OBJECTIVE--To study efficacy of treatment of filarial lymphoedema and elephantiasis with 5,6-benzo-alpha-pyrone. DESIGN--Randomised, double blind, placebo controlled study with matching for grade and duration of disease, age, and sex. Treatment was given for 367 days, and subjects were followed up for another year. SETTING--A town in Shandong Province, China. SUBJECTS--104 men and women with chronic unilateral filarial lymphoedema or elephantiasis of the leg: 64 were randomised to benzopyrone and 40 to placebo. By the end of the study 19 patients had dropped out of the treatment group and two out of the placebo group. INTERVENTIONS--Two 200 mg tablets of 5,6-benzo-alpha-pyrone or two placebo tablets given daily. MAIN OUTCOME MEASURES--Volumes of the affected and normal legs estimated every three months, and daily listing of any side effects. RESULTS--Benzopyrone reduced oedema for all grades of lymphoedema during the year of treatment (pW0.001) and the follow up year (p = 0.026). During treatment the mean monthly reductions in leg volume were 0.62% (95% confidence intervals 0.4% to 0.85%), 1.1% (0.71% to 1.6%), and 1.6% (0.89% to 2.3%) of the volume of the normal leg for grades 1, 2, and 3-5 (elephantiasis) of lymphoedema respectively. During follow up the mean monthly reductions were 0.18% (0.01% to 0.35%), 0.54% (0.27% to 0.82%), and 0.87% (0.51% to 1.2%). At the end of the trial the total reduction in oedema was 100%, 95%, and 45% for grades 1, 2, and 3-5. Symptoms and complications were considerably reduced, including attacks of secondary acute inflammation, while side effects were minor and disappeared after one month. In the placebo group there were no changes in the severity of lymphoedema. CONCLUSIONS--5,6-benzo-alpha-pyrone reduces the oedema and many symptoms of filarial lymphoedema and elephantiasis. It has few side effects, and its relatively slow action makes it ideal for use without compression garments.
Claudin-2 is a unique member of the claudin family of transmembrane proteins as its expression is restricted to the leaky epithelium in vivo and correlates with epithelial leakiness in vitro. However, recent evidence suggests potential functions of claudin-2 that are relevant to neoplastic transformation and growth. In accordance here we report, based upon analysis of mRNA and protein expression using a total of 309 patient samples, that claudin-2 expression is significantly increased in colorectal cancer and correlates with cancer progression. We also report similar increases in claudin-2 expression in inflammatory bowel disease (IBD)-associated colorectal cancer. Most importantly, we demonstrate that the increased claudin-2 expression in colorectal cancer is causally associated with tumor growth as forced claudin-2 expression in colon cancer cells that do not express claudin-2 resulted in significant increases in cell proliferation, anchorage-independent growth, and tumor growth in vivo. We further show that the colonic microenvironment regulates claudin-2 expression in a manner dependent on signaling through the EGF receptor (EGFR), a key regulator of colon tumorigenesis. In addition, claudin-2 expression is specifically decreased in the colon of waved-2 mice, naturally deficient in EGFR activation. Furthermore, genetic silencing of claudin-2 expression in Caco-2 , a colon cancer cell line, prevents the EGF-induced increase in cell proliferation. Taken together, these results uncover a novel role for claudin-2 in promoting colon cancer, potentially via EGFR transactivation.
Tight Junction; Claudin; EGFR; Colon Cancer; Proliferation
Viewing emotional pictures is associated with heightened perception and attention, indexed by a relative increase in visual cortical activity. Visual cortical modulation by emotion is hypothesized to reflect re-entrant connectivity originating in higher-order cortical and/or limbic structures. The present study used dense-array electroencephalography and individual brain anatomy to investigate functional coupling between the visual cortex and other cortical areas during affective picture viewing. Participants viewed pleasant, neutral, and unpleasant pictures that flickered at a rate of 10 Hz to evoke steady-state visual evoked potentials (ssVEPs) in the EEG. The spectral power of ssVEPs was quantified using Fourier transform, and cortical sources were estimated using beamformer spatial filters based on individual structural magnetic resonance images. In addition to lower-tier visual cortex, a network of occipito-temporal and parietal (bilateral precuneus, inferior parietal lobules) structures showed enhanced ssVEP power when participants viewed emotional (either pleasant or unpleasant), compared to neutral pictures. Functional coupling during emotional processing was enhanced between the bilateral occipital poles and a network of temporal (left middle/inferior temporal gyrus), parietal (bilateral parietal lobules), and frontal (left middle/inferior frontal gyrus) structures. These results converge with findings from hemodynamic analyses of emotional picture viewing and suggest that viewing emotionally engaging stimuli is associated with the formation of functional links between visual cortex and the cortical regions underlying attention modulation and preparation for action.
Dense-Array EEG; Emotion; Motivation; Arousal; Picture perception
Background & Aims
Mutational inactivation of APC is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. CRC progression also involves inactivation of signaling via transforming growth factor (TGF)β and bone morphenogenic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 loss on levels of β-catenin mRNA and Wnt signaling.
We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APCΔ1638/+) and (APCΔ1638/+)x(K19CreERT2Smad4lox/lox) mice using laser-capture microdissection.
In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APCΔ1638/+), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes, compared with adenomas from APCΔ1638/+mice that expressed Smad4.
Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among TGF-β, BMP, and Wnt signaling pathways in CRC progression.
Colon Cancer; Mouse Model; Intestinal Epithelium; signal transduction; carcinoma
Extensive research efforts have been directed toward strategies for predicting risk of developing Alzheimer’s disease (AD) prior to the appearance of observable symptoms. Existing approaches for early detection of AD vary in terms of their efficacy, invasiveness, and ease of implementation. Several non-invasive magnetic resonance imaging strategies have been developed for predicting decline in cognitively healthy older adults. This review will survey a number of studies, beginning with the development of a famous name discrimination task used to identify neural regions that participate in semantic memory retrieval and to test predictions of several key theories of the role of the hippocampus in memory. This task has revealed medial temporal and neocortical contributions to recent and remote memory retrieval, and it has been used to demonstrate compensatory neural recruitment in older adults, apolipoprotein E ε4 carriers, and amnestic mild cognitive impairment patients. Recently, we have also found that the famous name discrimination task provides predictive value for forecasting episodic memory decline among asymptomatic older adults. Other studies investigating the predictive value of semantic memory tasks will also be presented. We suggest several advantages associated with the use of semantic processing tasks, particularly those based on person identification, in comparison to episodic memory tasks to study AD risk. Future directions for research and potential clinical uses of semantic memory paradigms are also discussed.
Apolipoprotein E; Cognitive Activity; Functional Magnetic Resonance Imaging; Hippocampus; Physical Activity; fMRI; semantic memory; person identification; Alzheimer’s disease; cognitive decline; prediction
Previous studies suggest that task-activated fMRI can predict future cognitive decline among healthy older adults. The present fMRI study examined the relative sensitivity of semantic memory (SM) versus episodic memory (EM) activation tasks for predicting cognitive decline. Seventy-eight cognitively intact elders underwent neuropsychological testing at entry and after an 18-month interval, with participants classified as cognitively “Stable” or “Declining” based on ≥1.0 SD decline in performance. Baseline fMRI scanning involved SM (famous name discrimination) and EM (name recognition) tasks. SM and EM fMRI activation, along with APOE ε4 status, served as predictors of cognitive outcome using a logistic regression analysis. Twenty-seven (34.6%) participants were classified as Declining and 51 (65.4%) as Stable. APOE ε4 status alone significantly predicted cognitive decline (R2 = .106; C index = .642). Addition of SM activation significantly improved prediction accuracy (R2 = .285; C index = .787), whereas the addition of EM did not (R2 = .212; C index = .711). In combination with APOE status, SM task activation predicts future cognitive decline better than EM activation. These results have implications for use of fMRI in prevention clinical trials involving the identification of persons at-risk for age-associated memory loss and Alzheimer’s disease.
magnetic resonance imaging; aging; Apolipoprotein-E; memory loss; mild cognitive impairment; longitudinal study
Engagement in cognitively stimulating activities (CA) and leisure time physical activity (PA) have been associated with maintaining cognitive performance and reducing the likelihood of cognitive decline in older adults. However, neural mechanisms underlying protective effects of these lifestyle behaviors are largely unknown. In the current study, we investigated the effect of self-reported PA and CA on hippocampal volume and semantic processing activation during a fame discrimination task, as measured by functional magnetic resonance imaging (fMRI). We also examined whether possession of the apolipoprotein E (APOE) ε4 allele could moderate the effect of PA or CA on hippocampal structure or function.
Seventy-eight healthy, cognitively intact older adults underwent baseline neuropsychological assessment, hippocampal volume measurement via manually-traced structural MRI, and task-activated fMRI.
After 18 months, 27 participants declined by one standard deviation or more on follow-up neuropsychological testing. Logistic regression analyses revealed that CA alone or in combination with baseline hippocampal structure or functional activity did not predict the probability of cognitive decline. In contrast, PA interacted with APOE ε4 status such that engagement in PA reduced the risk of cognitive decline in APOE ε4 carriers only. Furthermore, the benefits of PA appeared to diminish with reduced functional activity or volume in the hippocampus.
Our findings suggest that increased leisure time PA is associated with reduced probability of cognitive decline in persons who are at high risk for AD. The beneficial effects of PA in this group may be related to enhancement of the functional and structural integrity of the hippocampus.
Apolipoprotein E; Cognitive Activity; Cognitive Decline; Functional Magnetic Resonance Imaging; Hippocampus; Physical Activity
Increased levels of cardio-enriched microRNAs (miRNAs) have been described in patients with myocardial infarction (MI). We wanted to evaluate the diagnostic and prognostic potential of cardio-enriched miRNAs in patients presenting with a suspected acute coronary syndrome (ACS).
Cardio-enriched miRNAs (miR-1, miR-208b and miR-499-5p) were measured using real time PCR in plasma samples from 424 patients with suspected ACS treated in a coronary care unit. miRNAs were assessed for discrimination of a clinical diagnosis of myocardial infarction and for association with 30-day mortality and diagnosis of heart failure. Correlation with left ventricular systolic dysfunction as measured by the ejection fraction (LVEF) was also assessed. To confirm myocardial origin miRNA was measured during coronary artery bypass surgery.
miRNAs were higher in MI patients and correlated with LVEF (p < 0.001). Discrimination of MI was accurate for miR-208b (AUC = 0.82) and miR-499-5p (AUC = 0.79) but considerable lower than for Troponin T (AUC = 0.95). Increased miRNA levels were strongly associated with increased risk of mortality or heart failure within 30 days for miR-208b (OR 1.79, 95% CI = 1.38-2.23, p = 1 × 10-5) and miR-499-5p (OR 1.70, 95% CI = 1.31-2.20, p = 5 × 10-5) but the association was lost when adjusting for Troponin T. During surgery miR-208b and miR-499-5p was released in the coronary sinus after cardioplegia-reperfusion to markedly higher levels than in a peripheral vein.
Our findings confirm increased levels of cardio-enriched miRNAs in the blood of MI patients and establish association of increased miRNA levels with reduced systolic function after MI and risk of death or heart failure.
MiRNA; Myocardial infarction; Acute coronary syndrome; Biomarker; Prognosis
Aim. At present there is no data looking at modern multislice computerised tomography (CT) in the investigation of occult hip fracture. The aim of this study was to retrospectively compare the reports of patients sent for magnetic resonance imaging (MRI) or CT with negative radiographs and a clinical suspicion of a fractured neck of femur. Methods. All patients presenting to the hospital with a clinical suspicion of a hip fracture but initial negative radiographs over a three-year period were included. Patients were either investigated with an MRI scan or CT scan. The presence of a fracture, the requirement for surgery, and any further requirement for imaging were recorded. Results. Over three years 92 patients were included of which 61 were referred for a CT and 31 for an MRI. Thirty-four patients were found to have a fracture. Of these, MRI picked up a fracture in 36% and CT in 38% of referrals. Discussion. Up to 10% of proximal femur fractures may be missed on initial radiographs. Current guidelines state patients should be offered MRI if hip fracture is suspected despite negative hip radiographs. Our findings show that modern multislice CT may be comparable with MRI for detecting occult fracture.
Our aim was to determine Trichomonas vaginalis prevalence using the Aptima Trichomonas vaginalis assay (ATV; Gen-Probe) and the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae coinfections in U.S. women undergoing screening for C. trachomatis/N. gonorrhoeae. Discarded urogenital samples from 7,593 women (18 to 89 years old) undergoing C. trachomatis/N. gonorrhoeae screening using the Aptima Combo 2 assay (Gen-Probe) in various clinical settings were tested with ATV. Overall, T. vaginalis, C. trachomatis, and N. gonorrhoeae prevalences were 8.7%, 6.7%, and 1.7%, respectively. T. vaginalis was more prevalent than C. trachomatis or N. gonorrhoeae in all age groups except the 18- to 19-year-old group. The highest T. vaginalis prevalence was in women ≥40 years old (>11%), while the highest C. trachomatis prevalence (9.2%) and N. gonorrhoeae prevalence (2.2%) were in women <30 years old. Coinfection prevalences were 1.3% for C. trachomatis/T. vaginalis, 0.61% for C. trachomatis/N. gonorrhoeae and N. gonorrhoeae/T. vaginalis, and 0.24% for C. trachomatis/N. gonorrhoeae/T. vaginalis and highest in women <30 years old. T. vaginalis prevalence differed by race/ethnicity, with the highest prevalence in black women (20.2%). T. vaginalis prevalence ranged from 5.4% in family planning clinics to 22.3% in jails. Multivariate analysis determined that ages of ≥40 years, black race, and patient locations were significantly associated with T. vaginalis infection. T. vaginalis is the most common sexually transmitted infection (STI) in women of >40 years, while C. trachomatis and N. gonorrhoeae prevalence is lowest in that age group. Higher T. vaginalis prevalence in women of >40 years is probably attributed to the reason for testing, i.e., symptomatic status versus routine screening in younger women. Coinfections were relatively low. High T. vaginalis prevalence in all age groups suggests that women screened for C. trachomatis/N. gonorrhoeae, whether asymptomatic or symptomatic, should be screened for T. vaginalis.
► Transcriptome of developing blood and vascular endothelial cells in zebrafish is described. ► 388 Novel genes expressed by blood and endothelial cells are identified. ► tmem88a and trim2a are novel genes required for primitive erythropoiesis and myelopoiesis.
In this paper, we use zebrafish embryos to characterise the transcriptome of the developing blood and endothelium, two cell types that are closely associated during development. High-throughput sequencing identified 754 genes whose transcripts are enriched threefold or more in blood and/or vascular endothelial cells compared with the rest of the embryo at 26–28 h post fertilisation. Of these genes, 388 were classified as novel to these cell types after cross-reference with PubMed and the zebrafish information network (ZFIN). Analysis by quantitative PCR and in situ hybridisation showed that 83% (n = 41) of these novel genes are expressed in blood or vascular endothelium. Of 10 novel genes selected for knockdown by antisense morpholino oligonucleotides, we confirmed that two, tmem88a and trim2a, are required for primitive erythropoiesis and myelopoiesis. Our results provide a catalogue of genes whose expression is enriched in the developing blood and endothelium in zebrafish, many of which will be required for the development of those cell types, both in fish and in mammals.
Zebrafish; Haematopoiesis; Endothelial cell; High-throughput sequencing
Standard approaches have found that rapid growth during the first 2 years of life is a risk factor for overweight in later childhood. Our objective was to test whether growth velocity, independent of concurrent size, was associated with overweight using a nonlinear random-effects model that allows for enhanced specifications and estimations.
Longitudinal data from a birth cohort in Mexico (n=586) were used to estimate growth trajectories over 0–24 months for body mass index (BMI), length and weight using the SuperImposition by Translation and Rotation (SITAR) models. The SITAR models use a nonlinear random-effects model to estimate an average growth curve for BMI, length and weight and each participant's deviation from this curve on three dimensions—size, velocity and timing of peak velocity. We used logistic regression to estimate the association between overweight status at 7–9 years and size, velocity and timing of BMI, length and weight trajectories during 0–24 months. We tested whether any association between velocity and overweight varied by relative size during 0–24 months or birth weight.
SITAR models explained the majority of the variance in BMI (73%), height (86%) and weight (85%) between 0–24 months. When analyzed individually, relative BMI/length/weight (size) and BMI/length/weight velocity during 0–24 months were each associated with increased odds of overweight in late childhood. Associations for timing of peak velocity varied by anthropometric measure. However, in the mutually adjusted models, only relative BMI/length/weight (size) remained statistically significant. We found no evidence that any association between velocity and overweight varied by size during 0–24 months or birth weight.
After mutual adjustment, size during 0–24 months of life (as opposed to birth size), but not velocity or timing of peak velocity, was most consistently associated with overweight in later childhood.
developmental origins; obesity; mexico; rapid growth; growth velocity; Superimposition by Translation and Rotation (SITAR)
We present a rare case of left ventricular myxoma discovered incidentally in an asymptomatic 16-year old male. The patient underwent the appropriate work-up and a robotic-assisted excision of the mass. The patient had an uneventful recovery and was discharged home at postoperative day 3. To our knowledge, this is the first case of robotic-assisted left ventricular myxoma excision in the literature. Robotic-assisted surgery of left ventricular myxomas is a safe and feasible method of excision.
Robotic surgery; Myxoma; Robotic-assisted cardiac surgery
Research in non-human animal (hereafter, animal) cognition has found strong evidence that some animal species are capable of meta-cognitively monitoring their mental states. They know when they know and when they do not know. In contrast, animals have generally not shown robust theory of mind (ToM) capabilities. Comparative research uses methods that are non-verbal, and thus might easily be labelled ‘implicit’ using the terminology of traditional human cognition. However, comparative psychology has developed several non-verbal methods that are designed to test for aspects of meta-cognition that – while perhaps not fully explicit – go beyond the merely implicit or associative. We believe similar methods might be useful to developmental researchers who work with young children, and may provide a sound empirical alternative to verbal reports. Comparative psychology has moved away from all-or-none categorical labels (e.g., ‘implicit’ vs. ‘explicit’) towards a theoretical framework that contains a spectrum of mental abilities ranging from implicit to explicit, and from associative to cognitive to fully conscious. We discuss how this same framework might be applied to developmental psychology when it comes to implicit versus explicit processing and ToM.
Recent theoretical and empirical developments in human category learning have differentiated an analytic, rule-based system of category learning from a nonanalytic system that integrates information across stimulus dimensions. The researchers applied this theoretical distinction to pigeons’ category learning. Pigeons learned to categorize stimuli varying in the tilt and width of their internal striping. The matched category problems had either a unidimensional (rule-based) or multidimensional (information-integration) solution. Whereas humans and nonhuman primates strongly dimensionalize these stimuli and learn rule-based tasks far more quickly than information-integration tasks, pigeons learned the two tasks equally quickly to the same accuracy level. Pigeons may represent a cognitive system in which the commitment to dimensional analysis and category rules was not strongly made. Their performance could suggest the character of the ancestral vertebrate categorization system from which that of primates emerged.
category learning; comparative cognition; pigeons; rules; analytic/nonanalytic cognition; implicit/explicit cognition
A central question in categorization research concerns the categories that animals and humans learn naturally and well. Here, the authors examined monkeys’ and humans’ learning of the important class of exclusive-or (XOR) categories. Both species exhibited—through a sustained level of ongoing errors—substantial difficulty learning XOR category tasks at three stimulus dimensionalities. Clearly, both species brought a linear-separability constraint to XOR category learning. This constraint illuminates the primate category-learning system from which that of humans arose, and it has theoretical implications concerning the evolution of cognitive systems for categorization. The present data also clarify the role of exemplar-specific processes in fully explaining XOR category learning, and suggest that humans sometimes overcome their linear-separability constraint through the use of language and verbalization.
categorization; learning; primates; comparative psychology; exclusive-or relation
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO2. In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.
We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution.
Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco.
Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP+ve neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.
Photoreceptor; Retina; Transplantation; Neurotrophic factors; Gene therapy; Stem cell
Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co-repressor functions for the MTG family, suggesting coordinate regulation of transcription by Kaiso/MTG complexes in cancer.
Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates.
To characterise genetic and clinical findings in patients with SIX3 mutations.
Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish.
In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%.
Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.
A systematic method for plant genome manipulation is a major aim of plant biotechnology. One approach to achieving this involves producing a double-strand DNA break at a genomic target site followed by the introduction or removal of DNA sequences by cellular DNA repair. Hence, a site-specific endonuclease capable of targeting double-strand breaks to unique locations in the plant genome is needed.
We engineered and tested a synthetic homing endonuclease, PB1, derived from the I-CreI endonuclease of Chlamydomonas reinhardtii, which was re-designed to recognize and cleave a newly specified DNA sequence. We demonstrate that an activity-optimized version of the PB1 endonuclease, under the control of a heat-inducible promoter, is capable of targeting DNA breaks to an introduced PB1 recognition site in the genome of Arabidopsis thaliana. We further demonstrate that this engineered endonuclease can very efficiently excise unwanted transgenic DNA, such as an herbicide resistance marker, from the genome when the marker gene is flanked by PB1 recognition sites. Interestingly, under certain conditions the repair of the DNA junctions resulted in a conservative pairing of recognition half sites to remove the intervening DNA and reconstitute a single functional recognition site.
These results establish parameters needed to use engineered homing endonucleases for the modification of endogenous loci in plant genomes.
Homing endonuclease; I-CreI; Targeted marker excision
Local drug delivery has substantial potential to prevent infections compared with systemic delivery. Although calcium sulfate (CaSO4) has been studied for local drug delivery and two types are commercially available, it is unknown whether they differentially release antibiotics.
We determined the differences between two sources of CaSO4 and the K2SO4 catalyst’s presence on the degradation, daptomycin elution, and activity against Staphylococcus aureus.
We formed pellets from synthetic and naturally sourced (from gypsum) CaSO4 and loaded with 5% daptomycin and 3% or 0% K2SO4. We used in vitro experiments to determine the daptomycin concentration and degradation profiles over 10 days. Turbidity assays were used to evaluate the activity of the daptomycin eluates against S. aureus.
All pellets exhibited a bolus release with the highest daptomycin concentration on Day 1 with the sourced CaSO4 pellets. The synthetic CaSO4 pellets with 3% K2SO4 exhibited a slower drug release compared with the synthetic CaSO4 pellets with 0% K2SO4, which degraded and eluted daptomycin too quickly to inhibit S. aureus. Turbidity assays demonstrated that all CaSO4 pellets inhibit S. aureus for expected lengths of time.
Our preliminary in vitro data suggest differences in the degradation, elution, and activity properties between sourced and synthetic CaSO4 pellets. The addition of K2SO4 appeared beneficial when using synthetic CaSO4. Synthetic CaSO4 may be effective when slow degradation and longer elution times are needed.
Local delivery of eluted daptomycin can be tailored through material selection and K2SO4 addition.
Despite continual efforts to develop prognostic and predictive models of colorectal cancer by using clinicopathological and genetic parameters, a clinical test that can discriminate between patients with good or poor outcome after treatment has not been established. Thus, the authors aim to uncover subtypes of colorectal cancer that have distinct biological characteristics associated with prognosis and identify potential biomarkers that best reflect the biological and clinical characteristics of subtypes.
Unsupervised hierarchical clustering analysis was applied to gene expression data from 177 patients with colorectal cancer to determine a prognostic gene expression signature. Validation of the signature was sought in two independent patient groups. The association between the signature and prognosis of patients was assessed by Kaplan–Meier plots, log-rank tests and the Cox model.
The authors identified a gene signature that was associated with overall survival and disease-free survival in 177 patients and validated in two independent cohorts of 213 patients. In multivariate analysis, the signature was an independent risk factor (HR 3.08; 95% CI 1.33 to 7.14; p=0.008 for overall survival). Subset analysis of patients with AJCC (American Joint Committee on Cancer) stage III cancer revealed that the signature can also identify the patients who have better outcome with adjuvant chemotherapy (CTX). Adjuvant chemotherapy significantly affected disease-free survival in patients in subtype B (3-year rate, 71.2% (CTX) vs 41.9% (no CTX); p=0.004). However, such benefit of adjuvant chemotherapy was not significant for patients in subtype A.
The gene signature is an independent predictor of response to chemotherapy and clinical outcome in patients with colorectal cancer.
Elevated Src expression correlates with malignant potential and metastatic disease in many tumors including pancreas cancer. We sought to characterize the molecular effects of Src kinase inhibition with dasatinib (BMS-354825) a novel, multi-targeted kinase inhibitor that targets Src family kinases, in pancreas ductal adenocarcinoma (PDA). We identified sensitive and resistant PDA cell lines to dasatinib treatment and tested the molecular effects of Src inhibition in vitro and in vivo. We show for the first time that cellular localization of Src expression impacts survival in patients with PDA. Pancreas tumors with increased membranous expression of Src result in decreased survival compared with tumors that have increased cytoplasmic Src expression. Src kinase inhibition with dasatinib markedly inhibits cell proliferation, migration, invasion, cell cycle progression and anchorage independent growth and stimulates apoptosis. This is accompanied by decreased phosphorylation of Src, FAK, paxillin, AKT, STAT3, ERK, JNK and MAPK, as well as decreased cyclinD1 expression in a time and concentration-dependent manner. Furthermore, siRNA to Src results in significant decrease in cell proliferation, invasion and migration of pancreas cancer cells. Dasatinib treatment also inhibits in vivo pancreas tumor growth. Mechanisms of resistance to Src inhibition appear to be related to a lack of inhibition of STAT3 and MAPK signaling. These results establish a mechanistic rationale for Src inhibition with dasatinib as a therapeutic target in the treatment of pancreas cancer and identify potential biomarkers of resistance to Src inhibition.
Src family kinase; Dasatinib; Pancreas cancer; Therapeutic target; Tumor growth