Nonlethal mechanical trauma causes cardiomyocyte apoptosis which contributes to posttraumatic cardiac dysfunction. Apoptosis is positively correlated with protein nitration in the traumatic heart. However, the mechanisms responsible for the cardiomyocyte protein nitration remain unclear. The present study was designed to identify whether myeloperoxidase may contribute to protein nitration in nonlethal mechanical trauma and subsequent cardiomyocyte apoptosis, and, if so, to determine the possible mechanisms responsible. We used Noble-Collip drum to make nonlethal traumatic mice models. Male adult C57B16/J mice were placed in the Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Then myeloperoxidase activity and release, protein nitration, cardiomyocyte apoptosis, endothelial function and intercellular adhesion molecule-1 expression were determined. Nonlethal mechanical trauma was characterized by the 100% survival rate during the first 24 h after trauma, the lack of circulatory shock and without direct heart injury. However, myeloperoxidase activity significantly increased 6 h after trauma, and reached a maximum level 12 h after trauma. Obviously, protein nitration and cardiomyocyte apoptosis increased 12h after trauma and could be blocked by administration of R15.7, a monoclonal antibody that blocks polymorphonuclear neutrophils adhesion. Moreover, endothelial dysfunction and intercellular adhesion molecule-1 upregulation were observed in traumatic mice. Our present study demonstrated for the first time that myeloperoxidase caused protein nitration and cardiomyocyte apoptosis in nonlethal traumatic mice. Inhibition of polymorphonuclear neutrophils adhesion and antinitration treatments may be novel measures in reducing posttraumatic cardiomyocyte apoptosis and secondary heart injury.

Hepatocellular carcinoma (HCC) is a major primary liver malignancy in adults. Despite the progress made, the outcome of the treatment to this disease is less than satisfactory as the post therapy tumor recurrence is almost inevitable. Accumulating pieces of evidence have suggested that the recurrence is due to the existence of a subpopulation of the HCC cells that possess the properties of stem cells and are resistant to radiation and chemotherapy. It is therefore important to understand the characteristics of this subpopulation of HCC cells, and which requires the establishment of an in vitro system to study these stem-like cancer cells. However, despite extensive efforts, the progress in establishing such an in vitro system has been slow largely due to the lack of definitive biomarkers in the isolation and expansion of these cells. In order to successfully maintain and expand HCC CSCs, we first optimized the culture system. We establish a novel medium system that allows the culture and enrichment of these hepatic stem-like cancer cells from both hepatoma cells and human primary HCC cells. These cells exhibited typical stem cell properties, such as enhanced stem cell markers, gain of EMT properties and drug resistance, and more importantly, stronger tumor-initiating capabilities. The medium may help to establish an in vitro model for hepatic cancer stem cell (HCSC) studies, which may contribute to the development of novel cell therapies and new drugs for the treatment of HCC.

This study aimed to address the status, role, and mechanism of sympathetic nerve infiltration in the progression of stomach adenocarcinoma (STAD).

Salvage radiotherapy (SRT) is the first-line treatment for prostate cancer patients with biochemical recurrence following radical prostatectomy, and new specific radiosensitizers are in urgent need to enhance SRT effect. MLN4924 (also known as Pevonedistat), a specific inhibitor of NEDD8-activating enzyme, has recently entered phase I/II clinical trials in several malignancies. By inhibiting cullin neddylation, MLN4924 inactivates Cullin-RING ligases (CRL), which have been validated as an attractive radiosensitizing target. In our study, we demonstrate that MLN4924 can be used as a potent radiosensitizer in hormone-resistant prostate cancer cells. We found that MLN4924 inhibited cullin neddylation and sensitized prostate cancer cells to irradiation (IR). Mechanistically, MLN4924 enhanced IR-induced G2 cell-cycle arrest, by inducing accumulation of WEE1/p21/p27, three well-known CRL substrates. Importantly, siRNA knockdown of WEE1/p21/p27 partially abrogated MLN4924-induced G2 cell-cycle arrest, indicating a causal role of WEE1/p21/p27 in MLN4924-induced radiosensitization. Further mechanistic studies revealed that induction of DNA damage and apoptosis also contributed to MLN4924 radiosensitization in hormone-resistant prostate cancer cells. Our findings lay the foundation for future application of MLN4924 as a potential radiosensitizer in hormone refractory prostate cancer (HRPC).

β3-Adrenoceptors (β3-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of β1- and β2-ARs. It has been previously demonstrated that autoantibodies against the β1/β2-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the β3-AR (β3-AR Abs) exist in the sera of patients with HF and to assess the effects of β3-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of β3-AR Abs in the sera of HF patients was screened by ELISA. β3-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of β3-AR Abs in the sera of HF patients were greatly increased, and β3-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. β3-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca(2+) transient and membrane L-type Ca(2+) current in cardiomyocytes. Our results demonstrated the existence of β3-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.

Autophagy is important in cells for removing damaged organelles, such as mitochondria. Insufficient autophagy plays a critical role in tissue injury and organ dysfunction under a variety of pathological conditions. However, the role of autophagy in nonlethal traumatic cardiac damage remains unclear. The aims of the present study were to investigate whether nonlethal mechanical trauma may result in the change of cardiomyocyte autophagy, and if so, to determine whether the changed myocardial autophagy may contribute to delayed cardiac dysfunction. Male adult rats were subjected to nonlethal traumatic injury, and cardiomyocyte autophagy, cardiac mitochondrial function, and cardiac function in isolated perfused hearts were detected. Direct mechanical traumatic injury was not observed in the heart within 24 h after trauma. However, cardiomyocyte autophagy gradually decreased and reached a minimal level 6 h after trauma. Cardiac mitochondrial dysfunction was observed by cardiac radionuclide imaging 6 h after trauma, and cardiac dysfunction was observed 24 h after trauma in the isolated perfused heart. These were reversed when autophagy was induced by administration of the autophagy inducer rapamycin 30 min before trauma. Our present study demonstrated for the first time that nonlethal traumatic injury caused decreased autophagy, and decreased autophagy may contribute to post-traumatic organ dysfunction. Though our study has some limitations, it strongly suggests that cardiac damage induced by nonlethal mechanical trauma can be detected by noninvasive radionuclide imaging, and induction of autophagy may be a novel strategy for reducing posttrauma multiple organ failure.

Intratumoral heterogeneity reflects subclonal diversity and accounts for a variety of clinically defined phenotypes including the development of drug resistance and recurrence. However, intratumoral heterogeneity of bile duct carcinoma (BDC) is rarely studied.

The consequences of sickle cell disease (SCD) include ongoing hematopoietic stress, hemolysis, vascular damage, and effect of chronic therapies, such as blood transfusions and hydroxyurea, on hematopoietic stem and progenitor cell (HSPC) have been poorly characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients, stratified by treatment and control cohorts. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34dim cells, a two to five-fold increase in CD34bright cells, a depletion in Megakaryocyte-Erythroid Progenitors, and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a as well as high levels CD49f antigen expression. These findings were present to varying degrees in all patients with SCD, including those on chronic therapy and those who were therapy naive. HU treatment appeared to normalize many of these parameters. Chronic stress erythropoiesis and inflammation incited by SCD and HU therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD.

Starch and sucrose metabolism plays a crucial role in the formation and development of bulbs in bulbous plants. However, these mechanisms remain unclear and unexplored in the corms of Freesia hybrida. Herein, we investigated the dynamics of the major form of carbohydrates and related enzyme activities and profiled the transcriptome of freesia corms at four developmental stages with the aim to reveal the relation between the expression of genes involved in the metabolism of carbohydrates and the accumulation of carbohydrates in corm developmental stages for further exploring the mechanism on the starch and sucrose metabolism regulating the formation and development of corms in F. hybrida. The content of starch, sucrose and soluble sugars followed an overall upward trend across the corm developmental stages. Activities of the adenosine diphosphoglucose pyrophosphorylase, starch branching enzyme and β-amylase generally followed the pattern of the starch and sucrose levels. Activities of sucrose phosphate synthase increased from corm formation till the initial swelling stage and subsequently reached a plateau. Activities of invertase and sucrose synthase peaked at the later rapid swelling stage. These suggested that the starch and sucrose dynamics paralleled corm swelling under the action of metabolic enzymes. A total of 100,999 unigenes were assembled in the transcriptomic analysis, and 44,405 unigenes of them were annotated. Analysis based on Clusters of Orthologous Groups suggested that carbohydrate transport and metabolism (9.34% of the sequences) was prominent across the corm developmental process. In total 3,427 differentially expressed genes (DEGs) were identified and the enrichment analysis detected starch and sucrose metabolism as a critical pathway in corm development, especially at the rapid swelling stage. Further, DEGs encoding key carbohydrate-metabolizing enzymes were identified and correlated to enzyme activities and carbohydrate accumulation. The results construct a valuable resource pool for further molecular-level studies, which are helpful for metabolic regulation of carbohydrates and improvement in F. hybrida.

Fragile X syndrome (FXS) is caused by CGG repeat expansion that leads to FMR1 silencing. Women with a premutation allele are at risk of having a full mutation child with FXS. To investigate the mechanism of repeat expansion, we examined the relationship between a single-nucleotide polymorphism (SNP) variant that is linked to repeat expansion in haplogroup D and a replication origin located ∼53 kb upstream of the repeats. This origin is absent in FXS human embryonic stem cells (hESCs), which have the SNP variant C, but present in the nonaffected hESCs, which have a T variant. The SNP maps directly within the replication origin. Interestingly, premutation hESCs have a replication origin and the T variant similar to nonaffected hESCs. These results suggest that a T/C SNP located at a replication origin could contribute to the inactivation of this replication origin in FXS hESCs, leading to altered replication fork progression through the repeats, which could result in repeat expansion to the FXS full mutation.

Lysine crotonylation (Kcr) is a newly identified protein translational modification and is involved in major biological processes including glycolysis, but its role in colorectal cancer (CRC) is unknown. Here, we found that the Kcr of α enolase (ENO1) was significantly elevated in human CRC tissues compared with the paratumoral tissues. CREB-binding protein (CBP) functioned as a crotonyltranferase of ENO1, and SIRT2 was involved in the decrotonylation of ENO1. Using quantitative mass spectrometry for crotonylomics analysis, we further found that K420 was the main Kcr site of ENO1 and ENO1 K420 Kcr promoted the growth, migration, and invasion of CRC cells in vitro by enhancing the activity of ENO1 and regulating the expression of tumor-associated genes. Our study reveals an important mechanism by which ENO1 regulates CRC through crotonylation.

Non-essential proteins for viral replication affect host cell metabolism, while the function of the UL43 protein of herpes simplex virus 1 (HSV-1) is not clear. Herein, we performed a comprehensive microarray analysis of HUVEC cells infected with HSV-1 and its UL43-deficient mutant and found significant variation in genes associated with cellular energy metabolic pathways. The localization of UL43 protein in host cells and how it affects cellular energy metabolism pathways were further investigated. Internalization analysis showed that the UL43 protein could be endocytosis-mediated by YPLF motif (aa144-147) and localized to mitochondria. At the same time, more ATP was produced by coupling with mitochondrial small G protein ARF-like 2 (ARL2) GTPase, which triggered the phosphorylation of ANT1 (SLC25A4) to affect the opening degree of mitochondrial permeability transition pore (mPTP), and significantly promoted the aerobic oxidation and oxidative phosphorylation of glucose. Our study shows that UL43 mediates the improvement of host cell metabolism after HSV-1 infection. Additionally, UL43 protein could be a valuable ATP-stimulating factor for mammalian cells.

It has been recognized that myocardial apoptosis is one major factor in the development of heart dysfunction and autophagy has been shown to influence the apoptosis. In previous studies, we reported that anti-β1-adrenergic receptor autoantibodies (β1-AABs) decreased myocardial autophagy, but the role of decreased autophagy in cardiomyocyte apoptosis remains unclear. In the present study, we used a β1-AAB-immunized rat model to investigate the role of decreased autophagy in cardiomyocyte apoptosis. We reported that the level of autophagic flux increased early and then decreased in an actively β1-AAB-immunized rat model. Rapamycin, an mTOR inhibitor, restored myocardial apoptosis in the presence of β1-AABs. Further, we found that the early increase of autophagy was an adaptive stress response that is possibly unrelated to β1-AR, and the activation of the β1-AR and PKA contributed to late decreased autophagy. Then, after upregulating or inhibiting autophagy with rapamycin, Atg5 overexpression adenovirus or 3-methyladenine in cultured primary neonatal rat cardiomyocytes, we found that autophagy decline promoted myocardial apoptosis effectively through the mitochondrial apoptotic pathway. In conclusion, the reduction of apoptosis through the proper regulation of autophagy may be important for treating patients with β1-AAB-positive heart dysfunction.

The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available.

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs-i.e., origin distribution, termination site location, and fork rate and direction-of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

Efficiency of reprogramming of human cells into induced pluripotent stem cells (iPSCs) has remained low. We report that individual adult human CD49f+ long-term hematopoietic stem cells (LT-HSCs) can be reprogrammed into iPSCs at close to 50% efficiency using Sendai virus transduction. This exquisite sensitivity to reprogramming is specific to LT-HSCs, since it progressively decreases in committed progenitors. LT-HSC reprogramming can follow multiple paths and is most efficient when transduction is performed after the cells have exited G0. Sequencing of 75 paired skin fibroblasts/LT-HSC samples collected from nine individuals revealed that LT-HSCs contain a lower load of somatic single-nucleotide variants (SNVs) and indels than skin fibroblasts and accumulate about 12 SNVs/year. Mutation analysis revealed that LT-HSCs and fibroblasts have very different somatic mutation signatures and that somatic mutations in iPSCs generally exist prior to reprogramming. LT-HSCs may become the preferred cell source for the production of clinical-grade iPSCs.

It has been recognized that changes in mitochondrial structure plays a key role in development of cardiac dysfunction, and autophagy has been shown to exert maintenance of mitochondrial homeostasis effects. Our previous study found that anti-β1-adrenergic receptor autoantibodies (β1-AABs) could lead to cardiac dysfunction along with abnormalities in mitochondrial structure. The present study tested the hypothesis that β1-AABs may induce the decline in mitochondrial membrane potential (ΔΨm) by suppression of cardiac autophagy, which contributed to cardiac dysfunction. Male adult rats were randomized to receive a vehicle or peptide corresponding to the second extracellular loop of the β1 adrenergic receptor (β1-AAB group, 0.4 μg/g every two weeks for 12 weeks) and treated with rapamycin (RAPA, an autophagy agonist) at 5 mg/kg/day for two days before detection. At the 4th week, 8th week and 12th week of active immunization, the rats were sacrificed and cardiac function and the levels of cardiac LC3 and Beclin-1 were detected. ΔΨm in cardiac myocytes was determined by myocardial radionuclide imaging technology and JC-1 staining. In the present study, β1-AABs caused cardiac dysfunction, reduced ΔΨm and decreased cardiac autophagy. Treatment with RAPA markedly attenuated β1-AABs-induced cardiac injury evidenced by recovered ΔΨm. Taken together, these results suggested that β1-AABs exerted significant decreased ΔΨm, which may contribute to cardiac dysfunction, most likely by decreasing cardiac autophagy in vivo. Moreover, myocardial radionuclide imaging technology may be needed to assess the risk in developing cardiac dysfunction for the people who have β1-AABs in their blood.

The present study aimed to reveal expression status of the neddylation enzymes in HNSCC and to elucidate the anticancer efficacy and the underlying mechanisms of inhibiting neddylation pathway.

Characterization of human cells that sustain blood cell production lifelong has historically been inferred from phenotypically defined subsets of cells assayed in vitro, in transplanted immunodeficient mice, or in patients transplanted with genetically marked cells. These approaches have led to the concept of a persistent complex hierarchical process of differentiation divisions originating from a rare population of CD34+CD38-CD45RA-CD90+CD49f+ cells with an average self-renewal potential of >0.5 and an ability to produce some or all blood cell types for >1 year. However, the role of these "49f" cells in the unperturbed adult has remained poorly understood. To address this gap, somatic single-nucleotide polymorphisms (SNVs) have recently been exploited as lineage tracing markers to enumerate and characterize active hematopoietic clones in normal adults using a capture and recapture approach. We show here that the use of somatic transversions to identify somatically acquired variant alleles enabled their detection in bulk populations at frequencies of approximately 1 in 80,000 cells. We then applied this method to blood cells isolated from two normal adults (aged 31 and 53 years) over a 1- to 3-year period. The results revealed in both donors a continued clonal output of both T- and B-lymphoid cells as well as myeloid cells identified by the same unique transversions found to distinguish single 49f cells isolated from the same donors' initial blood samples. These findings provide the first evidence of a continuing hematopoietic stem cell-derived source of all mature blood cell types in normal (unperturbed) adult humans.

A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter.