time of the day. We also recorded a relatively strong phylogenetic bias in the expression of this trait. Confirming our predictions, our results showed no differences in BR among natural, rural, and urban locations. Similarly, within species, there were no differences in BR between rural and urban locations. However, we also found that herbivores showed higher BR than omnivores. Overall, our results provide support to the pace-of-life hypothesis, but suggest acute stress responses can be diet-mediated, which may help to explain the marked decline of specialized trophic guilds around the world in response to anthropogenic disturbance.

Cells undergo metabolic adaptation during environmental changes by using evolutionarily conserved stress response programs. This metabolic homeostasis is exquisitely regulated, and its imbalance could underlie human pathological conditions. We report here that C9orf72, which is linked to the most common forms of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a key regulator of lipid metabolism under stress. Loss of C9orf72 leads to an overactivation of starvation-induced lipid metabolism that is mediated by dysregulated autophagic digestion of lipids and increased de novo fatty acid synthesis. C9orf72 acts by promoting the lysosomal degradation of coactivator-associated arginine methyltransferase 1 (CARM1), which in turn regulates autophagy-lysosomal functions and lipid metabolism. In ALS/FTD patient-derived neurons or tissues, a reduction in C9orf72 function is associated with dysregulation in the levels of CARM1, fatty acids, and NADPH oxidase NOX2. These results reveal a C9orf72-CARM1 axis in the control of stress-induced lipid metabolism and implicates epigenetic dysregulation in relevant human diseases.

Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes. Inorganic quantum dots (QDs) can overcome these problems but years of development have not yielded viable QD-FISH probes. Here we report that macromolecular size thresholds limit mRNA labeling in cells, and that a new generation of compact QDs produces accurate mRNA counts. Compared with dyes, compact QD probes provide exceptional photostability and more robust transcript quantification due to enhanced brightness. New spectrally engineered QDs also allow quantification of multiple distinct mRNA transcripts at the single-molecule level in individual cells. We expect that QD-FISH will particularly benefit high-resolution gene expression studies in three dimensional biological specimens for which quantification and multiplexing are major challenges.

Mice with histone deacetylase 6 (HDAC6) deficiency grow and develop normally but exhibit impaired immune response. The molecular mechanisms for this phenotype remain largely elusive. Methods: A mouse acute peritonitis model was used to study the infiltration of neutrophils and monocyte-derived macrophages. In vitro cell motility assays were performed to analyze monocyte/macrophage recruitment. Fluorescence microscopy and flow cytometry were performed to examine the phagocytic ability of macrophages. Immunofluorescence microscopy was used to investigate protein localization, protrusion formation, and microtubule acetylation. Results: HDAC6 deficiency does not affect neutrophil infiltration, but instead attenuates the infiltration of monocyte-derived macrophages into the peritoneal cavity. HDAC6 plays a specific role in monocyte/macrophage recruitment. Loss of HDAC6 suppresses the phagocytic capacity of macrophages challenged with E. coli. Lipopolysaccharide stimulation results in the translocation of HDAC6 and cortactin from the cytosol to the cell periphery, promotes the formation of filopodial protrusions, and enhances microtubule acetylation around the microtubule-organizing center, all of which are abrogated by HDAC6 deficiency. Conclusion: These findings implicate HDAC6 in the innate immune response and suggest that it may serve as a promising target for the treatment of macrophage-associated immune diseases.

Aging of white adipose tissue (WAT) is associated with reduced insulin sensitivity, which contributes to whole-body glucose intolerance. WAT aging in mice impairs cold-induced beige adipocyte recruitment (beiging), which has been attributed to the senescence of adipose progenitor cells. Tumor suppressor p53 has also been implicated in WAT aging. However, whether p53-related cellular aging in mature white adipocytes is causative of age-impaired WAT beiging remains unknown. It is also unclear whether transient p53 inhibition can rescue WAT beiging. Herein, we report that p53 increased in adipose tissues of 28-wk-old (aged) mice with impaired beiging capability. Cold exposure decreased p53 in beiging WAT of young mice but not in aged mice. In aged mice, inducible p53 ablation in differentiated adipocytes restored cold-induced WAT beiging and augmented whole-body energy expenditure and insulin sensitivity. Transient pharmacological inhibition of p53 led to the same beneficial effects. Mechanistically, cold exposure repressed autophagy in beiging WAT of young mice yet increased autophagy in aged WAT. p53-ablation reduced microtubule-associated protein light chain 3-mediated mitochondria clearance (mitophagy) and hence facilitated the increase of mitochondria during beiging. These findings suggest that p53-induced mitophagy in aged white adipocytes impedes WAT beiging and may be therapeutically targeted to improve insulin sensitivity in aged WAT.-Fu, W., Liu, Y., Sun, C., Yin, H. Transient p53 inhibition sensitizes aged white adipose tissue for beige adipocyte recruitment by blocking mitophagy.

Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24 h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.

Histone modifications influence higher-order chromatin structures at individual epigenomic states and chromatin environments to regulate gene expression. However, genome-wide higher-order chromatin structures shaped by different histone modifications remain poorly characterized. With stochastic optical reconstruction microscopy (STORM), we characterized the higher-order chromatin structures at their epigenomic states, categorized into three major types in interphase: histone acetylation marks form spatially segregated nanoclusters, active histone methylation marks form spatially dispersed larger nanodomains, and repressive histone methylation marks form condensed large aggregates. These distinct structural characteristics are also observed in mitotic chromosomes. Furthermore, active histone marks coincide with less compact chromatin and exhibit a higher degree of co-localization with other active marks and RNA polymerase II (RNAP II), while repressive marks coincide with densely packed chromatin and spatially distant from repressive marks and active RNAP II. Taken together, super-resolution imaging reveals three distinct chromatin structures at various epigenomic states, which may be spatially coordinated to impact transcription.