DECO: decompose heterogeneous population cohorts for patient stratification and discovery of sample biomarkers using omic data profiling.

MOTIVATION: Patient and sample diversity is one of the main challenges when dealing with clinical cohorts in biomedical genomics studies. During last decade, several methods have been developed to identify biomarkers assigned to specific individuals or subtypes of samples. However, current methods still fail to discover markers in complex scenarios where heterogeneity or hidden phenotypical factors are present. Here, we propose a method to analyze and understand heterogeneous data avoiding classical normalization approaches of reducing or removing variation. RESULTS: DEcomposing heterogeneous Cohorts using Omic data profiling (DECO) is a method to find significant association among biological features (biomarkers) and samples (individuals) analyzing large-scale omic data. The method identifies and categorizes biomarkers of specific phenotypic conditions based on a recurrent differential analysis integrated with a non-symmetrical correspondence analysis. DECO integrates both omic data dispersion and predictor-response relationship from non-symmetrical correspondence analysis in a unique statistic (called h-statistic), allowing the identification of closely related sample categories within complex cohorts. The performance is demonstrated using simulated data and five experimental transcriptomic datasets, and comparing to seven other methods. We show DECO greatly enhances the discovery and subtle identification of biomarkers, making it especially suited for deep and accurate patient stratification. AVAILABILITY AND IMPLEMENTATION: DECO is freely available as an R package (including a practical vignette) at Bioconductor repository (http://bioconductor.org/packages/deco/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells.

AIMS/HYPOTHESIS: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge. METHODS: We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications. RESULTS: This study reveals, for the first time, that activin/TGF-ß signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-ß signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9. CONCLUSION/INTERPRETATION: Our methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.

Can accelerometer ear tags identify behavioural changes in sheep associated with parturition?

On-animal sensor systems provide an opportunity to monitor ewes during parturition, potentially reducing ewe and lamb mortality risk. This study investigated the capacity of machine learning (ML) behaviour classification to monitor changes in sheep behaviour around the time of lambing using ear-borne accelerometers. Accelerometers were attached to 27 ewes grazing a 4.4 ha paddock. Data were then classified based on three different ethograms: (i) detection of grazing, lying, standing, walking; (ii) detection of active behaviour; and (iii) detection of body posture. Proportion of time devoted to performing each behaviour and activity was then calculated at a daily and hourly scale. Frequency of posture change was also calculated on an hourly scale. Assessment of each metric using a linear mixed-effects model was conducted for the 7 days (day scale) or 12 h (hour scale) before and after lambing. For all physical movements, regardless of the ethogram, there was a change in the days surrounding lambing. This involved either a decrease (grazing, lying, active behaviour) or peak (standing, walking) on the day of parturition, with most values returning to either pre-partum or near-pre-partum levels (all P < 0.001). Hourly changes also occurred for all behaviours (all P < 0.001), the most marked being increased walking behaviour and frequency of posture change. These findings indicate ewes were more restless around the time of parturition. Further application of this research should focus on development of algorithms that can be used to identify onset of lambing and/or time of parturition in pasture-based ewes.

Bilateral facial nerve palsy associated with HIV seroconversion illness.

Bilateral facial nerve palsy is a rare but recognised manifestation of HIV seroconversion illness. The pathophysiology of this clinical presentation is thought to be associated with the immune response of the dissemination of virus throughout the body. We describe a case of bilateral facial nerve palsy, subsequently diagnosed with HIV. Related medical literature is also reviewed. The case highlights the vigilance required in unusual, atypical signs and symptoms if a diagnosis of HIV infection is not to be missed. Clinicians should be aware of the potential presentation of HIV seroconversion illness to provide opportunity for early diagnosis and intervention.

Refractory Crohn's disease of the vulva treated with infliximab: a case report.

Crohn's disease (CD) is a chronic relapsing and remitting inflammatory disorder of the gastrointestinal tract. The common presentation includes abdominal pain, abdominal cramping and diarrhea. Many patients may exhibit systemic symptoms of fever and weight loss. Approximately 20% to 40% of patients will experience extraintestinal manifestations that involve the eyes, skin and joints. Women may experience a variety of gynecological manifestations, including vulvovaginal involvement, which is often not recognized and also difficult to treat. A case of refractory vulvovaginal CD is presented and the literature of gynecological manifestations of CD and its treatment are reviewed.

Effect of nicotinamide on the microregional heterogeneity of oxygen delivery within a murine tumor.

Nicotinamide, an agent previously reported to reduce hypoxia and increase the irradiation response of experimental tumors, has been evaluated for its effect on the occurrence of acute hypoxia in the murine squamous cell tumor SCCVII. Treatment of C3H mice bearing 500-750-mg subcutaneous tumors with nicotinamide (1.0 mg/g intraperitoneally) 1 hour prior to irradiation resulted in an enhancement ratio of 1.3 (+/- 0.1). We assessed the effect of nicotinamide on the response of acutely hypoxic cells in vivo using a recently developed fluorescence-activated-cell sorting technique. This technique employs the in vivo pharmacokinetic and DNA binding properties of the bisbenzamide stain Hoechst 33342. The results clearly show that nicotinamide, at the doses used, reduces the amount of acute hypoxia in these SCCVII tumors. We confirmed these findings using a histological technique that facilitates the assessment of functional tumor vasculature at two instances in time. This method shows that nicotinamide reduces the number of vessels opening and closing over a 20-minute period from 10.3% to 2.0%. The identification of a compound that can modify the dynamic fluctuations in microregional oxygen delivery in tumors could have important implications for radiation therapy.

Expression of apoptosis regulators in cutaneous malignant melanoma.

Metastatic malignant melanoma (MM) is usually incurable and responds poorly to chemotherapy. Because many cytotoxic drugs cause cell death by inducing apoptosis, an imbalance of apoptosis regulatory proteins may contribute to MM treatment resistance. We have previously shown reduced expression of Bcl-2 protein, a negative regulator of apoptosis, in MM as compared with benign nevi. It is hypothesized that other apoptosis regulators may be involved in survival of MM cells. We examined the expression of Bax, Bcl-2, Bcl-X, and Mcl-1 in human benign nevi, primary MM, and metastatic MM using immunohistochemistry. Results were confirmed with Western blotting. The proapoptotic protein, Bax, was surprisingly overexpressed in all MM samples compared with benign nevi. Interestingly, in most MM samples there was overexpression of Mcl-1 or Bcl-XL, both negative regulators of apoptosis. Increased expression of Mcl-1 and Bcl-XL was first observed in thin primary melanomas, suggesting that up-regulation of these proteins represents a relatively early event associated with malignant transformation in MM. As published previously, the majority of primary and metastatic MM exhibited reduced Bcl-2 levels. We conclude that the apoptosis inhibitors Bcl-XL or Mcl-1, alone or in combination, may circumvent the normal cell death pathway, contributing to the pathogenesis and treatment resistance in metastatic MM.

Leishmaniasis of the nasal cavity: a case report.

Establishing diagnosis of a granulomatous lesion of the nose is often difficult. Here we report a case of granulomatous lesion of the nose caused by Leishmania--an unlikely cause in the UK. The diagnosis and management of the case is discussed here.

Heat shock transcription factor-1 regulates heat shock protein-72 expression in human keratinocytes exposed to ultraviolet B light.

In response to ultraviolet radiation (UVR), skin keratinocytes increase expression of heat shock proteins that can protect cells from stress-induced damage. This heat shock response is known to be transcriptionally regulated in eukaryotic cells exposed to certain forms of environmental stress. In the skin, absorption of ultraviolet B light occurs primarily in the epidermis, and therefore, using primary cultures of normal human epidermal keratinocytes, we have examined whether transcriptional activation of the hsp72 gene occurs following UVB irradiation. Cultured keratinocytes were exposed to UVB (290-320 nm, 300 J per m2) and then incubated at 37 degrees C for various intervals before harvesting. Immediately following UV exposure, the heat shock transcription factor 1 (HSF1) dissociated from HSP72-HSF1 complexes, underwent trimerization and phosphorylation, and demonstrated DNA binding activity to the heat shock element in the promoter region of the hsp72 gene. UVB also increased hsp72 mRNA, with peak levels observed 1-3 h post-UVR. HSP72 protein was constitutively expressed in keratinocytes, and its expression was increased by UVB, with maximum levels at 6 h post-UVR. The stress response may be extremely important in the protection of human skin from UVB radiation, and modulation of heat shock protein expression and/or function offers a potential therapeutic target in the prevention of photoaging and skin cancer.

Effect of angiotensin II on intermittent tumour blood flow and acute hypoxia in the murine SCCVII carcinoma.

Effects of the vasoconstrictor angiotensin II on tumour microvascular perfusion and oxygenation were examined in the murine SCCVII carcinoma grown subcutaneously in C3H/He mice. Angiotensin II infusion (2 micrograms/kg/min) caused an increase in mouse arterial blood pressure from 85 (2) mm Hg (mean, S.D.) to 112 (7) mm Hg. During drug infusion, tumour red blood cell (RBC) flow (measured by laser doppler flowmetry) increased 206 (50%) (P less than 0.001) in unanaesthetised animals and 305 (90%) (P less than 0.001) in mice immobilised with ketamine and diazepam. As assessed using a fluorescent double-staining technique, angiotensin II reduced staining mismatch (indicative of intermittent blood flow) in SCCVII microvasculature from 8.1 (2.5%) of total vessels to 2.0 (1.3%) (P less than 0.001). However, a large proportion of this reduction could be attributed to volume loading. Angiotensin II reduced but did not completely eliminate the radiobiological acute hypoxia which results from intermittent tumour vessel non-perfusion. We propose that angiotensin II improves tumour microcirculatory flow distribution via its systemic actions, by elevating perfusion pressure, thereby preventing collapse and/or temporary flow stasis in tumour microvessels.

Measurement of oxygen diffusion distance in tumor cubes using a fluorescent hypoxia probe.

Oxygen diffusion distance was measured in solid tumor "cubes" prepared by excising the tumor from the mouse and incubating 1-2 mm sided tumor cubes in spinner culture flasks with fluorescent drugs (AF-2 or DM113) which bind to hypoxic cells. After incubation, frozen sections were prepared and examined for AF-2 or DM113 binding using a fluorescence image processing system. Alternatively, cubes were stained with a slowly penetrating fluorescent dye, Hoechst 33342, prior to disaggregation and measurement of AF-2 binding using flow cytometry. The distance from the cube surface to the AF-2 stained region (i.e., the thickness of the unstained rim) was used as an indication of oxygen diffusion distance, which depends on the square root of the tumor oxygen consumption rate. Oxygen diffusion distance was dependent on tumor type, external oxygen concentration, and temperature during incubation with AF-2, but was unaffected by tumor size up to 1.2 gm or position of the cube within the tumor. Oxygen diffusion distances (in planar geometry) measured for cubes prepared from SCCVII, RIF-1, Lewis lung or WiDr tumors were 107, 123, 153 and 193 microns, respectively. For SCCVII and WiDr tumors, the percentage of blood vessels separated by a distance greater than double the mean oxygen diffusion distance was used as an indication of the hypoxic fraction.

Histological evidence for nonperfused vasculature in a murine tumor following hydralazine administration.

The effect of the vasodilator hydralazine on tumor vascular function has been evaluated in C3H/He mice bearing subcutaneously implanted SCCVII squamous cell carcinoma. Changes in microregional perfusion following hydralazine administration were observed using a double fluorescent staining technique. Hydralazine-induced alterations in tumor blood flow were measured using laser Doppler flowmetry. The results obtained indicate that hydralazine causes a dose-dependent reduction in functional tumor vasculature implying complete flow stasis and/or vascular collapse in some vessels. Fifteen minutes after a dose of 10 mg/kg intravenously, perfusion in 36 +/- 5% (SEM) of tumor vessels was completely abolished. In addition to cessation of perfusion in individual vessels, hydralazine eliminated flow in large patches of vasculature distributed non-uniformly throughout the tumor. Hydralazine (10 mg/kg i.v.) resulted in a 67 +/- 5% (SEM) reduction in tumor red blood cell (RBC) flow as measured by laser Doppler techniques. The mean number of moving red blood cells declined by 35 +/- 8%, suggesting a reduction in microvascular volume. These results support the hypothesis that following hydralazine administration, perfusion stops completely in some blood vessels probably as a result of vascular collapse or flow stasis.

The use of fluorescent probes to identify regions of transient perfusion in murine tumors.

Sequential intravenous injection of two fluorescent stains, Hoechst 33342 and DiOC7(3), can be used to quantify transient perfusion in experimental tumors. Regions of unmatched staining, indicative of intermittent perfusion, occur when vessels open or close in the 20 minute interval between administration of the dyes. In the murine SCCVII carcinoma, 8.9 +/- 2.4% (SD) of vessels in 0.5 g subcutaneous tumors had labelling of adjacent cells with only one stain, suggesting complete vessel closure lasting at least 5 minutes. Regions of intermittent perfusion were not homogeneously distributed throughout the tumor and larger tumors exhibited more mismatch than smaller tumors. Transient perfusion was observed in both subcutaneous and intramuscular tumor implants and was not significantly affected by restraint of the animal or by ketamine/diazepam anesthesia.

The effect of hydralazine on blood flow and misonidazole toxicity in human tumour xenografts.

The effect of post-irradiation hypoxia induced by 5 or 30 mg/kg hydralazine has been studied in three human tumour xenografts (two rectocolic adenocarcinomas and one melanoma) treated with two doses of misonidazole similar to those used in patients (0.1 and 0.2 mg/g). Only a small sensitization was detected using an in vitro colony assay. These results are in marked contrast to the results obtained with rodent tumours. This difference between human tumour xenografts and rodent tumours might be explained by differences in the reduction of tumour blood flow after hydralazine administration (5 and/or 10 mg/kg). Using the laser Doppler technique, the tumour blood flow reduction was 33% and 25% of the control for NA11 and HRT18 tumours, respectively. In contrast, hydralazine induced a 60-70% reduction in blood flow in the murine SCCVII tumour. Using the fluorescent marker Hoechst 33342, the reduction in perfusion was again more pronounced in the murine tumour as compared to the Na11 and HRT18 xenografts. The differences between human tumour xenografts and rodent tumours are not linked to the mouse strain used (nude versus C3H) nor to a tumour bed effect.

Drug induced perturbations in tumor blood flow: therapeutic potential and possible limitations.

Chemical modulation of tumor blood flow has until recently received relatively little attention as a therapeutic tool. Developments in the last few years, both in technology and in drug development, have changed this perspective. Fluorescence activated cell sorting techniques have provided evidence for the existence of acutely hypoxic cells resulting from transient fluctuations in microregional tumor blood flow in experimental tumor systems. We have used such techniques to assess the effects of three systemically administered agents, nicotinamide, flunarazine and Flusol-DA, on the amount of acute hypoxia in the SCCVII tumor. The most effective agent identified in this study is the benzamide analog nicotinamide. We suggest that compounds which modulate such hypoxia could well have a role in radiation therapy, particularly when combined with techniques which increase the oxygen carrying capacity of the blood. The potential of tumor blood flow reduction to improve the effectiveness of bioreductive agents administered alone or in combination with radiation and/or hyperthermia, is well established in experimental systems. Further data are presented, which show that combining hydralazine and the beta-blocker propranolol can provide greater reduction in tumor blood flow than observed with hydralazine alone. Potential limitations of drug induced reduction in tumor blood flow are discussed including the possibility of inducing hypoxia in normal tissues.

Oxygen enhancement ratio of fractionated regimens in vitro.

The oxygen enhancement ratio (OER) of proliferating and nonproliferating cells grown in vitro was measured using accelerated fractionated regimens. Irradiations were performed either twice daily or three times per day, with a minimum of 6 h between the consecutive fractions. The dose delivered was 2.3 Gy per fraction. Two significant observations were made: (i) the OER of accelerated fractionation regimens for proliferating cells is lower than that obtained from single-exposure experiments at 2.3 Gy (approximately 1.4 vs 2.4, respectively), while for nonproliferating cells it is approximately the same (2.3); (ii) the fractionated regimen does not spare proliferating cells irradiated under hypoxic conditions, and thus the fractionated survival curve lies below the single-exposure curve. For cells irradiated under aerobic conditions or for nonproliferating cells, irradiated under either hypoxic or aerobic conditions, the fractionated survival curve lies above the single-exposure curves as expected.

Localized chrysiasis induced by laser therapy.

BACKGROUND: Chrysiasis is a rare blue-gray skin discoloration that occurs in sun-exposed sites of some patients who receive gold salts. A unique case of localized chrysiasis developed immediately after Q-switched ruby laser (694 nm) irradiation for postinflammatory hyperpigmentation secondary to granuloma faciale in a patient who was receiving long-term gold sodium thiomalate therapy for psoriatic arthritis. Skin biopsy specimens showed striking changes in the ultrastructural characteristics of cutaneous gold deposits following laser treatment. OBSERVATIONS: A blue-gray skin discoloration developed immediately after laser exposure and persisted unchanged after 1 year. Transmission electron microscopy of skin biopsy specimens showed electron-dense gold deposits. Before laser irradiation, these deposits were 106 +/- 35 (mean +/- SD) nm in diameter and faceted, consistent with a crystalline structure. Posttreatment deposits were round, smaller, measured 16 +/- 4 nm, and resembled colloidal gold. Identical findings were observed in an area of sun-protected skin treated with the Q-switched ruby laser; irradiation with a pulsed dye laser at 585 nm had no effect. CONCLUSIONS: Localized chrysiasis was induced in a patient receiving parenteral gold therapy who underwent treatment with a Q-switched ruby laser. This form of chrysiasis resulted from a structural alteration in dermal gold deposits. A similar physiochemical modification in gold deposits induced by UV light may explain the localization of chrysiasis to sun-exposed skin in affected patients.

Expression of cell cycle regulators in human cutaneous malignant melanoma.

We postulate that genes involved in the control of cell proliferation are important determinants of melanoma growth and/or transformation. Using Western blot analysis, we compared the expression of nine key cell cycle regulators in metastatic melanomas with that in benign acquired naevi. Among the cyclin-dependent kinases (CDKs) examined, CDK2 was consistently and significantly overexpressed (three- to eight-fold) in metastatic melanomas compared with naevi. CDK1 and CDK4 exhibited no significant difference in expression between benign naevi and metastatic melanomas. CDK6 expression was variable, with four out of 10 metastatic melanomas showing higher expression than naevi. All the cyclins examined, especially cyclins A and D, were expressed more in metastatic melanomas than in naevi. Cyclin E was not detected in benign naevi, but was easily detectable in most of the metastatic melanomas. In addition, there was significantly greater expression of CDC25A, a tyrosine phosphatase that activates CDK kinases, in the metastatic melanomas. Over-expression of CDK2, CDK6, CDC25A and cyclin A was confirmed in melanoma cell lines. These cell cycle regulators may play an important role in melanoma growth and/or transformation.

Spontaneous torsion of the right lung: a case report.

Pulmonary torsion is an uncommon event and may occur spontaneously or after trauma or surgery. It may involve the entire lung or individual lobes. Early recognition with prompt intervention is required to prevent hemorrhagic infarction or gangrene and salvage parenchyma. Surgical options of detorsion or resection are dependent upon tissue viability. A case of spontaneous torsion of the entire right lung is presented that reflects the first successful nonresectional management of this entity to be reported. Guidelines for appropriate management are discussed based on an understanding of the etiology, pathophysiology, and natural history.

Postpneumonectomy chylothorax: a logical approach to successful management.

Chylothorax is unusual as a postoperative complication following pneumonectomy. Although rare today, it can still manifest life-threatening consequences if not recognized and treated in a timely and appropriate manner. Management options range from a conservative approach to immediate re-exploration, each of which has been reported as successful. However, treatment of this entity has been characterized as ill-defined or controversial despite the fact that most approaches to this clinical problem are similar. A successful outcome of this unusual complication is presented based on early recognition and institution of logical principles of conservative management. Treatment should be neither ill-defined nor controversial.