Clinical implementation of artificial-intelligence-assisted detection of breast cancer metastases in sentinel lymph nodes: the CONFIDENT-B single-center, non-randomized clinical trial.

Pathologists' assessment of sentinel lymph nodes (SNs) for breast cancer (BC) metastases is a treatment-guiding yet labor-intensive and costly task because of the performance of immunohistochemistry (IHC) in morphologically negative cases. This non-randomized, single-center clinical trial (International Standard Randomized Controlled Trial Number:14323711) assessed the efficacy of an artificial intelligence (AI)-assisted workflow for detecting BC metastases in SNs while maintaining diagnostic safety standards. From September 2022 to May 2023, 190 SN specimens were consecutively enrolled and allocated biweekly to the intervention arm (n = 100) or control arm (n = 90). In both arms, digital whole-slide images of hematoxylin-eosin sections of SN specimens were assessed by an expert pathologist, who was assisted by the 'Metastasis Detection' app (Visiopharm) in the intervention arm. Our primary endpoint showed a significantly reduced adjusted relative risk of IHC use (0.680, 95% confidence interval: 0.347-0.878) for AI-assisted pathologists, with subsequent cost savings of ~3,000 €. Secondary endpoints showed significant time reductions and up to 30% improved sensitivity for AI-assisted pathologists. This trial demonstrates the safety and potential for cost and time savings of AI assistance.

Psychometric properties of two implementation measures: Normalization MeAsure Development questionnaire (NoMAD) and organizational readiness for implementing change (ORIC).

Background: Effective interventions need to be implemented successfully to achieve impact. Two theory-based measures exist for measuring the effectiveness of implementation strategies and monitor implementation progress. The Normalization MeAsure Development questionnaire (NoMAD) explores the four core concepts (Coherence, Cognitive Participation, Collective Action, Reflexive Monitoring) of the Normalization Process Theory. The Organizational Readiness for Implementing Change (ORIC) is based on the theory of Organizational Readiness for Change, measuring organization members' psychological and behavioral preparedness for implementing a change. We examined the measurement properties of the NoMAD and ORIC in a multi-national implementation effectiveness study. Method: Twelve mental health organizations in nine countries implemented Internet-based cognitive behavioral therapy (iCBT) for common mental disorders. Staff involved in iCBT service delivery (n = 318) participated in the study. Both measures were translated into eight languages using a standardized forward-backward translation procedure. Correlations between measures and subscales were estimated to examine convergent validity. The theoretical factor structures of the scales were tested using confirmatory factor analysis (CFA). Test-retest reliability was based on the correlation between scores at two time points 3 months apart. Internal consistency was assessed using Cronbach's alpha. Floor and ceiling effects were quantified using the proportion of zero and maximum scores. Results: NoMAD and ORIC measure related but distinct latent constructs. The CFA showed that the use of a total score for each measure is appropriate. The theoretical subscales of the NoMAD had adequate internal consistency. The total scale had high internal consistency. The total ORIC scale and subscales demonstrated high internal consistency. Test-retest reliability was suboptimal for both measures and floor and ceiling effects were absent. Conclusions: This study confirmed the psychometric properties of the NoMAD and ORIC in multi-national mental health care settings. While measuring on different but related aspects of implementation processes, the NoMAD and ORIC prove to be valid and reliable across different language settings.

Why was the study done?: Effective interventions need to be implemented successfully to achieve impact. Reliable measurement instruments are needed to determine if an implementation was successful or not. Two theory-based instruments exist for measuring the effectiveness of implementation strategies and monitor progress. The NoMAD measures aspects of normalization related to sense-making, willingness to implement, the work people do, and reflection. The Organizational Readiness for Implementing Change (ORIC) measures organization members' preparedness for implementing a change. What did we do?: This study examined whether the NoMAD and ORIC measure what they are supposed to measure. We translated the instruments from English to eight languages (Albanian, Danish, Dutch, French, German, Italian, and Spanish/Catalan) We applied various statistical methods to confirm the measurement properties, including correlations of scales, factor structures, test­retest reliability, consistency and floor and ceiling effects. 318 mental health professionals from nine countries participated in the study. What did we find?: For both instruments, total scores can be used as well as the subscale scores. Internal consistency for ORIC was high and for NoMAD adequate. Test­retest reliability was demonstrated, and floor and ceiling effects were rare. What does this mean?: NoMAD and ORIC are reliable instruments for measuring implementation processes and outcomes across mental health care settings in different countries and languages. They measure related but different aspects of implementation processes and outcomes. The measures are brief, and theory supported. However, more work is to be done on interpreting scores in relation to implementation success and regarding changes over time.

Confocal laser endomicroscopy for upper tract urothelial carcinoma: validation of the proposed criteria and proposal of a scoring system for real-time tumor grading.

PURPOSE: Confocal laser endomicroscopy (CLE) is a fluorescence-based fiber-optic imaging technique with the potential for intraoperative grading of upper tract urothelial carcinoma (UTUC). This study aims to (1) investigate the prevalence of the previously proposed CLE criteria for bladder cancer in papillary UTUC, (2) estimate the diagnostic value of CLE for UTUC grading and (3) propose a scoring system for a more quantifiable approach of CLE-based grading of UTUC. MATERIALS AND METHODS: Ureteroscopic CLE was performed in patients with UTUC. Following CLE imaging, co-localized biopsies were taken for histopathologic comparison. Postoperatively, two blinded raters assessed the CLE images. RESULTS: Fifty-three papillary UTUCs (34 low grade and 19 high grade) were imaged with CLE in 36 patients. All the previously described CLE criteria were identifiable in varying proportions. After excluding 10 non-diagnostic recordings (5 low grade and 5 high grade) due to insufficient image quality, the histopathologic grade was correctly identified with CLE in 26 low-grade UTUCs (90%) and in 12 high-grade UTUCs (86%). The most prevalent CLE criteria with the highest diagnostic potential were cellular organization, morphology and cohesiveness of cells. A scoring system was proposed with these criteria, which yielded similar diagnostic accuracies. CONCLUSIONS: Based on the previously proposed criteria, CLE enables accurate grading of papillary UTUC at a non-diagnostic rate of 19%. The most prevalent CLE criteria with the highest diagnostic potential for grading of papillary UTUC are cellular organization, morphology and cohesiveness of cells. The proposed scoring system may simplify the assessment of CLE images for UTUC grading but external validation is required.

Fluorescence in situ hybridization in 1 mL of selective urine for the detection of upper tract urothelial carcinoma: a feasibility study.

Kidney-sparing surgery of upper tract urothelial carcinoma (UTUC) requires a stringent follow-up with frequent ureteroscopies. Triage testing could reduce the number of follow-up ureteroscopies and hence minimize the invasiveness of follow-up. The use of urine-based markers for triage seems appealing but should be feasible with selective urine from outpatient cystoscopy to maximize the reduction of invasiveness. In this study, the feasibility of UroVysion® fluorescence in situ hybridization (FISH) for the detection of UTUC in 1 mL of selective urine is investigated. Ten consecutive patients with biopsy-proven UTUC and five patients with negative diagnostic ureteroscopy findings were included in this case-control study. During ureteroscopy, 1 mL of selective urine was collected passively with a ureteral splint for Urovysion® FISH. The FISH rater was blinded to any clinical information. The results of FISH were compared to the findings of concomitantly collected selective urine cytology and the patients' UTUC status. FISH was feasible in all samples with a sensitivity of 90% and a specificity of 80% for UTUC. In comparison, selective cytology resulted in a diagnostic yield of 87% with a sensitivity of 80% and a specificity of 67%. In conclusion, UTUC detection is feasible with FISH in 1 mL of passively collected selective urine. Thus from a technical point of view, FISH could be used as an outpatient triage test to decide if follow-up ureteroscopy is necessary after kidney-sparing surgery of UTUC. Evaluation of the diagnostic accuracy of FISH for the suggested pathway deserves further attention.

Advanced three-dimensional culture of equine intestinal epithelial stem cells.

BACKGROUND: Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. OBJECTIVES: To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. STUDY DESIGN: Descriptive study. METHODS: Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. RESULTS: Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. MAIN LIMITATIONS: Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. CONCLUSIONS: The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).

How to assess communication skills? Development of the rating scale ComOn Check.

BACKGROUND: Good communication is a core competency for all physicians. Thus, medical students require adequate preparation in communication skills. For research purposes, as well as for evaluation in teaching, there is a clear need for reliable assessment tools. We analyzed the shortcomings of existing instruments and saw a need for a new rating scale. The aim of this publication is to describe the development process for, and evaluation of, a new rating scale. METHODS: First, we developed the rating scale in 10 steps. Then, two raters evaluated the newly developed rating scale by rating 135 videotaped consultations of medical students with standardized patients. Additionally, standardized patients evaluated students' performance, which was used as an outside criterion to validate ratings. RESULTS: Our rating scale comprises six domains with 13 specific items evaluated on a five-point Likert scale: initiating conversation, patient's perception, structure of conversation, patient's emotions, end of conversation, and general communication skills. Item-total correlation coefficients between the checklist items ranged from 0.15 to 0.78. Subscale consistency was calculated for domains comprised of more than one item and Cronbach's α ≥ 0.77, indicating acceptable consistency. Standardized patients' global evaluation correlated moderately with overall expert ratings (Spearman's ρ = .40, p < .001). CONCLUSION: Our rating scale is a reliable and applicable assessment tool. The rating scale focuses on the evaluation of general communication skills and can be applied in research as well as in evaluations, such as objective structured clinical examinations (OSCE). ABBREVIATIONS: CST: Communication skills training; ICC: Intra-class correlation coefficient; OSCE: Objective structured clinical examination; SP: Standardized patients; SD: Standard deviation; M: Mean.

Association between exploratory activity and social individuality in genetically identical mice living in the same enriched environment.

We previously reported that inbred, genetically identical mice living in one enriched environment develop individual behavioral trajectories, indicating increasingly different levels of spatial exploratory behavior as quantified by roaming entropy. Cumulative roaming entropy (cRE) correlated positively with adult hippocampal neurogenesis, a type of plasticity involved in the flexible integration of new information into existing contexts (Freund et al., 2013). The study on which we report here was done in parallel to that first experiment, but here we acquired detailed observational data on the behavior of individual mice. Roaming entropy (RE) was again assessed in real-time with an antenna-based system over the entire experimental period of 3months. Compared to the least active mice in the enclosure (low number of antenna contacts), the most active animals showed tendencies of increased socially interactive behavior in the final observation block whereas least active mice displayed more self-related behavior (non-social local exploration and play). When looking at roaming behavior, we discovered that RE correlated negatively with latent factors representing social exploratory and non-social exploratory and play behavior. Adult neurogenesis could not be studied in the present cohort but we do know that under identical conditions, cumulative RE correlated positively with adult hippocampal neurogenesis. We can thus hypothesize that the mice with more exploratory experience in terms of areal coverage (as quantified by RE) and related greater levels of adult hippocampal plasticity, might also be the ones that were less involved in interactions within the group and, hence, more individualistic. While this remains to be confirmed experimentally, the present data suggest that the described mechanism of individualization, which has previously been shown to be hippocampus-dependent, has a social component.

Broader expression of the mouse platelet factor 4-cre transgene beyond the megakaryocyte lineage.

BACKGROUND: Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100-kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown. OBJECTIVES/METHODS: To characterize the expression of this transgene, we used double-fluorescent cre reporter mice. RESULTS: In the bone marrow, Pf4-cre-mediated recombination had occurred in all CD42-positive megakaryocytes as early as stage I of maturation, and in rare CD42-negative cells. In circulating blood, all platelets had recombined, along with only a minor fraction of CD45-positive cells. However, we found that all tissues contained recombined cells of monocyte/macrophage origin. When recombined, these cells might potentially modify the function of the tissues under particular conditions, especially inflammatory conditions, which further increase recombination in immune cells. Unexpectedly, a subset of epithelial cells from the distal colon showed signs of recombination resulting from endogenous Pf4-cre expression. This is probably the basis of the unexplained colon tumors developed by Apc(flox/flox) ;Pf4-cre mice, generated in a separate study on the role of Apc in platelet formation. CONCLUSION: Altogether, our results indicate early recombination with full penetrance in megakaryopoiesis, and confirm the value of Pf4-cre mice for the genetic engineering of megakaryocytes and platelets. However, care must be taken when investigating the role of platelets in processes outside hemostasis, especially when immune cells might be involved.

A FIB induced boiling mechanism for rapid nanopore formation.

Focused ion beam (FIB) technology is widely used to fabricate nanopores in solid-state membranes. These nanopores have desirable thermomechanical properties for applications such as high-throughput DNA sequencing. Using large scale molecular dynamics simulations of the FIB nanopore formation process, we show that there is a threshold ion delivery rate above which the mechanism underlying nanopore formation changes. At low rates nanopore formation is slow, with the rate proportional to the ion flux and therefore limited by the sputter rate of the target material. However, at higher fluxes nanopores form via a thermally dominated process, consistent with an explosive boiling mechanism. In this case, mass is rapidly rearranged via bubble growth and coalescence, much more quickly than would occur during sputtering. This mechanism has the potential to greatly speed up nanopore formation.

Regulation of the tumor suppressor homeogene Cdx2 by HNF4α in intestinal cancer.

The gut-specific homeotic transcription factor Cdx2 is a crucial regulator of intestinal development and homeostasis, which is downregulated in colorectal cancers (CRC) and exhibits a tumor suppressor function in the colon. We have previously established that several endodermal transcription factors, including HNF4α and GATA6, are involved in Cdx2 regulation in the normal gut. Here we have studied the role of HNF4α in the mechanism of deregulation of Cdx2 in colon cancers. Crossing Apc(Δ14/+) mice prone to spontaneous intestinal tumor development with pCdx2-9LacZ transgenic mice containing the LacZ reporter under the control of the 9.3-kb Cdx2 promoter showed that this promoter segment contains sequences recapitulating the decrease of Cdx2 expression in intestinal cancers. Immunohistochemistry revealed that HNF4α, unlike GATA6, exhibited a similar decrease to Cdx2 in genetic (Apc(min/+) and Apc(Δ14/+)) and chemically induced (Azoxymethane (AOM) treatment) models of intestinal tumors in mice. HNF4α and Cdx2 also exhibited a comparable deregulated pattern in human CRC. Correlated patterns were observed between HNF4α and Cdx2 in several experimental models of human colon cancer cell lines: xenografts in nude mice, wound healing and glucose starvation. Furthermore, Cdx2 decreased by knocking down HNF4α in human colon cancer cells using siRNA and in the colon of mice conditionally knocked out for the Hnf4α gene in the adult intestine (Hnf4α(f/f);VilCre(ERT2) mice). Finally, the conditionally knocked out mice Hnf4α(f/f);VilCre(ERT2) treated with the carcinogen AOM developed colorectal tumors earlier than wild-type mice, as previously reported for mice with a reduced Cdx2 expression. In conclusion, this study provides evidence that the downregulation of HNF4α is an important determinant of the reduced expression of the Cdx2 tumor suppressor gene in intestinal cancers. Consistently, similar to Cdx2, HNF4α exerts a tumor suppressor function in the colon in that its loss of function facilitates tumor progression.

Increasing the oxygen load by treatment with myo-inositol trispyrophosphate reduces growth of colon cancer and modulates the intestine homeobox gene Cdx2.

Preventing tumor neovascularisation is one of the strategies recently developed to limit the dissemination of cancer cells and apparition of metastases. Although these approaches could improve the existing treatments, a number of unexpected negative effects have been reported, mainly linked to the hypoxic condition and the subsequent induction of the pro-oncogenic hypoxia inducible factor(s) resulting from cancer cells' oxygen starvation. Here, we checked in vivo on colon cancer cells an alternative approach. It is based on treatment with myo-inositol trispyrophosphate (ITPP), a molecule that leads to increased oxygenation of tumors. We provide evidence that ITPP increases the survival of mice in a model of carcinomatosis of human colon cancer cells implanted into the peritoneal cavity. ITPP also reduced the growth of subcutaneous colon cancer cells xenografted in nu/nu mice. In the subcutaneous tumors, ITPP stimulated the expression of the homeobox gene Cdx2 that is crucial for intestinal differentiation and that also has an anti-tumoral function. On this basis, human colon cancer cells were cultured in vitro in hypoxic conditions. Hypoxia was shown to decrease the level of Cdx2 protein, mRNA and the activity of the Cdx2 promoter. This decline was unrelated to the activation of HIF1α and HIF2α by hypoxia. However, it resulted from the activation of a phosphatidylinositol 3-kinases-like mitogen-activated protein kinase pathway, as assessed by the fact that LY294002 and U0126 restored high Cdx2 expression in hypoxia. Corroborating these results, U0126 recapitulated the increase of Cdx2 triggered by ITPP in subcutaneous colon tumor xenografts. The present study provides evidence that a chemical compound that increases oxygen pressure can antagonize the hypoxic setting and reduce the growth of human colon tumors implanted in nu/nu mice.

Enteropathogenic e.coli sustains iodoacetamide-induced ulcerative colitis-like colitis in rats: modulation of IL-1ß, IL-6, TNF-α, COX-2, and apoptosisi.

Pathogenic or non-pathogenic bacteria from flora may play a key role in inflammatory bowel disease (IBD) pathogenesis. However, a specific infectious agent causing IBD has not been identified. This study assessed the impact of enteropathogenic E. coli (EPEC) on the modulation of IL-1beta, IL-6, TNF- alpha, COX-2, BAX and Bcl-2 expression, in sustaining inflammation of a rat colitis model. Two hundred male Sprague-Dawley rats (4 groups) were inoculated weekly or bi-weekly for 70 days, with 1 percent methylcellulose (MC), (b) 6 percent iodoacetamide (IA) in 1 percent MC, (c) 4x108 CFU of EPEC, and (d) IA+EPEC. After a month, treatment was stopped in half of the animals in each group. IL-1beta, IL-6, TNF-alpha, COX-2, BAX and Bcl-2 expression were measured in colonic mucosa scrapings. IL-1beta, IL-6, TNF-alpha, and COX-2 were significantly increased in colonic mucosa of the IA+EPEC group and to a lesser but significant level in the IA group compared to controls, or EPEC alone, both in continued and discontinued treatment groups. Additionally, the BAX/Bcl-2 ratio decreased, indicating less apoptosis in the IA+EPEC group which exhibited more necrosis. These effects increased with experiment duration. This work provides new arguments favouring the role of bacteria in IBD pathogenesis.

The effect of high-intensity intermittent exercise on body composition of overweight young males.

To determine the effect of a 12-week high intensity intermittent exercise (HIIE) intervention on total body, abdominal, trunk, visceral fat mass, and fat free mass of young overweight males. Participants were randomly assigned to either exercise or control group. The intervention group received HIIE three times per week, 20 min per session, for 12 weeks. Aerobic power improved significantly (P < 0.001) by 15% for the exercising group. Exercisers compared to controls experienced significant weight loss of 1.5 kg (P < 0.005) and a significant reduction in total fat mass of 2 kg (P < 0.001). Abdominal and trunk adiposity was also significantly reduced in the exercising group by 0.1 kg (P < 0.05) and 1.5 kg (P < 0.001). Also the exercise group had a significant (P < 0.01) 17% reduction in visceral fat after 12 weeks of HIIE, whereas waist circumference was significantly decreased by week six (P < 0.001). Fat free mass was significantly increased (P < 0.05) in the exercising group by 0.4 kg for the leg and 0.7 kg for the trunk. No significant change (P > 0.05) occurred in levels of insulin, HOMA-IR, and blood lipids. Twelve weeks of HIIE resulted in significant reductions in total, abdominal, trunk, and visceral fat and significant increases in fat free mass and aerobic power.

A family history of type 2 diabetes increases risk factors associated with overfeeding.

AIMS/HYPOTHESIS: The purpose of the study was to test prospectively whether healthy individuals with a family history of type 2 diabetes are more susceptible to adverse metabolic effects during experimental overfeeding. METHODS: We studied the effects of 3 and 28 days of overfeeding by 5,200 kJ/day in 41 sedentary individuals with and without a family history of type 2 diabetes (FH+ and FH- respectively). Measures included body weight, fat distribution (computed tomography) and insulin sensitivity (hyperinsulinaemic-euglycaemic clamp). RESULTS: Body weight was increased compared with baseline at 3 and 28 days in both groups (p < 0.001), FH+ individuals having gained significantly more weight than FH- individuals at 28 days (3.4 +/- 1.6 vs 2.2 +/- 1.4 kg, p < 0.05). Fasting serum insulin and C-peptide were increased at 3 and 28 days compared with baseline in both groups, with greater increases in FH+ than in FH- for insulin at +3 and +28 days (p < 0.01) and C-peptide at +28 days (p < 0.05). Fasting glucose also increased at both time points, but without a significant group effect (p = 0.1). Peripheral insulin sensitivity decreased in the whole cohort at +28 days (54.8 +/- 17.7 to 50.3 +/- 15.6 micromol min(-1) [kg fat-free mass](-1), p = 0.03), and insulin sensitivity by HOMA-IR decreased at both time points (p < 0.001) and to a greater extent in FH+ than in FH- (p = 0.008). Liver fat, subcutaneous and visceral fat increased similarly in the two groups (p < 0.001). CONCLUSIONS: Overfeeding induced weight and fat gain, insulin resistance and hepatic fat deposition in healthy individuals. However, individuals with a family history of type 2 diabetes gained more weight and greater insulin resistance by HOMA-IR. The results of this study suggest that healthy individuals with a family history of type 2 diabetes are predisposed to adverse effects of overfeeding. TRIAL REGISTRATION: ClinicalTrials.gov NCT00562393 FUNDING: The study was funded by the National Health and Medical Research Council (NHMRC), Australia (no. #427639).

Shock-induced bubble jetting into a viscous fluid with application to tissue injury in shock-wave lithotripsy.

Shock waves in liquids are known to cause spherical gas bubbles to rapidly collapse and form strong re-entrant jets in the direction of the propagating shock. The interaction of these jets with an adjacent viscous liquid is investigated using finite-volume simulation methods. This configuration serves as a model for tissue injury during shock-wave lithotripsy, a medical procedure to remove kidney stones. In this case, the viscous fluid provides a crude model for the tissue. It is found that for viscosities comparable to what might be expected in tissue, the jet that forms upon collapse of a small bubble fails to penetrate deeply into the viscous fluid "tissue." A simple model reproduces the penetration distance versus viscosity observed in the simulations and leads to a phenomenological model for the spreading of injury with multiple shocks. For a reasonable selection of a single efficiency parameter, this model is able to reproduce in vivo observations of an apparent 1000-shock threshold before wide-spread tissue injury occurs in targeted kidneys and the approximate extent of this injury after a typical clinical dose of 2000 shock waves.

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2.

BACKGROUND AND AIMS: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. METHODS: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. RESULTS: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and beta-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of Apc(Min/+) and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. CONCLUSIONS: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

A multiscale crater function model for ion-induced pattern formation in silicon.

The ion-induced formation of nanometer-scale ripples on semiconductors, long known as the sputter erosion surface instability, is explained using a coupled atomistic-continuum framework. Molecular dynamics simulations of individual medium energy ion impacts on an amorphous silicon target show that the average effect of an incident ion is to leave an ångström-scale crater-like impression on the surface, complete with a crater rim. The summation of many such impacts on a micron-scale surface, combined with the smoothing effect of surface diffusion, leads to the formation of surface ripples aligned perpendicular to the projected ion beam direction. The same numerical approach can be used to evaluate the standard analytical model for this process, known as the Bradley-Harper model. Both Bradley-Harper surface evolution and the atomistically determined crater function surface evolution are computed over time under conditions similar to those for known experimental data. The results show that the surface mass rearrangement associated with the finite atomistic crater rims explains a key experimental observation, ripple amplitude saturation, which cannot be accurately explained using the Bradley-Harper model or any other known numerical or analytical model for the sputter erosion surface instability.

Enhanced droplet spreading due to thermal fluctuations.

The lubrication equation that governs the dynamics of thin liquid films can be augmented to account for stochastic stresses associated with the thermal fluctuations of the fluid. It has been suggested that under certain conditions the spreading rate of a liquid drop on a surface will be increased by these stochastic stresses. Here, an atomistic simulation of a spreading drop is designed to examine such a regime and provide a quantitative assessment of the stochastic lubrication equation for spreading. It is found that the atomistic drop does indeed spread faster than the standard lubrication equations would suggest and that the stochastic lubrication equation of Grün et al (2006 J. Stat. Phys. 122 1261-91) predicts the spread rate.

Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines.

Helicobacter pylori infection induces intestinal metaplasia of the stomach, a preneoplastic lesion associated with an increased risk for gastric cancer development. Intestinal metaplasia is induced by the intestine-specific transcription factor CDX2 but the mechanisms responsible for this ectopic expression have never been described. We hypothesized that the BMP/SMAD pathway has a role in CDX2 regulation, in this context, for the following reasons: (1) the BMP pathway is crucial for normal intestinal differentiation and (2) there is an influx of BMP2 and BMP4-producing cells to the stomach upon Helicobacter pylori infection. We evaluated the expression of key elements of the BMP pathway in human stomach specimens with IM. Growth factor treatments, with BMP2 and BMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was done using RNAi. We showed overexpression in IM of BMP2/4, BMPR1A, and SMAD4 in 56% of IM foci, and pSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In vitro, treatment of AGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as the intestinal differentiation markers MUC2 and LI-cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2, and LI-cadherin in AGS. Treatment of the SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wild-type cells. A 9.3 kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in a cell-dependent manner. In conclusion, we identified for the first time that the BMP pathway is active in intestinal metaplasia and that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiation through the canonical signal transducers.

Cdx1, a dispensable homeobox gene for gut development with limited effect in intestinal cancer.

The homeobox gene Cdx1 is involved in anteroposterior patterning in embryos and its expression selectively persists in the intestinal epithelium throughout life. In human colon cancers, Cdx1 is overexpressed in few cases and lost in the majority of adenocarcinomas. We used mouse models of gain and loss-of-function to investigate the role of Cdx1 in intestinal development and cancers. Transgenic mice overexpressing Cdx1 and knockout mice exhibited a morphologically normal intestine. Cell proliferation, specification into the four differentiated lineages and migration along the crypt-villus axis were unchanged compared to wild-type mice. Changing Cdx1 caused an inverse and dose-dependent modification of the expression of the paralogous gene Cdx2, indicating that Cdx1 fine-tunes Cdx2 activity. Transgenenic and knockout mice failed to spontaneously develop tumours. Overexpressing Cdx1 was without incidence on the frequency of intestinal tumours induced chemically by azoxymethane treatment or genetically in Apc(Delta14/+) mice. However, it augmented the severity of the tumours in Apc(Delta14/+) mice. Inversely, the loss-of-function of Cdx1 in knockout mice was without incidence on the growth of tumours induced by azoxymethane. We conclude that Cdx1 is dispensable for intestinal development and that its overexpression could increase malignancy in early stages of tumourigenesis.