Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-beta could target to nerve cells and improve nerve regeneration.

In disparate organisms adaptation to thermal stress has been linked to changes in the expression of genes encoding heat-shock proteins (Hsp). The underlying genetics, however, remain elusive. We show here that two AT-rich sequence elements in the promoter region of the hsp70 gene of the fly Liriomyza sativae that are absent in the congeneric species, Liriomyza huidobrensis, have marked cis-regulatory consequences. We studied the cis-regulatory consequences of these elements (called ATRS1 and ATRS2) by measuring the constitutive and heat-shock-induced luciferase luminescence that they drive in cells transfected with constructs carrying them modified, deleted, or intact, in the hsp70 promoter fused to the luciferase gene. The elements affected expression level markedly and in different ways: Deleting ATRS1 augmented both the constitutive and the heat-shock-induced luminescence, suggesting that this element represses transcription. Interestingly, replacing the element with random sequences of the same length and A+T content delivered the wild-type luminescence pattern, proving that the element's high A+T content is crucial for its effects. Deleting ATRS2 decreased luminescence dramatically and almost abolished heat-shock inducibility and so did replacing the element with random sequences matching the element's length and A+T content, suggesting that ATRS2's effects on transcription and heat-shock inducibility involve a common mechanism requiring at least in part the element's specific primary structure. Finally, constitutive and heat-shock luminescence were reduced strongly when two putative binding sites for the Zeste transcription factor identified within ATRS2 were altered through site-directed mutagenesis, and the heat-shock-induced luminescence increased when Zeste was over-expressed, indicating that Zeste participates in the effects mapped to ATRS2 at least in part. AT-rich sequences are common in promoters and our results suggest that they should play important roles in regulatory evolution since they can affect expression markedly and constrain promoter DNA in at least two different ways.

For a long time, honey was purportedly helpful to prevent drunkenness and relieve hangover symptoms. However, few of the assertions have experienced scientific assessment. The present study examined the effects of honey on intoxicated male mice.

8-Prenylnaringenin (8-PN) is a phytoestrogen with the highest estrogenic activity. The objective of the present study was to confirm the superiority of 8-PN on bone metabolisms and the estrogen receptor (ER) subtype mediating effects of 8-PN. The osteoblast MC3T3-E1 and osteoclast-like cell line RAW264.7 were treated with 17β-estradiol (10(-8) mol/L), genistein (10(-5) mol/L), daidzein (10(-5) mol/L), 8-PN (10(-5) mol/L) alone or in the presence of ERα antagonist MPP (10(-7) mol/L) and ERβ antagonist PTHPP (1.5 × 10(-7) mol/L). It has been found that 8-PN did not affect osteoblast proliferation, and that 8-PN increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) concentrations, and the mineralized nodules. 8-PN inhibited RAW264.7 differentiating into osteoclasts and reduced the pit area of bone resorption. 8-PN could also inhibit the protein and mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts, and conversely promote the expression of osteoprotegerin (OPG). These effects of 8-PN were mainly inhibited not by PTHPP but by MPP and they were weaker than estrogen's effects but stronger than those of genistein and daidzein. In conclusion, the effects of 8-PN on promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption were mediated by ERα instead of ERβ and the efficacy was more potent than that of the two classic phytoestrogens: genistein and daidzein.

Many stem cells grow into three-dimensional (3D) spheres or colonies, such as neural progenitor cells (NPCs) and embryonic stem cells (ESCs). Sphere morphology helps maintaining the stemness of stem cells. Our previous study demonstrated that forced growth of RT4 and HEK293 cells into 3D sphere on low attachment surface could induce stem cell properties. The close relationship between 3D sphere morphology and stem cell stemness drives us to hypothesize that 3D sphere formation induces fibroblasts reprogramming. The key gene Sox2 for reprogramming fibroblasts into NPCs was found to be overexpressed in 3D sphere cultured mouse fibroblasts. These cells exhibited similar morphological and molecular features to NPCs in vitro, were capable of differentiating into neurons, astrocytes and oligodendrocytes, and could generate long-term expandable neurospheres while maintaining differentiation capability. When engrafted into hippocampus of adult rat brain, the 3D sphere cells differentiated into neural cells. Thus, NPCs can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Obesity is associated with hypertriglyceridemia and elevated circulating free fatty acids (FFA), resulting in endothelial dysfunction. Endoplasmic reticulum (ER) stress has been implicated in many of these processes. To determine if ER stress participates in palmitate-induced apoptosis, we investigated the effects of diet-induced obesity and palmitate on mouse aortic endothelial cells (MAEC) in vivo and in vitro.

Revealing the mechanisms of cell fate regulation is important for scientific research and stem cell-based therapy. The traditional two-dimensional (2D) cultured mES cells are in a very different 2D niche from the in vivo equivalent-inner cell mass (ICM). Because the cell fate decision could be regulated by many cues which could be impacted by geometry, the traditional 2D culture system would hamper us from understanding the in vivo situations correctly. Three-dimensional (3D) scaffold was believed to provide a 3D environment closed to the in vivo one. In this work, three different scaffolds were prepared for cell culture. Several characters of mES cells were changed under 3D scaffolds culture compared to 2D, and these changes were mainly due to the alteration in geometry but not the matrix. The self-renewal of mES cells was promoted by the introducing of dimensionality. The stemness maintenance of mES was supported by all three 3D scaffolds without feeder cells in the long-time culture. Our findings demonstrated that the stemness maintenance of mES cells was promoted by the 3D geometry of scaffolds and this would provide a promising platform for ES cell research.

Reprogramming energy metabolism has been an emerging hallmark of cancer cells. MicroRNAs play important roles in glucose metabolism.

Circulating tumor cells (CTCs), which have stem cell-like characteristics, might play a crucial role in cancer metastasis. CD44 has been identified as gastric cancer (GC) stem cell (CSC) marker. Here, the prognostic significance of CD44-positive CTCs in GC patients was investigated. CTCs were detected in 27 of 45 GC patients. The presence of CTCs was significantly associated with lymph node metastasis, distant metastasis, and recurrence (P = 0.007, P = 0.035, and P = 0.035, resp.). Nineteen of the 27 CTC-positive patients had CD44-positive CTCs. These patients were more likely to develop metastasis and recurrence than patients with CD44-negative CTCs. CD44-positive CTC counts were higher in recurrent patients than in the nonrecurrent ones (means 4.8 and 1.9, resp.; P = 0.010). Furthermore, 13 of 19 patients with CD44-positive CTCs developed recurrent disease, and the mean time to recurrence was shorter than that in patients with CD44-negative CTCs (10.54 ± 5.55 and 19.13 ± 9.72 months, resp.; P = 0.04). COX proportional hazards model indicated that the presence of CD44-positive CTCs and TNM stage were independent predictors of recurrence for GC (P = 0.030 and 0.008). So identifying the stem cell-like CTC subset may provide more clinically useful prognostic information than only detecting CTCs.

Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β(1) -AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β(1) -AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed β(1) -AR expression at the protein level, without affecting β(1) -AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β(1) -AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β(1) -AR expression in rats, whereas len-AMO-let-7e up-regulated β(1) -AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β(1) -AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β(1) -AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β(1) -AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β(1) -AR blocker. Down-regulation of let-7e contributes to the adverse increase in β(1) -AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β(1) -AR up-regulation and treating AMI-induced arrhythmia.

Immunization strategies that elicit antibodies capable of neutralizing diverse virus strains will likely be an important part of a successful vaccine against HIV. However, strategies to promote robust humoral responses against the native intact HIV envelope trimer structure are lacking. We recently developed chemically cross-linked lipid nanocapsules as carriers of molecular adjuvants and encapsulated or surface-displayed antigens, which promoted follicular helper T-cell responses and elicited high-avidity, durable antibody responses to a candidate malaria antigen. To apply this system to the delivery of HIV antigens, Env gp140 trimers with terminal his-tags (gp140T-his) were anchored to the surface of lipid nanocapsules via Ni-NTA-functionalized lipids. Initial experiments revealed that the large (409 kDa), heavily glycosylated trimers were capable of extracting fluid phase lipids from the membranes of nanocapsules. Thus, liquid-ordered and/or gel-phase lipid compositions were required to stably anchor trimers to the particle membranes. Trimer-loaded nanocapsules combined with the clinically relevant adjuvant monophosphoryl lipid A primed high-titer antibody responses in mice at antigen doses ranging from 5 μg to as low as 100 ng, whereas titers dropped more than 50-fold over the same dose range when soluble trimer was mixed with a strong oil-in-water adjuvant comparator. Nanocapsule immunization also broadened the number of distinct epitopes on the HIV trimer recognized by the antibody response. These results suggest that nanocapsules displaying HIV trimers in an oriented, multivalent presentation can promote key aspects of the humoral response against Env immunogens.

The clinical use of doxorubicin, an effective anticancer drug, is severely hampered by its cardiotoxicity. 23-Hydroxybetulinic acid (23-HBA), isolated from Pulsatilla chinensis, enhances the anticancer effect of doxorubicin while simultaneously reducing its cardiac toxicity, but does not affect the concentration of doxorubicin in the plasma and heart. As the metabolite doxorubicinol is more potent than doxorubicin at inducing cardiac toxicity, in the present study we aimed to clarify the role of doxorubicinol in the protective effect of 23-HBA.

Hirschsprung disease (HSCR), or colonic aganglionosis, is a congenital disorder characterized by the absence of intramural ganglia along variable lengths of the colon, resulting in intestinal obstruction. It is the most common cause of congenital intestinal obstruction, with an incidence of 1 in 5,000 live births. N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. In the current study, a novel mutant mouse with aganglionic megacolon and coat color spotting was generated by ENU-induced mutagenesis. Histological and acetylcholinesterase (AChE) whole-mount staining analysis showed a lack of ganglion cells in the colon in mutant mice. The mutation was mapped to chromosome 14 between markers rs30928624 and D14Mit205 (Chr 14 positions 103723921 bp and 105054651 bp). The Ednrb (Chr 14 position 103814625-103844173 bp) was identified as a potential candidate gene in this location. Mutation analysis revealed a T>C missense mutation at nucleotide 857 of the cDNA encoding endothelin receptor B (EDNRB) in which a proline was substituted for the highly conserved Lys-286 residue (L286P) in the fifth transmembrane (TM V) domain of this G protein-coupled receptor. The mutant mouse was named Ednrb(m1yzcm) (Ednrb; mutation 1, Yangzhou University Comparative Medicine Center). The results of the present study implicate the structural importance of the TM V domain in Ednrb function, and the Ednrb(m1yzcm) mouse represents a valuable model for the study of HSCR in humans.

Modern lifestyles, such as shift work, nocturnal social activities, and jet lag, disturb the circadian rhythm. The interaction between mammals and the co-evolved intestinal microbiota modulates host physiopathological processes. Radiotherapy is a cornerstone of modern management of malignancies; however, it was previously unknown whether circadian rhythm disorder impairs prognosis after radiotherapy. To investigate the effect of circadian rhythm on radiotherapy, C57BL/6 mice were housed in different dark/light cycles, and their intestinal bacterial compositions were compared using high throughput sequencing. The survival rate, body weight, and food intake of mice in diverse cohorts were measured following irradiation exposure. Finally, the enteric bacterial composition of irradiated mice that experienced different dark/light cycles was assessed using 16S RNA sequencing. Intriguingly, mice housed in aberrant light cycles harbored a reduction of observed intestinal bacterial species and shifts of gut bacterial composition compared with those of the mice kept under 12 h dark/12 h light cycles, resulting in a decrease of host radioresistance. Moreover, the alteration of enteric bacterial composition of mice in different groups was dissimilar. Our findings provide novel insights into the effects of biological clocks on the gut bacterial composition, and underpin that the circadian rhythm influences the prognosis of patients after radiotherapy in a preclinical setting.

Hepatocellular carcinoma (HCC) patients with main portal vein tumor thrombus have a median survival time of only about 4 months. We therefore compared the safety and efficacy of endovascular brachytherapy (EVBT) and sequential three-dimensional conformal radiotherapy (3-DCRT). From a cohort of 176 patients, we treated 123 with EVBT using iodine-125 seed strands (group A) and the remaining 53 with sequential 3-DCRT (group B). Overall survival, progression free survival and stent patency characteristics were compared between the two groups. Our analysis demonstrated a median survival of 11.7 ± 1.2 months in group A versus 9.5 ± 1.8 months in group B (p = 0.002). The median progression free survival was 5.3 ± 0.7 months in groupA versus 4.4 ± 0.4 months in group B (p = 0.010). The median stent patency period was 10.3 ± 1.1 months in group A versus 8.7 ± 0.7 months in group B (p = 0.003). Therefore, as compared to sequential 3-DCRT, EVBT combined with portal vein stenting and TACE improved overall survival of HCC patients with main portal vein tumor thrombus.

Intron evolution, including its dynamics in the evolutionary transitions and diversification of eukaryotes, remains elusive. Inadequate taxon sampling due to data shortage, unclear phylogenetic framework, and inappropriate outgroup application might be among the causes. Besides, the integrity of all the introns within a gene was often neglected previously. Taking advantage of the ancient conserved triosephosphate isomerase gene (tim), the relatively robust phylogeny of Metazoa, and choanoflagellates as outgroup, the evolutionary dynamics of tim intron location pattern (ILP) in Metazoa was investigated. From 133 representative species of ten phyla, 30 types of ILPs were identified. A most common one, which harbors the maximum six intron positions, is deduced to be the common ancestral tim ILP of Metazoa, which almost had formed in their protozoan ancestor and was surprisingly retained and passed down till to each ancestors of metazoan phyla. In the subsequent animal diversification, it underwent different evolutionary trajectories: within Deuterostomia, it was almost completely retained only with changes in a few species with relatively recently fast-evolving histories, while within the rapidly radiating Protostomia, besides few but remarkable retention, it usually displayed extensive intron losses and a few gains. Therefore, a common ancestral exon-intron arrangement pattern of an animal gene is definitely discovered; besides the 'intron-rich view' of early animal genes being confirmed, the novel insight that high exon-intron re-arrangements of genes seem to be associated with the relatively recently rapid evolution of lineages/species/genomes but have no correlation with the ancient major evolutionary transitions in animal evolution, is revealed.

The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) have shown remarkable antitumor activity in an increasing number of cancers. When combined, ipilimumab and nivolumab have demonstrated superior activity in patients with metastatic melanoma (CHECKMATE-067). Here we describe the preclinical development strategy that predicted these clinical results. Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells accumulated at the tumor margin and infiltrated the tumor mass in response to the combination therapy, resulting in favorable effector and regulatory T-cell ratios, increased pro-inflammatory cytokine secretion, and activation of tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and nivolumab showed enhanced cytokine secretion in superantigen stimulation of human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a cynomolgus macaque toxicology study, dose-dependent immune-related gastrointestinal inflammation was observed with the combination therapy; this response had not been observed in previous single agent cynomolgus studies. Together, these in vitro assays and in vivo models comprise a preclinical strategy for the identification and development of highly effective antitumor combination immunotherapies.

The purpose of this study was to investigate the effects and mechanisms of dihydrotestosterone (DHT)-induced expression of sterol regulatory element binding protein-1 (SREBP-1), and the synthesis and secretion of lipids, in HaCaT cells. HaCaT cells were treated with DHT and either the phosphoinositide 3-kinase inhibitor LY294002 or the extracellular-signal-regulated kinase (ERK) inhibitor PD98059. Real time-PCR, Western blot, Oil Red staining and flow cytometry were employed to examine the mRNA and protein expressions of SREBP-1, the gene transcription of lipid synthesis, and lipid secretion in HaCaT cells.

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 μl (5 × 10(4) conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 μl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.