Aberration in DNA replication is a major cause to genome instability that is a hallmark of cancer cells. Cell division cycle 6 (Cdc6) and c-Myc have a critical role in the initiation of DNA replication. However, whether their interaction induces epithelial-mesenchymal transition (EMT) and promotes tumorigenesis in in vivo animal model remains unclear. Since using zebrafish as a cancer model has been restricted by the late onset of tumorigenesis and extreme difficulty in transformation on skin, we tried to establish a novel non-melanoma skin model in zebrafish to study their role in tumorigenesis. A stable transgenic zebrafish was created by using tol2 transposon, in which cdc6 and c-myc were co-overexpressed in epidermis driven by a skin-specific krt4 promoter. Intriguingly, co-overexpression of cdc6 and c-myc in transgenic zebrafish skin triggered tumor-like transformation, apoptosis attenuation, genomic instability, and EMT, hallmarks of malignant tumorigenesis. Our findings and other characteristics of zebrafish, including optical clarity and small molecule treatment, provide the future utility of this model for easy and non-invasive detection and for identification of new anti-cancer drug.
Cdc6; c-Myc; zebrafish; genomic instability; epithelial to mesenchymal transition
A 20 mo old male child, born at 31 weeks, presented to an out-of-hours General Practitioner (GP) with a 7 d history of cough and fever. There was history of post-tussive vomiting. On assessment a pernasal swab was cultured from which B. pertussis was isolated. This child had received a complete course of primary immunization. In the light of recent increase in Pertussis cases in the United Kingdom we would like to use this case report to increase the awareness among clinicians to the possibility of a diagnosis of whooping cough even in children with completed primary immunization and particularly in preterm infants.
Bordetella pertussis; whooping cough; vaccine failure; vaccination; United Kingdom
Many animal species besides humans show evidence of individuality. Knowing how a risk-taker differs from its stay-at-home counterpart could not only help humans live more easily with our fellow creatures, says Lee Dugatkin of the University of Louisville, but also tell us a few things about ourselves and how we got this way.
Gliomas are the most common tumor in the central nervous system. High-grade glioblastomas are characterized by their high invasiveness and resistance to radiotherapy, leading to high recurrence rate and short median survival despite radical surgical resection. Characterizations of gliomas at molecular level have revealed aberrations of various growth factor receptors, receptor tyrosine kinases, and tumor suppressor genes that lead to deregulation of multiple signaling pathways, thereby contributing to abnormal proliferation, invasion, and resistance to apoptosis in cancer cells. Recently, accumulating evidence points to the emerging role of axon guidance molecules in glioma progression. Notably, many signaling events harnessed by guidance molecules to regulate cell migration and axon navigation during development are also found to be involved in the modulation of deregulated pathways in gliomas. This paper focused on the signalings triggered by the guidance molecule semaphorins and their receptors plexins and neuropilins, and how their crosstalk with oncogenic pathways in gliomas might modulate cancer progression. The emerging role of semaphorins and plexins as tumor suppressors or oncogenes is also discussed.
This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non-small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155 and let-7a was determined by quantitative real-time PCR in paraffin embedded formalin fixed tumor specimens from 639 IALT patients. Prognostic and predictive value of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathological prognostic factors and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed association of TP53 mutation status and miR-34a/b/c expression, EGFR and KRAS mutation status and miR-21 and Let-7a expression, respectively. Finally, association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 where a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the miRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort of radically resected NSCLC randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only.
non–small cell lung cancer; adjuvant chemotherapy; randomized trial; biomarker; drug resistance; microRNA
Burn injuries continue to cause morbidity and mortality internationally. Despite international collaborations and preventative measures, there are still many cases reported in high- and low-income countries. The treatment of these patients is often protracted and requires extensive resources. The adequate resuscitation of these patients coupled with meticulous wound care can have a huge impact on their outcome. The authors present a simple guideline for the initial management of severe burns which is utilised by the South African Burn Society and is based on the guidelines of the American Burn Association and the Australian and New Zealand Burn Association.
C2 laminar screws have become an increasingly used alternative method to C2 pedicle screw fixation. However, the outcome of this technique has not been thoroughly investigated. A total of 35 cases with upper cervical spinal instability undergoing C2 laminar screw fixation were reviewed. All cases had symptoms of atlantoaxial instability, such as craniocervical junction pain, and were fixed with the Vertex cervical internal fixation system. A total of 68 screws were placed and hybrid constructs (a C2 translaminar screw combined with a C2 pars screw) were incorporated in two patients. In this series, there were no intraoperative complications and no cases of neurological worsening or vascular injury from hardware placement. Computed tomographic scans demonstrated a partial dorsal laminar breach in ten patients. None of these resulted in neurological symptoms. None of the patients was found to have a breach of the ventral laminar cortex. All the C2 laminar screws fixations were performed successfully. There was no instability seen on the films with no evidence of hardware failure or screw loosening during the follow-up period in all patients. In conclusion, C2 laminar screw technique is straightforward and easily adopted; it can efficiently and reliably restore upper cervical stability. It is an alternative method to C2 pedicle screw fixation, especially in patients with unilateral occlusion of vertebral artery and pedicle deformity of C2.
C2 translaminar screws; Atlantoaxial stabilization; Cervical fusion; Screw fixation; Posterior instrumentation
Much work in behavioral ecology has shown that animals fight over resources such as food, and that they make strategic decisions about when to engage in such fights. Here, we examine the evolution of one, heretofore unexamined, component of that strategic decision about whether to fight for a resource. We present the results of a computer simulation that examined the evolution of over- or underestimating the value of a resource (food) as a function of an individual's current hunger level. In our model, animals fought for food when they perceived their current food level to be below the mean for the environment. We considered seven strategies for estimating food value: 1) always underestimate food value, 2) always overestimate food value, 3) never over- or underestimate food value, 4) overestimate food value when hungry, 5) underestimate food value when hungry, 6) overestimate food value when relatively satiated, and 7) underestimate food value when relatively satiated. We first competed all seven strategies against each other when they began at approximately equal frequencies. In such a competition, two strategies–“always overestimate food value,” and “overestimate food value when hungry”–were very successful. We next competed each of these strategies against the default strategy of “never over- or underestimate,” when the default strategy was set at 99% of the population. Again, the strategies of “always overestimate food value” and “overestimate food value when hungry” fared well. Our results suggest that overestimating food value when deciding whether to fight should be favored by natural selection.
The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown.
Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug.
It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h.
In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.
The high attrition rate of in vitro human embryo culture presents a major obstacle in the treatment of clinical infertility by in vitro fertilization (IVF). Physical and genetic requirements are not well understood for human or mouse pre-implantation embryo development. Group culture is an established requirement for optimal embryo development in the mouse model. However, conventional microdrop culture limitations hinder investigations of the effects of physical parameters on in vitro embryo development. We report a microfluidics platform that enables embryo culture in precisely defined, sub-microliter volumes (5 – 500nL) which cannot be investigated using conventional methods. Groups of two embryos per microfluidic well successfully developed to the blastocyst stage, at a rate of over 80%, which is comparable to those cultured in 20μL microdrops. This system can be used to dissect physical requirements of in vitro single or group embryo culture, and be made highly parallel to increase experimental throughput.
Compared to the emerging embryonic stem cell (ESC) gene network, little is
known about the dynamic gene network that directs reprogramming in the early
embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is
also highly expressed at the 1- to 2-cell stages in embryos, may be a
critical regulator of the earliest gene network in the embryo.
Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we
show that Oct4 is required for development prior to the blastocyst stage.
Specifically, Oct4 has a novel and critical role in regulating genes that
encode transcriptional and post-transcriptional regulators as early as the
2-cell stage. Our data suggest that the key function of Oct4 may be to
switch the developmental program from one that is predominantly regulated by
post-transcriptional control to one that depends on the transcriptional
network. Further, we propose to rank candidate genes quantitatively based on
the inter-embryo variation in their differential expression in response to
Oct4 knockdown. Of over 30 genes analyzed according to
this proposed paradigm, Rest and Mta2,
both of which have established pluripotency functions in ESCs, were found to
be the most tightly regulated by Oct4 at the 2-cell stage.
We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early
embryo development is large and distinct from its established network in
ESCs. Further, our experimental approach can be applied to dissect the gene
regulatory network of Oct4 and other pluripotency regulators to deconstruct
the dynamic developmental program in the early embryo.
We examined the impact of winner and loser effects on dominance hierarchy formation when individuals are capable of estimating their opponent's resource holding power (RHP). The accuracy of such estimates was a variable in our simulations, and we considered cases in which all individuals err within the same bounds, as well as cases in which some individuals consistently overestimate, while others consistently underestimate their opponent's fighting RHP. In all cases, we found a clearly defined linear hierarchy. In most simulations, the vast majority of interactions were ‘attack–retreats’, and the remainder of interactions were almost all ‘fights’. Error rates had no effect on the linearity of the hierarchy or the basic attack–retreat nature of interactions, and consistent over and underestimation did not affect the ultimate position of an individual in a hierarchy.
winner effect; loser effect; individual recognition
We present the results of an individual agent-based model of antibiotic resistance in bacteria. Our model examines antibiotic resistance when two strategies exist: “producers”–who secrete a substance that breaks down antibiotics–and nonproducers (“cheats”) who do not secrete, or carry the machinery associated with secretion. The model allows for populations of up to 10,000, in which bacteria are affected by their nearest neighbors, and we assume cheaters die when there are no producers in their neighborhood. Each of 10,000 slots on our grid (a torus) could be occupied by a producer or a nonproducer, or could (temporarily) be unoccupied. The most surprising and dramatic result we uncovered is that when producers and nonproducers coexist at equilibrium, nonproducers are almost always found on the edges of clusters of producers.
DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.
Gingival overgrowth and fibrosis is a side effect of certain medications and occurs in non-drug induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin, the least fibrotic lesions are caused by cyclosporin A, and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. In the present study, the hypothesis was investigated that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies show that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real time PCR analyses of RNA extracted from control and drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analyzed for the basal and TGF-β1 or lysophosphatidic acid stimulated CTGF/CCN2 expression at the protein and RNA levels. Data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues and cultured gingival epithelial cells express CTGF/CCN2, and lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.
gingival overgrowth; fibrosis; connective tissue growth factor; epithelium; fibroblast; epithelial-mesenchymal transition
The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.
Winner and loser effects have now been documented in a number of species. To our knowledge, experimental work, however, has focused exclusively on pairwise interactions, and not the extent to which winner and loser effects impact hierarchy formation. We report the results of experimentally manipulated winner and loser effects on hierarchy formation in a socially living species, the green swordtail, Xiphophorus helleri. Our results demonstrate that randomly chosen winners in pairwise aggressive contests were more likely to emerge as top-ranked individuals in a hierarchy, whereas randomly chosen losers were more likely to emerge as the lowest-ranking individuals, and that 'winner-neutral-loser' hierarchies were significantly overrepresented.
Winner and loser effects are defined as an increased probability of winning an aggressive interaction at time T, based on victories at time T-1, T-2, etc., and an increased probability of losing at time T, based on losses at time T-1, T-2, etc., respectively. Prior theoretical work on dominance hierarchy formation has demonstrated that when players are not capable of individual recognition, loser effects always produce a clear top-ranked (alpha) individual, but all other ranks in a group remain unclear; whereas winner effects always produce strict linear hierarchies in which the rank of each individual is clear. Paradoxically, however, when individual recognition--a phenomenon long thought to stabilize hierarchies--is possible, winner and loser effects have no impact on the probability of forming strict linear hierarchies.
The evolution of group-beneficial traits potentially allows the survival of ‘cheaters’ that would otherwise be unfit. Here we describe experimental work on group-beneficial traits and the consequences of frequency-dependent selection in the context of bacterial antibiotic resistance. We constructed a ‘self-limited antibiotic resistant’ (SLAR) strain of Escherichia coli in which a TEM-1 β-lactamase was anchored to the inner membrane. In pairwise competition experiments between the SLAR strain and ampicillin-sensitive strains, only the SLAR strain survived in the presence of ampicillin. We also constructed a ‘shared antibiotic resistant’ (SAR) strain in which TEM-1 β-lactamase protected both the SAR strain and nearby sensitive cells, thus acting as a model for a genetically defined group-beneficial trait. In pairwise competition experiments of the SAR strain against two different sensitive strains of E. coli, we found that the sensitive strains maintained themselves at frequencies of 5–12% in the presence of ampicillin. When the relative cost of the SAR strain was lowered, its equilibrial frequency rose. Sensitive strains also arose from pure cultures of the SAR strain. In these cases, too, the sensitive ‘cheaters’ were maintained in ampicillin at frequencies comparable to those observed in the previous competitions. These results suggest that traits which benefit other group members can permit survival of genotypes that otherwise would be eliminated by natural selection, and allow the maintenance of greater genetic variation upon which evolution can operate.
frequency dependence; group-beneficial traits; antibiotic resistance
Indolicidin, a l3-residue antimicrobial peptide-amide, which is unusually rich in tryptophan and proline, is isolated from the cytoplasmic granules of bovine neutrophils. In this study, the structures of indolicidin in 50% D3-trifluoroethanol and in the absence and presence of SDS and D38-dodecylphosphocholine were determined using NMR spectroscopy. Multiple conformations were found and were shown to be due to different combinations of contact between the two WPW motifs. Although indolicidin is bactericidal and able to permeabilize bacterial membranes, it does not lead to cell wall lysis, showing that there is more than one mechanism of antimicrobial action. The structure of indolicidin in aqueous solution was a globular and amphipathic conformation, differing from the wedge shape adopted in lipid micelles, and these two structures were predicted to have different functions. Indolicidin, which is known to inhibit DNA synthesis and induce filamentation of bacteria, was shown to bind DNA in gel retardation and fluorescence quenching experiments. Further investigations using surface plasmon resonance confirmed the DNA-binding ability and showed the sequence preference of indolicidin. Based on our biophysical studies and previous results, we present a diagram illustrating the DNA-binding mechanism of the antimicrobial action of indolicidin and explaining the roles of the peptide when interacting with lipid bilayers at different concentrations.
Aggressive contests probably occur in networking environments where information about fighting ability is conveyed both to an opponent and to individuals peripheral to the fight itself, the bystanders. Our primary aim was to investigate the relative influences of eavesdropping and prior social experience on the dynamics of aggressive contests in Xiphophorus helleri. A bystander's ability to witness an encounter was manipulated using clear, one-way mirror, and opaque partitions. After watching (or not watching) the initial contest, the bystander encountered either the winner or loser of the bout. Treatment comparisons of bystander-winner or bystander-loser contest dynamics indicated the presence or absence of winner, loser, or eavesdropping effects. Winner and loser effects had negligible influences on bystander contest dynamics. Eavesdropping significantly reduced the bystander's propensity to initiate aggression, escalate, and win against seen winners regardless of whether the watched bout had escalated or not. Though eavesdropping had relatively little effect on bystander-loser contest dynamics, bystanders were less prone to initiate aggression and win against losers that had escalated in the witnessed bout. Thus, bystanders appear to preferentially retain and utilize information gained about potentially dangerous opponents (winners or persistent losers). Our data lend clear support for the importance of eavesdropping in visually based aggressive signalling systems.