Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P-values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P< 5 × 10−8, and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19–1.40) and P= 7.63 × 10−10. An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8/ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8. Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.
To assess the roles of extracellular signal-regulated kinase (ERK), p38, and CD151-integrin complexes on proliferation, migration, and tube formation activities of CD151-induced human umbilical vein endothelial cells (HUVECs).
CD151, anti-CD151 and CD151-AAA mutant were inserted into recombinant adeno-associated virus (rAAV) vectors and used to transfect HUVECs. After transfection, the expression of CD151 was measured. Proliferation was assessed using the 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell migration was evaluated in Boyden transwell chambers using FBS as the chemotactic stimulus. The tube formation assay was performed on matrigel. The potential involvement of various signaling pathways was explored using selective inhibitors.
CD151 gene delivery increased the expression of CD151 at both the mRNA and protein levels. Overexpression of CD151 promoted cell proliferation, migration and tube formation in vitro, and phosphorylation of ERK was also increased. Further, CD151-induced cell proliferation, migration, and tube formation were attenuated by the ERK inhibitor PD98059 (20 μmol/L) but not by a p38 inhibitor (SB203580, 20 μmol/L). Moreover, there was no significant difference in CD151 protein expression between the CD151 group and the CD151-AAA group, but the CD151-AAA mutant abrogated cellular proliferation, migration, and tube formation and decreased the phosphorylation of ERK.
This study suggests that activation of the ERK signaling pathway may be involved in the angiogenic effects of CD151. Activation of ERK was dependent on the formation of CD151-integrin complexes. Therefore modulation of CD151 may be as a novel therapeutic strategy for regulating angiogenesis.
CD151; cell migration; cell proliferation; angiogenesis; ERK; p38 MAPK; human umbilical vein endothelial cells
Two atmospheric circulation systems, the mid-latitude Westerlies and the Asian summer monsoon (ASM), play key roles in northern-hemisphere climatic changes. However, the variability of the Westerlies in Asia and their relationship to the ASM remain unclear. Here, we present the longest and highest-resolution drill core from Lake Qinghai on the northeastern Tibetan Plateau (TP), which uniquely records the variability of both the Westerlies and the ASM since 32 ka, reflecting the interplay of these two systems. These records document the anti-phase relationship of the Westerlies and the ASM for both glacial-interglacial and glacial millennial timescales. During the last glaciation, the influence of the Westerlies dominated; prominent dust-rich intervals, correlated with Heinrich events, reflect intensified Westerlies linked to northern high-latitude climate. During the Holocene, the dominant ASM circulation, punctuated by weak events, indicates linkages of the ASM to orbital forcing, North Atlantic abrupt events, and perhaps solar activity changes.
Co-infections of HBV and HIV are frequent due to similar routes of transmission. In that transmission through blood is an important route for both HBV and HIV, evaluation of the prevalence of HBV in HIV infected blood donors may be important for transfusion safety. In addition, because the epidemiological characteristics of HBV in HIV infected patients and blood donors may differ from each other, understanding of it could be significant for therapy and prevention of HBV in HIV infected adults. However, data reported on these in Chinese people remains limited.
614 HIV confirmed positive samples were collected from blood donors and patients and were screened for HBsAg and HBV DNA. The samples screened reactive for HBsAg or positive for HBV DNA were tested for the other serological markers of HBV including anti-HBs, HBeAg, anti-HBe and anti-HBc. For the samples tested positive for HBV DNA, the S region of HBV was amplified by nested PCR and the HBV genotypes were determined.
HBV coinfections were found in 12.9% (79/614) HIV infected individuals including 42/417(10.1%) blood donors and 37/197 (18.8%) AIDS patients. In the HBsAg positive individuals, 80.0% were HBeAg negative in which 10.0% were HBV DNA negative and 38.3% with HBV DNA lower than 2000 IU/ml. The average HBV DNA levels were lower in donors than in patients. In the HBV DNA positive populations, HBV genotypes B, A and C accounted for 48.1%, 22.8% and 8.86% respectively. Mutations related to the failure of HBsAg detection were found in 2 of the 4 HBsAg-/HBV DNA + subjects.
High prevalence of HBV in HIV infected individuals was found in this study. Hence, we recommend routine testing of HBV for patients newly diagnosed with HIV/AIDS in China. Some HIV-HBV co-infected patients remain undiagnosed if only conventional serological markers for HBV are used and it’s important to detect HBV DNA for HIV infected patients. HBV DNA levels were relatively low in HBeAg negative patients, thus this serologic marker may be useful in prioritizing patients on their need for HBV treatment in settings in which HBV DNA is not available.
HIV; HBV; Blood donors; AIDS patients
Although the genetic variability of hepatitis B virus (HBV) in HBV-infected patients has been extensively studied, reports on genotypes, subtypes and mutations in the S region of HBV strains from Chinese blood donors are limited. In this study, 245 blood samples from HBsAg-positive blood donors were collected from five geographically diverse blood centers in China. The S region of HBV was amplified, and the HBV genotype and subtype were determined. The amino acid sequences of the S region were aligned, and mutations related to the failure of immunization and HBsAg detection were determined. Of the 245 samples, 228 (93 %) were genotyped successfully. We found that genotypes B, C, D and A accounted for 58.8 %, 21.9 %, 6.6 % and 3.95 % of the isolates, respectively. The distribution of HBV antigen subtypes was as follows: adw (67.6 %), adr (23.3 %) and ayw (8.7 %). Mutations were present in 39 (17.1 %) of 228 samples in the major hydrophilic region (MHR) of the S region. This study demonstrated that HBV genotype/subtype B/adw was the most frequent strain circulating in HBV-infected Chinese blood donors, followed by C/adr. The occurrence of MHR mutants in HBV-infected blood donors and the potential failure to detect some of them in collected units poses a threat to transfusion safety.
Pancreatic carcinoma is one common cancer with gradually increasing incidence during the past several decades. However, currently the candidate drugs to suppress pancreatic cancer remain lacking. This research was carried out to investigate if zerumbone, a natural cyclic sesquiterpene isolated from Zingiber zerumbet Smith, will produce the anticancer effects on pancreatic carcinoma cell lines. The results showed that zerumbone concentration, and time, dependently produced inhibitory actions on cell viability of PANC-1 cells. In addition, Hoechst 33342, AO/EB, TUNEL staining, and caspase-3 activity assay further showed that zerumbone induced apoptosis of PANC-1 cells. The expression of p53 protein was markedly upregulated, and the p21 level was also obviously elevated in zerumbone-treated PANC-1 cells. Moreover, ROS production was increased by about 149% in PANC-1 cells treated by zerumbone 30 μM. Zerumbone also produced the same antitumor activity in pancreatic carcinoma cell lines SW1990 and AsPC-1. In summary, we found that zerumbone was able to induce apoptosis of pancreatic carcinoma cell lines, indicating to be a promising treatment for pancreatic cancer.
Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.
The fungus Arthrobotrys oligospora has multiple lifestyles. It's not only a nematode pathogen, but also a saprophyte, a pathogen of other fungi, and a colonizer of plant roots. As a nematode pathogen, A. oligospora forms adhesive networks to capture nematodes and is a model organism for understanding the interaction between these fungi and their host nematodes. In this study, the whole genome sequence of A. oligospora was reported. Our analyses of the proteome profiles of intracellular proteins from cells treated with nematode extracts for 10 h and 48 h revealed a key set of genes involved in trap formation. The changes in protein levels for some trap formation related genes were further confirmed by qPCR. The combined genome and proteome analysis identified the major genetic and metabolic pathways involved in trap formation in A. oligospora. Our results provide the first glimpse into the genome and proteome of this fascinating group of carnivorous fungi. The data should serve as a roadmap for further investigations into the interaction between nematode-trapping fungi and their host nematodes, providing broad foundations for research on the biocontrol of pathogenic nematodes.
The tuberous sclerosis complex (TSC)–mammalian target of rapamycin (mTOR) pathway is a key regulator of cellular metabolism. We used conditional deletion of Tsc1 to address how quiescence is associated with the function of hematopoietic stem cells (HSCs). We demonstrate that Tsc1 deletion in the HSCs drives them from quiescence into rapid cycling, with increased mitochondrial biogenesis and elevated levels of reactive oxygen species (ROS). Importantly, this deletion dramatically reduced both hematopoiesis and self-renewal of HSCs, as revealed by serial and competitive bone marrow transplantation. In vivo treatment with an ROS antagonist restored HSC numbers and functions. These data demonstrated that the TSC–mTOR pathway maintains the quiescence and function of HSCs by repressing ROS production. The detrimental effect of up-regulated ROS in metabolically active HSCs may explain the well-documented association between quiescence and the “stemness” of HSCs.
Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms.
Methods and results
First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects.
Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway.
Tollip; Cardiac remodelling; Pressure overload; AKT; Cardiomyocyte hypertrophy
The epidemiology of hepatitis D virus (HDV) in China is fairly unknown. The mechanisms whereby HDV leads to accelerated liver disease in hepatitis B virus (HBV)/HDV co-infected patients and the histological characteristics of chronic hepatitis D (CHD) patients need further investigation.
The prevalence of HDV was retrospectively evaluated in all consecutive hospitalized patients with chronic HBV infection from May 2005 to October 2011. HBV/HDV co-infected patients and HBV mono-infected patients were compared clinically and histologically. Significant histological abnormality was defined as significant necroinflammation (grade ≥A2) and/or significant fibrosis (stage ≥ F2).
6.5% of patients (426/6604) tested positive for IgM anti-HDV. HDV was more common in patients over 50 years old than those under 50 (11.7% vs. 5.1%, P<0.001). HBV/HDV co-infected patients had higher frequencies of end-stage liver disease (ESLD) than HBV mono-infected patients, and HDV co-infection was an independent risk factor for ESLD (OR: 1.428, 95%CI: 1.116–1.827; P = 0.005). The HBV DNA levels in the HBV/HDV group were significantly lower than the HBV group in chronic hepatitis patients (median: 6.50 log10copies/mL vs 6.80 log10copies/mL, P = 0.003), but higher than the HBV group in ESLD patients (median: 5.73 log10copies/mL vs 5.16 log10copies/mL, P<0.001). When stratified by alanine aminotransferase (ALT) level, 46.7%, 56.5% and 80.5% of CHD patients had significant necroinflammation and 86.7%, 87.0% and 90.3% had significant fibrosis with ALT 1–2×upper limit normal (ULN), 2–5×ULN and>5×ULN respectively.
The prevalence of HDV is not low in patients with chronic HBV infection. HDV may contribute to progression to ESLD through late-phase HBV DNA reactivation.
During August 2011–February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China.
A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment.
One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8–1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5–12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February–May, 2012, and the strain has not been isolated since.
High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary.
Sexually transmitted infections (STIs) increase HIV infectivity through local inflammatory processes. Prevalent and incident STIs among people who live with HIV/AIDS (PLWHA) are indicators of high-risk sexual behaviors and imply potential spread of HIV. Little is known about the prevalence and incidence of concurrent syphilis and associated risk behaviors among PLWHA in China.
A retrospective cohort study was conducted among PLWHA who attended the outpatient clinic of a designated AIDS treatment hospital in Shenyang, China, between March 2009 and May 2013. Physical examinations and syphilis serology were conducted at each visit. A questionnaire on demographic characteristics was also collected.
A total of 1010 PLWHA were enrolled, of whom 77.0% were men who have sex with men (MSM). The baseline syphilis prevalence among PLWHA was 19.8% (95% confidence interval [CI]:17.3–22.3%). During follow-up, 78.3% retained in the cohort, and contributed a median follow-up of 9.4 months (interquartile range: 5.9-18.7 months). Syphilis incidence among PLWHA was 18.7 (95% CI: 15.5–21.8) per 100 person years. Mulitvariate logistic analysis showed that receiving antiretroviral therapy (ART) (adjusted OR [aOR] = 0.48), older age (≥40 years vs. ≤24 years, aOR = 2.43), being MSM (aOR = 2.30) and having higher baseline HIV viral load (>100000 copies/mL vs. ≤100000 copies/mL, aOR = 1.56) were independent predictors for syphilis infection among PLWHA at enrollment (p < 0.05 for all). Mulivariate Cox regression found that receiving ART (adjusted hazard ratio [aHR] = 1.81), older age (≥40 years vs. ≤24 years, aHR: 5.17) and MSM status (aHR = 2.68) were independent risk factors for syphilis seroconversion (each p < 0.05).
Syphilis prevalence and incidence were high among PLWHA in Shenyang. A campaign focusing on detection and treatment of syphilis among PLWHA is urgently needed, especially one with a focus on MSM who are at a higher risk for syphilis.
Retrospective cohort study; Syphilis; Prevalence; Incidence; Antiretroviral therapy (ART)
Lactose is a widely used pharmaceutical excipient, sometimes irreplaceable. Traces of residual proteins left during production of lactose are potential allergen to body. The present paper describes a sensitive and specific LC-MS method for the determination of α-lactalbumin (α-La) in lactose samples. Chromatographic separation was performed on an Acquity UPLC BEH300 C18 column (2.1 × 150 mm, 1.7 μm) with an isocratic mobile phase consisting of water containing 0.1% TFA and acetonitrile containing 0.1% TFA (80 : 20, v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using selected ion monitoring of m/z 2364 for α-La. The calibration curve was linear from 0.2 to 10 µg/mL. The intra- and interday precisions were less than 7.6% and the accuracy ranged from 96.4 to 104.5%. The limit of quantification (LOQ) was 0.15 µg/mL and the limit of detection (LOD) was 0.05 µg/mL. This method was then successfully applied to investigate 6 different lactose samples. The application can provide technical preparation for the development of specification of lactose.
SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.
Objective. To evaluate the efficacy and safety of CHDI in the treatment of influenza infection. Method. A randomized double-blind, double dummy trial was conducted. Influenza patients with a positive influenza A rapid test diagnosis were randomized to receive CHDI or oseltamivir. Primary outcome was assessed by the median fever alleviation time and clearance time, and secondary outcome was total scores of influenza symptoms. Results. One hundred thirty-nine participants were screened and 34 had a RT-PCR laboratory confirmation of influenza virus infection. Fever alleviation time was 2.5 and 5 hours in CHDI and oseltamivir, respectively, and fever clearance time was 32.5 and 49 hours. The HR of fever alleviation and clearance time shows no significant difference between two groups. Total scores of influenza symptoms descended significantly in both groups after treatment and descended more in CHDI than oseltamivir on day 2. Similar to total symptoms severity score, fever severity score descend more significantly in CHDI than oseltamivir on day 2, and there were no differences on other symptoms. Conclusions. CHDI have a similar effect to oseltamivir in reducing the duration of influenza illness. CHDI was well tolerated, with no serious adverse events noted during the study period.
The aim of this study was to evaluate the outcomes of multiple advanced surgical treatments (i.e. microsurgery, laparoscopic surgery and endoscopic surgery) for acquired obstructive azoospermia. We analyzed the surgical outcomes of 51 patients with suspected acquired obstructive azoospermia consecutively who enrolled at our center between January 2009 and May 2013. Modified vasoepididymostomy, laparoscopically assisted vasovasostomy and transurethral incision of the ejaculatory duct with holmium laser were chosen and performed based on the different obstruction sites. The mean postoperative follow-up time was 22 months (range: 9 months to 52 months). Semen analyses were initiated at four postoperative weeks, followed by trimonthly (months 3, 6, 9 and 12) semen analyses, until no sperm was found at 12 months or until pregnancy was achieved. Patency was defined as >10,000 sperm ml−1 of semen. The obstruction sites, postoperative patency and natural pregnancy rate were recorded. Of 51 patients, 47 underwent bilateral or unilateral surgical reconstruction; the other four patients were unable to be treated with surgical reconstruction because of pelvic vas or intratesticular tubules obstruction. The reconstruction rate was 92.2% (47/51), and the patency rate and natural pregnancy rate were 89.4% (42/47) and 38.1% (16/42), respectively. No severe complications were observed. Using multiple advanced surgical techniques, more extensive range of seminal duct obstruction was accessible and correctable; thus, a favorable patency and pregnancy rate can be achieved.
azoospermia; endoscopy; laparoscopy; male infertility; microsurgery
The distinguishing of intrapulmonary metastases from multiple primaries is of great clinical importance. Although comprehensive histological assessment (CHA) was recommended for addressing this problem, the limitations of CHA have been addressed. We hypothesized that a nonmucinous lepidic component with mild nuclear atypia (NLCMA) may be one of the important sign suggesting primary lesions. In this study, we measured the value of NLCMA in distinguishing multiple primaries from intrapulmonary metastases. We retrospectively analyzed a cohort of 54 patients with 116 lesions (70 comparisons). Intrapulmonary metastases and multiple primaries were differentiated on the basis of CHA (Method I) and CHA combined with the assessment of NLCMA (Method II), respectively. Then, the results of two methods were compared with survival analysis. 33 cases were defined as multiple primaries and 21 cases as metastases by Method I, while 41 cases as multiple primaries and 13 cases as metastases by Method II. On univariate analysis, there was a better DFS in patients with a tumor ≤ 3 cm (P=0.012), female gender (P=0.011), highest N0 (P=0.002), absent micropapillary (P=0.013), multiple primaries (P=0.008 by method I, P < 0.001 by method II). A multivariate analysis adjusting for gender, tumor size, micropapillary and multiple primaries/metastases (by methodI and method II, respectively) indicated that multiple primaries (by method II) was an independent predictors for DFS. The presence of NLCMA may indicate that a lesion should be defined as primary in multifocal adenocarcinoma.
Lepidic; multifocal lung adenocarcinoma; comprehensive histological assessment
Type 2 diabetes is characterized by a deficit in β-cell function and mass, and its incidence increases with age. Autophagy is a highly regulated intracellular process for degrading cytoplasmic components, particularly protein aggregates and damaged organelles. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Autophagy may be necessary to maintain structure, mass, and function of pancreatic β-cells. In this study, we investigated the effects of age on β-cell function and autophagy in pancreatic islets of 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats. We found that islet β-cell function decreased gradually with age. Protein expression of the autophagy markers LC3/Atg8 and Atg7 exhibited a marked decline in aged islets. The expression of Lamp-2, a good indicator of autophagic degradation rate, was significantly reduced in the islets of old rats, suggesting that autophagic degradation is decreased in the islets of aged rats. However, protein expression of beclin-1/Atg6, which plays an important role in the induction and formation of the pre-autophagosome structure by associating with a multimeric complex of autophagy regulatory proteins (Atg14, Vps34/class 3 PI3 kinase, and Vps15), was most prominent in the islets of adult rats, and was higher in 24-month-old islets than in 4-month-old islets. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the functions of autophagy and proteasomal degradation, were increased in aging islets. 8-Hydroxydeoxyguanosine, a marker of mitochondrial and nuclear DNA oxidative damage, exhibited strong immunostaining in old islets. Analysis by electron microscopy demonstrated swelling and disintegration of cristae in the mitochondria of aged islets. These results suggest that β-cell and autophagic function in islets decline simultaneously with increasing age in Wistar rats, and that impaired autophagy in the islets of older rats may cause accumulation of misfolded and aggregated proteins and reduce the removal of abnormal mitochondria in β-cells, leading to reduced β-cell function. Dysfunctional autophagy in islets during the aging process may be an important mechanism leading to the development of type 2 diabetes.
Autophagy; Islet; Aging; Mitochondria; Oxidative damage
Cancer-associated inflammation is a key determinant of disease progression and survival in most cancers. The aim of our study was to assess the predictive value of preoperative inflammatory markers, such as the neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio, red cell distribution width (RDW), and mean platelet volume, for survival in breast cancer patients. In total, 608 breast cancer patients operated on between January 2009 and December 2011 were included in this observational study. The association between preoperative inflammatory markers and survival outcomes was analyzed. Patients with high NLR (>2.57) or high RDW (>13.45%) showed a significantly lower overall survival rate than those with lower NLR (≤2.57) or lower RDW (≤13.45%). NLR and RDW, along with node stage and molecular subtypes, were independent prognostic factors. There was a significant survival difference according to NLR in the luminal A and triple-negative subtypes (93.3% versus 99.3%, P=0.001; 68.8% versus 95.1%, P=0.000, respectively). The triple-negative subtype was the only subtype in which higher RDW patients showed significantly poor prognosis (81.3% versus 95.5%, P=0.025). Pre-operation NLR and RDW is a convenient, easily measured prognostic indicator for patients with breast cancer, especially in patients with the triple-negative subtype.
neutrophil-to-lymphocyte ratio; NLR; red cell distribution width; RDW; overall survival
μ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the analgesic effects of synthetic μ-TRTX-Hhn1b on animal models of pain. The peptide was first synthesized and then successfully refolded/oxidized. The synthetic peptide had the same inhibitory effect on human Nav1.7 current transiently expressed in HEK 293 cells as the native toxin. Furthermore, the analgesic potentials of the synthetic peptide were examined on models of inflammatory pain and neuropathic pain. μ-TRTX-Hhn1b produced an efficient reversal of acute nociceptive pain in the abdominal constriction model, and significantly reduced the pain scores over the 40-min period in the formalin model. The efficiency of μ-TRTX-Hhn1b on both models was equivalent to that of morphine. In the spinal nerve model, the reversal effect of μ-TRTX-Hhn1b on allodynia was longer and higher than mexiletine. These results demonstrated that μ-TRTX-Hhn1b efficiently alleviated acute inflammatory pain and chronic neuropathic pain in animals and provided an attractive template for further clinical analgesic drug design.
μ-TRTX-Hhn1b; Nav1.7; analgesic; inflammatory pain; neuropathic pain
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) in human immune system. DC-based tumor vaccine has met with some success in specific malignancies, inclusive of breast cancer. In this study, we electrofused MDA-MB-231 breast cancer cell line with day-3 DCs derived from peripheral blood monocytes, and explored the biological characteristics of fusion vaccine and its anti-tumor effects in vitro. Day-3 mature DCs were generated from day-2 immature DCs by adding cocktails composed of TNF-α, IL-1β, IL-6 and PEG2. Day-3 mature DCs were identified and electofused with breast cancer cells to generate fusion vaccine. Phenotype of fusion cells were identified by fluorescence microscope and flow cytometer. The fusion vaccine was evaluated for T cell proliferation, secretion of IL-12 and IFN-γ, and induction of tumor-specific CTL response. Despite differences in morphology, day-3 and day-7 DC expressed similar surface markers. The secretion of IL-12 and IFN-γ in fusion vaccine group was much higher than that in the control group. Compared with control group, DC-tumor fusion vaccine could better stimulate the proliferation of allogeneic T lymphocytes and kill more breast cancer cells (MDA-MB-231) in vitro. Day-3 DCs had the same function as the day-7 DCs, but with a shorter culture period. Our findings suggested that day-3 DCs fused with whole apoptotic breast cancer cells could elicit effective specific antitumor T cell responses in vitro and may be developed into a prospective candidate for adoptivet immunotherapy.
Age-related declines in hematopoietic stem cell (HSC) function may contribute to anemia, poor response to vaccination, and tumorigenesis. Here, we show that mammalian target of rapamycin (mTOR) activity is increased in HSCs from old mice compared to those from young mice. mTOR activation through conditional deletion of Tsc1 in the HSC of young mice mimicked the phenotype of HSC from aged mice in various ways. These included increased abundance of the mRNA encoding the CDK inhibitors p16Ink4a, p19Arf, and p21Cip1, a relative decrease in lymphopoiesis, and impaired capacity to reconstitute the hematopoietic system. In the old mice, rapamycin increased the life span, restored the self renewal and hematopoiesis of HSC, and enabled effective vaccination against a lethal challenge with influenza virus. Together, our data implicate mTOR signaling in HSC aging and demonstrate the potential of mTOR inhibitors for restoring hematopoiesis in the elderly.
Background. Acetone, β-hydroxybutyric acid, and acetoacetic acid are three types of ketone body that may be found in the breath, blood, and urine. Detecting altered concentrations of ketones in the breath, blood, and urine is crucial for the diagnosis and treatment of diabetic ketosis. The aim of this study was to evaluate the advantages of different detection methods for ketones, and to establish whether detection of the concentration of ketones in the breath is an effective and practical technique. Methods. We measured the concentrations of acetone in the breath using gas chromatography-mass spectrometry and β-hydroxybutyrate in fingertip blood collected from 99 patients with diabetes assigned to groups 1 (−), 2 (±), 3 (+), 4 (++), or 5 (+++) according to urinary ketone concentrations. Results. There were strong relationships between fasting blood glucose, age, and diabetic ketosis. Exhaled acetone concentration significantly correlated with concentrations of fasting blood glucose, ketones in the blood and urine, LDL-C, creatinine, and blood urea nitrogen. Conclusions. Breath testing for ketones has a high sensitivity and specificity and appears to be a noninvasive, convenient, and repeatable method for the diagnosis and therapeutic monitoring of diabetic ketosis.
Polyethylenimine (PEI) has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF), a natural protein rich in arginyl-glycyl-aspartic acid (RGD) peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney) 293 and HCT (human colorectal carcinoma) 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH) release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors.
Antheraea pernyi silk fibroin; PEI (polyethylenimine); gene transfection
Histologic categories and related growth pattern proposed by IASLC/ATS/ERS classification has been reported to be prognostically important in lung adenocarcinoma. Thyroid transcription factor-1 (TTF1) and Napsin A have been investigated as potential prognostic parameters with conflicting results. A total of 211 cases with stage I lung adenocarcinoma were analyzed according to the IASLC/ATS/ERS classification with slight modifications. Expression levels of TTF1 and Napsin A were evaluated by immunohistochemistry. In univariate analyses, we found female sex (p=0.009), lepidic growth pattern (P=0.011) and lack of micropapillary pattern (P=0.048) were favorable predictor significantly associated with disease-free survival (DFS). Lack of mitosis (P=0.044) and Napsin A expression (P=0.031) were favorable predictors for overall survival (OS). Tumors with a maximum diameter ≤2 cm had both longer DFS (P=0.008) and OS (P=0.020). Negative TTF1 expression indicated increased risk of death, but failure in statistical significance (P=0.215). After multivariate analysis, histologic subtype, tumor size and gender were identified as independent predictor for DFS (RR: 0.343, 3.697, 0.494; P=0.006, 0.029, 0.019), no feature was found as an independent predictor for overall survival (P>0.05). To conclude, lepidic growth pattern, female sex and tumor size ≤2 cm are independent favorable predictors for tumor recurrence, tumors with more than 5% percentage of lepidic growth pattern will have a better prognosis than absence, in early-stage lung adenocarcinoma.
Stage I; lung adenocarcinoma; IASLC/ATS/ERS classification; TTF1; Napsin A; prognosis