Tumor necrosis factor-α (TNFα) activates both cell death and cell survival pathways. The activation of survival pathway renders most cancer cells resistant to TNF-induced cytotoxicity. We found that pretreatment with digitoflavone, a plant flavonoid, greatly sensitized TNFα-induced apoptotic cell death in several human pancreatic cancer cells. In search of the molecular basis of the sensitization effect of digitoflavone, digitoflavone was found to inhibit TNFα-induced activation of nuclear transcription factor-kappa B (NF-κB) which is the main survival factor in TNFα signaling. NF-κB suppression occurred through inhibition of IκBα kinase activation, IκBα phosphorylation, IκBα degradation, and NF-κB nuclear translocation. This inhibition correlated with suppression of NF-κB-dependent genes involved in antiapoptosis (mcl-1, bcl-2, bcl-xl, c-iap1, c-iap2, flip, and survivin), proliferation (c-myc, cyclin d1), and angiogenesis (vegf, cox-2, and mmp-9). In addition, digitoflavone can activate JNK through inhibition of NF-κB signaling, provide a continuous blockade of the feed-back inhibitory mechanism by JNK-induced NF-κB activation. This study found a novel function of digitoflavone and enhanced the value of digitoflavone as an anticancer agent.
SELEX-Seq is now the optimal high-throughput technique for characterizing DNA-binding specificities of transcription factors. In this study, we introduced an improved EMSA-based SELEX-Seq strategy with several advantages. The improvements of this strategy included: (1) using a FAM-labeled probe to track protein-DNA complex in polyacrylamide gel for rapidly recovering the protein-bound dsDNA without relying on traditional radioactive labeling or ethidium bromide staining; (2) monitoring the specificity of SELEX selection by detecting a positive and negative sequence doped into the input DNAs used in each round with PCR amplification; (3) using nested PCR to ensure the specificity of PCR amplification of the selected DNAs after each round; (4) using the nucleotides added at the 5′ end of the nested PCR primers as the split barcode to code DNAs from various rounds for multiplexing sequencing samples. The split barcode minimized selection times and thus greatly simplified the current SELEX-Seq procedure. The reliability of the strategy was demonstrated by performing a successful SELEX-Seq of a well-known transcription factor, NF-κB. Therefore, this study provided a useful SELEX-Seq strategy for characterizing DNA-binding specificities of transcription factors.
Intramuscular fat (IMF) is an important trait influencing meat quality, and preadipocyte differentiation is a key factor affecting IMF deposition. Here we compared the transcriptome profiles of porcine intramuscular and subcutaneous preadipocytes during differentiation to gain insight into specific molecular and cellular events associated with intramuscular stromal vascular cell (MSVC) differentiation. RNA-Seq was used to screen for differentially expressed genes (DEGs) during the in vitro differentiation of MSVC and subcutaneous stromal vascular cell (ASVC) on days 0, 2 and 4. A total of 985 DEGs were identified during ASVC differentiation and 1469 DEGs during MSVC differentiation. Among these DEGs, 409 genes were specifically expressed during ASVC differentiation, 893 genes were specifically expressed during MSVC differentiation, and 576 DEGs were co-expressed during ASVC and MSVC differentiation. The expression profiles of DEGs during ASVC or MSVC differentiation were determined by cluster analysis based on Short Time-series Expression Miner (STEM). Four significant STEM profiles (profiles 1, 4, 5, and 14) were determined during ASVC differentiation, and four significant STEM profiles (profiles 1, 4, 11, and 14) were determined during MSVC differentiation. Gene ontology (GO) analysis indicated that DEGs related to adipocyte differentiation were identified to be significantly enriched in both adipose and muscle profile 14. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in adipose profile 14 and muscle profiles 11 and 14 (STEM clustered them into one cluster) showed that the PPAR signaling pathway was significantly enriched in these profiles and four signaling pathways were specifically enriched in muscle profiles 11 and 14. Furthermore, analysis of transcription factor binding sites (TFBS) in the gene set revealed two over-represented transcription factors (NR3C4 and NR3C1), which were specifically significantly enriched in the promoter regions of genes within muscle gene expression profiles 11 and 14.
Biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents could improve the clinical efficiency. The sulfatases(SULFs) play a critical role in the pathogenesis of a variety of human cancers. Here, we focused our investigation on the prognostic and predictive impact of SULF2 methylation in gastric cancer.
Promoter CpG island methylation of SULF2 was analyzed in 100 gastric cancer samples. The in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed were determined by histoculture drug response assay(HDRA). Additionally, 56 gastric cancer patients treated with a modified FOLFOX regimen(biweekly oxaliplatin plus 5-FU and folinic acid) were retrospectively analyzed to further evaluate the prognostic and predictive impact of SULF2 methylation in gastric cancer.
Methylated SULF2(SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2(SULF2U, methylation rate: 28%). Samples with SULF2U were more sensitive to cisplatin than those with SULF2M(inhibition rate: 48.80% vs. 38.15%, P = 0.02), while samples with SULF2M were more sensitive to irinotecan than SULF2U(inhibition rate: 53.61% vs. 40.92%, P = 0.01). There were no association between SULF2 methylation and in vitro sensitivity to docetaxel, gemcitabine and pemetrexed. SULF2 methylation was found to have a significant association with cisplatin efficacy(SULF2M: 57.14%, SULF2U: 80.56%, P = 0.02) and irinotecan efficacy(SULF2M: 89.29%, SULF2U: 62.50%, P = 0.01). Among the 56 patients receiving the modified FOLFOX regimen, a significant association was observed between survival and SULF2 methylation status(SULF2M: 309 days, 95% CI = 236 to 382 days; SULF2U: 481 days, 95% CI = 418 to 490 days; P = 0.02). Multivariate analysis revealed that SULF2 methylation was an independent prognostic factor of overall survival in gastric cancer patients treated with platinum-based chemotherapy.
SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation may be a novel prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy.
Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.
Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.
I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.
The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.
The Src homology 2 domain-containing tyrosine phosphatase 2 (SHP-2) has been reported to have both tumor-promoting and tumor-suppressing roles in tumorigenesis. However, the role of SHP-2 in tumor immunity remains unclear. Here we observed progressively lower levels of phosphorylated SHP-2 in tumor-associated CD4+ T cells during melanoma development in a murine model. Similarly, the levels of phosphorylated SHP-2 in the CD4+ T cells of human melanoma specimens revealed a decrease paralleling cancer development. The CD4+ T cell-specific deletion of SHP-2 promoted melanoma metastasis in mice. Furthermore, SHP-2 deficiency in CD4+ T cells resulted in the increased release of inflammatory cytokines, especially IL-6, and the enhanced accumulation of tumor-promoting myeloid-derived suppressor cells (MDSCs) in tumor-bearing mice. An IL-6-neutralizing antibody reduced MDSC accumulation and inhibited tumor growth in CD4+ T-cell-specific SHP-2-knockout mice. Our results suggest that SHP-2 in CD4+ T cells plays an important role in preventing melanoma progression and metastasis.
Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.
The outbreak of human infections with an emerging avian influenza A (H7N9) virus occurred in China in early 2013. It remains unknown what and how the underlying risk factors were involved in the bird-to-human cross-species transmission. To illustrate the dynamics of viral spread, we created a thematic map displaying the distribution of affected counties and plotted epidemic curves for the three most affected provinces and the whole country. We then collected data of agro-ecological, environmental and meteorological factors at the county level, and used boosted regression tree (BRT) models to examine the relative contribution of each factor and map the probability of occurrence of human H7N9 infection. We found that live poultry markets, human population density, irrigated croplands, built-up land, relative humidity and temperature significantly contributed to the occurrence of human infection with H7N9 virus. The discriminatory ability of the model was up to 97.4%. A map showing the areas with high risk for human H7N9 infection was created based on the model. These findings could be used to inform targeted surveillance and control efforts in both human and animal populations to reduce the risk of future human infections.
Scaffolded DNA origami is a widely used technology for self-assembling precisely structured nanoscale objects that contain a large number of addressable features. Typical scaffolds are long, single strands of DNA (ssDNA) that are folded into distinct shapes through the action of many, short ssDNA staples that are complementary to several different domains of the scaffold. However, sources of long single stranded DNA are scarce, limiting the size and complexity of structures that can be assembled. Here we demonstrated that dsDNA scaffolds can be directly used to fabricate integrated DNA origami structures that incorporate both of the constituent ssDNA molecules. Two basic principles were employed in the design of scaffold folding paths – folding path asymmetry and periodic convergence of the two ssDNA scaffold strands. Asymmetry in the folding path minimizes unwanted complementarity between staples, and incorporating an offset between the folding paths of each ssDNA scaffold strand reduces the number of times that complementary portions of the strands are brought into close proximity with one another, both of which decrease the likelihood of dsDNA scaffold recovery. Meanwhile, the folding paths of the two ssDNA scaffold strands were designed to periodically converge to promote the assembly of a single, unified structure rather than two individual ones. Our results reveal that this basic strategy can be used to reliably assemble integrated DNA nanostructures from dsDNA scaffolds.
DNA origami; double stranded DNA scaffold; self-assembly; DNA nanotechnology; scale up
Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.
Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans.
Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS.
Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.
Inexorable lethality is still commonly attributed to rabies infection, although there is increasing evidence for non-lethal infection and even recovery from clinical rabies in various animal species and humans. This paper reports non-lethal infection in dogs. The striking difference between dogs that survived a wt RABV infection and dogs that succumbed to the infection is that the surviving dogs showed high level of VNA in the serum and in the CSF, as well as mild immune cell accumulation in the CNS, whereas dogs that succumbed to disease showed little or no VNA in the serum or in the CSF and developed severe CNS inflammation. Considering the role of VNA in clearing the virus from the CNS, production of VNA within the CNS or infiltration of VNA from the periphery into the CNS across the blood-brain barrier appears to be important for clearing the virus from CNS thereby preventing a lethal rabies infection.
CUL4B up-regulates CDK2 by repressing miR-372 and miR-373, leading to increased phosphorylation and stabilization of CDC6, thus promoting replication licensing.
Cullin-RING ubiquitin ligases (CRLs) participate in the regulation of diverse cellular processes including cell cycle progression. Mutations in the X-linked CUL4B, a member of the cullin family, cause mental retardation and other developmental abnormalities in humans. Cells that are deficient in CUL4B are severely selected against in vivo in heterozygotes. Here we report a role of CUL4B in the regulation of replication licensing. Strikingly, CDC6, the licensing factor in replication, was positively regulated by CUL4B and contributed to the loading of MCM2 to chromatin. The positive regulation of CDC6 by CUL4B depends on CDK2, which phosphorylates CDC6, protecting it from APCCDH1-mediated degradation. Thus, aside being required for cell cycle reentry from quiescence, CDK2 also contributes to pre-replication complex assembly in G1 phase of cycling cells. Interestingly, the up-regulation of CDK2 by CUL4B is achieved via the repression of miR-372 and miR-373, which target CDK2. Our findings thus establish a CUL4B–CDK2–CDC6 cascade in the regulation of DNA replication licensing.
As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs) have a great impact on biological behaviors of hepatocellular carcinoma (HCC). In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium) exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT) and upregulation of cancer stem cell (CSC) -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF) into the CM. HGF receptor tyrosine kinase mesenchymal–epithelial transition factor (Met) was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.
Age-related changes in cancer mortality risk are important for understanding the processes of disease and aging interaction. The extent to which these age changes differ by sex further contributes to this understanding but has not been well studied to date. We conducted a systematic examination of dynamics and heterogeneity of age changes in cancer mortality rates for the top 14 cancer sites using vital statistics from the NCHS and SEER between 1969 and 2007. We assessed patterns of age changes in site-specific mortality rates in terms of both increase (age slope) and acceleration (change in age slope) as measured by the log-log acceleration rate (LLA). We assessed sex differences in mortality rates through sex mortality rate ratios and sex differences in age changes through comparisons of the LLA by sex. The logged male-to-female mortality ratios are positive but vary substantially with age in magnitude. And the age patterns of sex ratios also vary across sites. The LLA values show similar declines and hence slowdowns of mortality increment into or during old age for both sexes for most sites and periods. Post-reproductive changes in sex differences in cancer mortality are not entirely consistent with the estrogenic hypothesis about the anticarcinogenic effects of sex hormones and suggest the utility of the multistage model of disease progression for some tumor sites. Analysis of age dynamics and sex differences in cancer mortality may modify extant aging-related theories of carcinogenesis and frame future searches for specific explanatory factors.
aging; sex differences; cancer mortality; multistage progression; estrogen
Obesity prevalence stabilized in the U.S. in the first decade of the 2000s. However, obesity prevalence may resume increasing if younger generations are more sensitive to the obesogenic environment than older generations.
We estimated cohort effects for obesity prevalence among young adults born in the 1980s. Using data collected from the National Health and Nutrition Examination Survey between 1971 and 2008, we calculated obesity for respondents aged between 2 and 74 years. We used the median polish approach to estimate smoothed age and period trends; residual non-linear deviations from age and period trends were regressed on cohort indicator variables to estimate birth cohort effects.
After taking into account age effects and ubiquitous secular changes, cohorts born in the 1980s had increased propensity to obesity versus those born in the late 1960s. The cohort effects were 1.18 [95% CI: 1.01, 1.07] and 1.21 [95% CI: 1.02, 1.09] for the 1979–1983 and 1984–1988 birth cohorts, respectively. The effects were especially pronounced in Black males and females but appeared absent in White males.
Our results indicate a generational divergence of obesity prevalence. Even if age-specific obesity prevalence stabilizes in those born before the 1980s, age-specific prevalence may continue to rise in the 1980s cohorts, culminating in record high obesity prevalence as this generation enters its ages of peak obesity prevalence.
Age factors; Obesity; Young adult; Developmental origins; Epidemiology; Models; statistical
A major portion of influenza disease burden during the 2009 pandemic was observed among young people.
We examined the effect of age on the transmission of influenza-like illness associated with the 2009 pandemic influenza A (H1N1) virus (pH1N1) for an April–May 2009 outbreak among youth-camp participants and household contacts in Washington State.
An influenza-like illness attack rate of 51% was found among 96 camp participants. We observed a cabin secondary attack rate of 42% (95% confidence interval = 21%–66%) and a camp local reproductive number of 2.7 (1.7–4.1) for influenza-like illness among children (less than 18 years old). Among the 136 contacts in the 41 households with an influenza-like illness index case who attended the camp, the influenza-like illness secondary attack rate was 11% for children (5%–21%) and 4% for adults (2%–8%). The odds ratio for influenza-like illness among children versus adults was 3.1 (1.3–7.3).
The strong age effect, combined with the low number of susceptible children per household (1.2), plausibly explains the lower-than-expected household secondary attack rate for influenza-like illness, illustrating the importance of other venues where children congregate for sustaining community transmission. Quantifying the effects of age on pH1N1 transmission is important for informing effective intervention strategies.
C9orf86 which is a novel subfamily within the Ras superfamily of GTPases, is overexpressed in the majority of primary breast tumors. Few functional studies have focused on the C9orf86 protein; therefore, in this study, we explored the role of C9orf86 in breast carcinogenesis. In our study, we found that silencing of C9orf86 by siRNA in MCF-7 and SK-BR-3 cells resulted in suppressed cell proliferation as well as in vitro cell invasion capabilities. Moreover, knockdown of C9orf86 inhibited tumor growth in nude mice. Cell cycle and apoptotic assays showed that the anti-proliferative effect of C9orf86-siRNA was mediated by arresting cells in the G1 phase and promoting apoptosis. In addition, we found that patients with high levels of C9orf86 expression showed a significant trend towards worse survival compared to patients with low C9orf86 expression (P = 0.002). These results provide evidence that C9orf86 represents a novel and clinically useful biomarker for BC patients and plays an important role during the progression of BC.
Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research.
In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4.
Hypertrichosis refers to increased vellus hair growth and is independent to androgen excess. The acquired localized hypertrichosis (ALH) is one of the typical hypertrichosis, which mainly results from chronic irritation, inflammation, friction, and occlusion by plaster of Paris. Here, we report a young boy who had ALH on his right hand following a closed fracture with internal fixation and plaster cast application. The case is unusual because the hairy area is limited to the operative region of internal fixation. We suggest that the local vascular changes and skin inflammation induced by internal fixation and plaster cast application may be associated with ALH.
Casts; Hyperemia; Hypertrichosis; Internal fixators; Localized; Surgical
Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.
The accurate diagnosis of periprosthetic joint infections (PJI) is crucial for therapy and the prevention of complications. No diagnostic test of PJI is 100% accurate. The aim of this study was to assess the use of anti-granulocyte scintigraphy using 99 mTc-labeled monoclonal antibodies to diagnose PJI after total joint arthroplasty. A systematic search of all relevant studies published through January 2013 was conducted using the MEDLINE, EMBASE, OVID, and ScienceDirect databases. Observational studies that assessed the accuracy of the anti-granulocyte scintigraphy with monoclonal antibodies or antibody fragments labeled with technetium 99 m in diagnosis for PJI and provided data on specificity and sensitivity were identified. Standard methods recommended for meta-analysis of diagnostic accuracy were used. Nineteen studies were eligible for inclusion. The results demonstrated that the area under the summary receiver operator curve was 0.88, and the diagnostic accuracy (Q*) was 0.81. Additionally, the diagnostic odds ratio (DOR) was 18.76 with a corresponding 95% confidence interval of 10.45–33.68. The pooled sensitivity and specificity of the diagnostic method for the diagnosis of PJI were 83% and 79%, respectively, while the pooled positive likelihood ratio (PLR) was 3.56, and the negative likelihood ratio (NLR) was 0.26. Anti-granulocyte scintigraphy using 99 mTc-labeled monoclonal antibodies has a reasonable role in the diagnosis of PJI after total joint arthroplasty. Due to the limitations of the present meta-analysis, additional high-quality original studies are required to confirm the predictive value.
Objectives. To investigate how Xiao-Ai-Ping injection, a traditional Chinese medicine and an ancillary drug in tumor treatment, enhances the antitumor effects of cisplatin on Lewis lung cancer (LLC) cells. Methods. LLC-bearing mice were daily intraperitoneally injected with various doses of cisplatin, Xiao-Ai-Ping, or cisplatin plus Xiao-Ai-Ping, respectively. Body weight and tumor volumes were measured every three days. Results. Combination of Xiao-Ai-Ping and cisplatin yielded significantly better antigrowth and proapoptotic effects on LLC xenografts than sole drug treatment did. In addition, we found that Xiao-Ai-Ping triggered the infiltration of CD8+ T cells, a group of cytotoxic T cells, to LLC xenografts. Furthermore, the mRNA levels of interferon-γ (ifn-γ), perforin-1 (prf-1), and granzyme B (gzmb) in CD8+ T cells were significantly increased after combination treatment of Xiao-Ai-Ping and cisplatin. In vitro studies showed that Xiao-Ai-Ping markedly upregulated the mRNA levels of ifn-γ, prf-1, and gzmb in CD8+ T cells in a concentration-dependent manner, suggesting that Xiao-Ai-Ping augments the function of CD8+ T cells. Conclusions. Xiao-Ai-Ping promotes the infiltration and function of CD8+ T cells and thus enhances the antigrowth effects of cisplatin on LLC xenografts, which provides new evidence for the combination of Xiao-Ai-Ping and cisplatin in clinic in China.
The barrier functions of the stratum corneum (SC) and the epidermal layers present a tremendous challenge in achieving effective transdermal delivery of drug molecules. Although a few reports have shown that poly(amidoamine) (PAMAM) dendrimers are effective skin penetration enhancers, little is known regarding the fundamental mechanisms behind the dendrimer-skin interactions. In this paper, we have performed a systematic study to better elucidate how dendrimers interact with skin layers depending on their size and surface groups. Franz diffusion cells and confocal microscopy were employed to observe dendrimer interactions with full-thickness porcine skin samples. We have found that smaller PAMAM dendrimers (generation 2 (G2)) penetrate the skin layers more efficiently than the larger ones (G4). We have also found that G2 PAMAM dendrimers that are surface modified by either acetylation or carboxylation exhibit increased skin permeation and likely diffuse through an extracellular pathway. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. In addition, conjugation of oleic acid (OA) to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here we report that size, surface charge, and hydrophobicity directly dictate the permeation route and efficiency of dendrimer translocation across the skin layers, providing a design guideline for engineering PAMAM dendrimers as a potential transdermal delivery vector.
PAMAM dendrimer; transdermal delivery; surface modification; oleic acid; partition coefficient
Angiogenesis is essential for the growth and metastasis of cancer. Although anti-angiogenic agents, particularly vascular endothelial growth factor (VEGF) inhibitors, have exhibited single-agent activity, there is considerable interest in combining these novel drugs with conventional chemotherapy reagents to achieve an optimal clinical efficacy. The objective of this study was to evaluate the benefits of the combination therapy of vascular endothelial growth factor trap (VEGF-Trap) with gemcitabine in a lung tumor model.
A luciferase-expressing Lewis lung carcinoma (LLC) model was established in C57BL/6J mice and tumor-bearing mice were randomized into control, VEGF-Trap, gemcitabine and VEGF-Trap/gemcitabine combination groups. Tumor growth and animal survival were monitored. Tumor microvessel density and cell proliferation were evaluated by CD31 and Ki-67 immunohistochemical analysis. TUNEL assay was performed to detect apoptotic cells. The protein levels of Cyclin D1, Pro-Caspase-3, Bcl-2, MMP2 and MMP9 in tumor extracts were examined by western blot.
VEGF-Trap in combination with gemcitabine showed significantly enhanced inhibition of tumor growth and prolonged mouse survival compared to the VEGF-Trap or gemcitabine monotherapy. The VEGF-Trap/gemcitabine combination therapy not only potently inhibited tumor angiogenesis and cell proliferation, but also increased cellular apoptosis within tumor tissues. In addition, the combination treatment markedly down-regulated the expression of proliferation, anti-apoptosis and invasion related proteins.
Combination therapy using VEGF-Trap and gemcitabine resulted in improved anti-tumor efficacy in a lung cancer model and VEGF-Trap/gemcitabine combination might represent a promising strategy in the treatment of human lung cancer.