Label-free electrochemical biosensor for direct detection of Oncostatin M (OSM) inflammatory bowel diseases (IBD) biomarker in human serum.

Oncostatin M (OSM) is an interleukin-6 (IL-6) member family cytokine implicated in the pathogenesis of chronic diseases including inflammatory bowel disease (IBD). OSM is a novel diagnostic biomarker over-expressed in the serum of IBD patients. This paper reports on the first electrochemical OSM immunosensor, developed using a multistep fabrication process aimed at covalently immobilizing OSM antibodies on a mixed self-assembled monolayer coated gold working electrode. Cyclic voltammetry, atomic force microscopy (AFM), IR spectroscopy and optical characterizations were used to validate the sensor functionalization protocol. Electrochemical impedance spectroscopy (EIS) measurements were performed to assess the reliability of the immunosensor preparation and to verify the antibody-antigen complexes formation. The label-free immunosensor showed high sensitivity identifying OSM at clinically relevant concentrations (37-1000 pg mL-1) with low detection limit of 2.86 pg mL-1. Both sensitivity and selectivity of the proposed immunosensor were also demonstrated in human serum in the presence of interfering biomarkers, making it an innovative potential platform for the OSM biomarker detection in IBD patients' serum.

Homologous control of protein signaling networks.

In a previous paper we introduced a method called augmented sparse reconstruction (ASR) that identifies links among nodes of ordinary differential equation networks, given a small set of observed trajectories with various initial conditions. The main purpose of that technique was to reconstruct intracellular protein signaling networks. In this paper we show that a recursive augmented sparse reconstruction generates artificial networks that are homologous to a large, reference network, in the sense that kinase inhibition of several reactions in the network alters the trajectories of a sizable number of proteins in comparable ways for reference and reconstructed networks. We show this result using a large in-silico model of the epidermal growth factor receptor (EGF-R) driven signaling cascade to generate the data used in the reconstruction algorithm. The most significant consequence of this observed homology is that a nearly optimal combinatorial dosage of kinase inhibitors can be inferred, for many nodes, from the reconstructed network, a result potentially useful for a variety of applications in personalized medicine.

Platinum-gold nanoclusters as catalyst for direct methanol fuel cells.

Nanosized platinum-gold alloys clusters have been deposited on gas diffusion electrode by sputter deposition. The deposits were characterized by FE-SEM, TEM and XPS in order to verify the formation of alloy nanoparticles and to study the influence of deposition technique on the nanomorphology. The deposition by sputtering process allowed a uniform distribution of metal particles on porous surface of carbon supports. Typical island growth mode was observed with the formation of a dispersed metal nanoclusters (mean size about 5 nm). Cyclic voltammetry was used to determine the electrochemical active surface and the electrocatalytic performance of the PtAu electrocatalysts for methanol oxidation reaction. The data were re-calculated in the form of mass specific activity (MSA). The sputter-catalyzed electrodes showed higher performance and stability compared to commercial catalysts.

Mesenchymal differentiation of glioblastoma stem cells.

Glioblastoma multiforme is a severe form of cancer most likely arising from the transformation of stem or progenitor cells resident in the brain. Although the tumorigenic population in glioblastoma is defined as composed by cancer stem cells (CSCs), the cellular target of the transformation hit remains to be identified. Glioma stem cells (SCs) are thought to have a differentiation potential restricted to the neural lineage. However, using orthotopic versus heterotopic xenograft models and in vitro differentiation assays, we found that a subset of glioblastomas contained CSCs with both neural and mesenchymal potential. Subcutaneous injection of CSCs or single CSC clones from two of seven patients produced tumor xenografts containing osteo-chondrogenic areas in the context of glioblastoma-like tumor lesions. Moreover, CSC clones from four of seven cases generated both neural and chondrogenic cells in vitro. Interestingly, mesenchymal differentiation of the tumor xenografts was associated with reduction of both growth rate and mitotic index. These findings suggest that in a subclass of glioblastomas the tumorigenic hit occurs on a multipotent stem cell, which may reveal its plasticity under specific environmental stimuli. The discovery of such biological properties might provide considerable information to the development of new therapeutic strategies aimed at forcing glioblastoma stem cell differentiation.

Fabrication and characterization of AlN-based flexible piezoelectric pressure sensor integrated into an implantable artificial pancreas.

This study reports on the fabrication and characterization of an event detection subsystem composed of a flexible piezoelectric pressure sensor and the electronic interface to be integrated into an implantable artificial pancreas (IAP) for diabetic patients. The developed sensor is made of an AlN layer, sandwiched between two Ti electrodes, sputtered on Kapton substrate, with a preferential orientation along c-axis which guarantees the best piezoelectric response. The IAP is made of an intestinal wall-interfaced refilling module, able to dock an ingestible insulin capsule. A linearly actuated needle punches the duodenum tissue and then the PDMS capsule to transfer the insulin to an implanted reservoir. The device is located at the connection of the needle with the linear actuator to reliably detect the occurred punching of the insulin-filled capsule. Finite Element Analysis (FEA) simulations were performed to evaluate the piezoelectric charge generated for increasing loads in the range of interest, applied on both the sensor full-area and footprint area of the Hamilton needle used for the capsule punching. The sensor-interface circuit was simulated to estimate the output voltage that can be obtained in real operating conditions. The characterization results confirmed a high device sensitivity during the punching, in the low forces (0-4 N) and low actuator speed (2-3 mm/s) ranges of interest, meeting the requirement of the research objective. The choice of a piezoelectric pressure sensor is particularly strategic in the medical field due to the request of self-powered implantable devices which do not need any external power source to output a signal and harvest energy from natural sources around the patient.

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition.

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients' samples and by in silico analysis on an independent patient cohort from Taylor's platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients' risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.

Metabolic/Proteomic Signature Defines Two Glioblastoma Subtypes With Different Clinical Outcome.

Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Development of tearing in preterm and term neonates.

BACKGROUND: Although term and preterm infants have the capacity to secrete tears, the relative contribution of basal and reflex secretion of tears has not been previously assessed together in a prospective study. This information potentially has practical clinical importance. OBJECTIVES: To measure basal and reflex tear secretion in preterm (30-37 weeks after conception) and term (38-42 weeks) newborns and to determine the developmental pattern of tear production. METHODS: Tear secretion was evaluated by applying Schirmer tear test strips to the inferior fornix for 5 minutes before (reflex plus basal secretion) and after (basal secretion) applying a topical anesthetic agent. RESULTS: Seventy infants (36 preterm and 34 term) were tested. Mean (+/- SD) basal tear secretion was 6.2 (+/- 4.5) mm in preterm and 9.2 (+/- 4.3) mm in term infants and increased progressively with increasing weight (P<.001) for all newborns. Mean (+/- SD) reflex tear secretion was 7.4 (+/- 4.8) mm in preterm and 13.2 (+/- 6.5) mm in term infants and also increased with increasing weight (P<.001) for all newborns. CONCLUSIONS: Preterm infants have reduced reflex and basal tear secretion. This may mask the diagnosis of a nasolacrimal duct obstruction, concentrate topically applied medications, and allow corneas to quickly become dry during ophthalmological examination and treatment. By term, tear production in newborns is similar to that in adults.

Otx genes in the development and evolution of the vertebrate brain.

Most of the gene candidates for the control of developmental programmes that underlie brain morphogenesis in vertebrates are the orthologues of Drosophila genes coding for signalling molecules or transcription factors. Among these, the orthodenticle group, including the Drosophila orthodenticle (otd) and the vertebrate Otx1 and Otx2 genes, is mostly involved in fundamental processes of anterior neural patterning. In mouse, Drosophila and intermediate species otd/Otx genes have shown a remarkable similarity in expression pattern suggesting that they could be part of a conserved control system operating in the brain and different from that coded by the HOX complexes controlling the hindbrain and spinal cord. In order to verify this hypothesis, a series of mouse models have been generated in which the functions of the murine Otx genes were: (i) fully inactivated, (ii) replaced with each other, and (iii) replaced with the Drosophila otd gene. The data obtained highlight a crucial role for the Otx genes in specification, regionalization and terminal differentiation of rostral central nervous system and lead to hypothesize that modification of their regulatory control may have influenced the morphogenesis and evolution of the brain.

Use of the HARK autorefractor in children.

PURPOSE: We investigated the reliability, accuracy, and repeatability of an autorefractor with the capability of over-refracting and measuring visual acuity for use in children in a prospective study. METHODS: Before and after cycloplegia, 68 children (mean +/- SD age, 10 +/- 3 years, range 5-16 years) underwent autorefraction twice with the HARK 599 Autorefractor (Humphrey Instruments Inc., San Leandro, CA), subjective over-refraction through the HARK autorefractor, and subjective refraction using a phoro-optometer. After cycloplegia, retinoscopy was performed. Results are reported for one eye (left) of each child. RESULTS: For 68 eyes of 68 children, before and after cycloplegia, correlation coefficients (R) for autorefraction reproducibility exceeded 0.95 for all comparisons of sphere and cylinder. R for spherical values for autorefraction vs. over-refraction was 0.93 and vs. subjective refraction 0.83 before cycloplegia and 0.94 and 0.97 after cycloplegia. Comparing values before and after cycloplegia, autorefraction, over-refraction, and subjective refraction, the data correlated > 0.81 for sphere and 0.75 to 0.87 for cylinder. Cycloplegic retinoscopy compared with autorefraction, over-refraction, and subjective refraction had R > 0.86 for sphere and cylinder for all comparisons except one. Cycloplegia increased the proportion of spherical equivalent values within 0.625 D of the subjective refraction from 41 of 68 eyes (61%) for auto- and over-refraction to 64 (94%) and 51 (75%) of the 68 eyes, respectively. A visual acuity of 20/30 or better was produced in 50 of 68 (73%) eyes with automated refraction before and after cycloplegia and in 62 (92%) with subjective refraction before cycloplegia and subjective refraction and retinoscopy after cycloplegia. Subjective over-refraction did not significantly improve the visual acuity. CONCLUSIONS: In children, HARK autorefraction improved in accuracy, when compared to subjective refraction, and the level of visual acuity improved after cycloplegia. Over-refraction through the instrument did not improve the results before or after cycloplegia.

A double application approach to ophthalmia neonatorum prophylaxis.

AIMS: To investigate if a second drop of 2.5% povidone-iodine ophthalmic solution placed within the first postnatal day would achieve better prophylaxis against ophthalmia neonatorum than a single drop applied at birth. METHODS: A masked, prospective, controlled trial was conducted over a 2 year period in a Kenyan hospital. Randomisation was achieved by alternating weeks of one or two eye drop application to both eyes. All 719 neonates received one drop of the povidone-iodine solution to both eyes at birth, while 317 received a second drop at hospital discharge or 24 (SD 4) hours after delivery, whichever was first. All infants developing conjunctivitis within a month after birth underwent microbiological analysis using Gram and Giemsa stains, direct fluorescent antibody assay for Chlamydia trachomatis, and culture. RESULTS: Of the neonates receiving the one eye drop application, 18.4% returned with a red eye with discharge, 4.0% had organisms found on the initial smear, and 8.2% had a positive culture. The corresponding proportions for the multidrop group were 24.3%, 4.7%, and 10.4%. Of those returning with an inflamed eye, there were no cases of Neisseria gonorrhoeae, 4.2% in the single dose group and 3.9% in the double dose group were positive for C trachomatis, and 5.4% and 6.5% respectively for Staphylococcus aureus. At discharge, the eyelid oedema score of the double dose group was mildly greater than the single dose group (1.4 (0.67) v 1.2 (0.73), p=0.0002). There was no statistically significant difference between the groups in any other category. CONCLUSION: There is no advantage to administering povidone-iodine prophylaxis against ophthalmia neonatorum twice in the first postnatal day over a single application at birth.

Non-invasive measurement of blood pressures in the Yucatan micropig (Sus scrofa domestica), with and without midazolam-induced sedation.

Current literature suggests that the effects of midazolam, a water-soluble benzodiazepine, on blood pressure in swine are minimal. The hypothesis of the study reported here was that a light sedative dose would induce a decrease in blood pressure in this species. Healthy female Yucatan Micropigs (n = 20), 16 to 30 (mean, 22) kg, aged four six months, were individually placed in a humane restraint sling and allowed to acclimate. Systolic (SBP), diastolic (DBP), and mean (MBP) blood pressures (mmHg) and heart rate (HR; beats per min [bpm]) were measured by use of oscillometry. The pressure cuff was placed at the base of the tail, and five sets of values were recorded at five-min intervals, beginning at 10 and ending 30 min after cuff placement. Following a three- to four-day rest period, this procedure was repeated with the addition of a dose of 0.5 mg of midazolam HCl/kg of body weight given intramuscularly at the time of cuff placement. A paired one-way Student's t-test was used to compare the means of the five measures between control and midazolam treatment. Mean (+/- SD) differences for SBP, DBP, MBP, and HR were 18.9 (+/- 3.97), 17.8 (+/- 5.27), and 18.6 (+/- 5.09) mmHg and 20.7 (+/- 3.73) bpm, respectively. All four parameters were significantly reduced in the midazolam-sedated group (P < 0.001). The maximal decrease in SBP, DBP, and MBP occurred at 15 and 20 min after dosing. Mean values based on the means of the five measures were 128 (+/- 12.6), 80 (+/- 9.4), and 99 (+/- 9.2) mmHg and 135 (+/- 17.4) bpm, and 109 (+/- 15.4), 63 (+/- 12.6), and 80 (+/- 13.6) mmHg and 115 (+/- 15.5) bpm for SBP, DBP, MBP, and HR in the control (n = 20) and midazolam (n = 20) groups, respectively. The control values can serve as normal oscillometric values for this age, sex, and breed of Micropig. We conclude that midazolam, given intramuscularly at a sedative dosage, negatively affects cardiovascular parameters measured by use of a blood pressure cuff, in sexually mature female Micropigs, compared with values in untreated pigs, which is similar to reports for humans.

Abnormal "low grade" transformation zone: current diagnostic gold standard.

The aim of this work was to examine different methods of investigation in the diagnosis of the abnormal "low grade" transformation zone of the portio. Over a period of one year 41 patients subjected to colposcopic examination underwent exo-endocervical sampling for oncologic evaluation and for detection of viral and bacterial infections (HPV, HSV, adenovirus, mycoplasmas and chlamydia trachomatis), as well as portio biopsy. A 65.8% correlation was found between cytology and the HPV-DNA test results, while histology and the presence of the HPV virus agreed in 51.4% of cases. In those cases in which minimal histological alterations were found (koilocytosis) a high percentage of HPV negativity was found. In discordant negative cytologic tests that were however positive for HPV by PCR, the genotypes identified were always 6 and 11.

[Critical review of colpo-histological results in cervix pathology]. / Revisione critica dei risultati colpo-istologici nella patologia cervicale.

BACKGROUND: The objective of this paper was to evaluate the role of squamous metaplasia in the determination of certain colposcopic appearances. METHODS: One thousand four hundred and six infertile outpatients, attending assisted reproduction techniques, underwent a "first level" colposcopy. Two hundred fifty nine abnormal transformation zones were biopsied and the histologic diagnoses were subdivided as follows: squamous metaplasia, squamous metaplasia + koilocytosis, isolated koilocytosis, condyloma, CIN + HPV, cervicitis. RESULTS: Two hundred forty seven cases out of 259 biopsied colposcopic findings (95.4%) were colposcopically classified as grade 1 abnormal transformation zone (thin white epithelium, regular mosaic and punctuation). The correlation between 247 grade 1 abnormal transformation zone colposcopic patterns and histologic diagnosis revealed 105 (42.5%) histologic findings described as squamous metaplasia that resulted immature in 63% of these samples. Between 132 (53.4%) cases that presented a pattern of human papillomavirus infection (condyloma, squamous metaplasia + koilocytosis or isolated koylocitosis), quite two thirds (62%) were described as condylomas, one third (31%) as squamous metaplasia associated with koylocitosis and only 7% as isolated koylocitosis. In conclusion, 42.5% of target biopsies performed on low grade abnormal transformation of the cervix revealed squamous metaplasia, more than half of them revealed one of HPV infection forms, while only 2% represented cervical intraepithelial neoplasia. CONCLUSIONS: Among the low risk female population, one out of two cases of colposcopically low grade pattern should be considered indicative of squamous metaplasia. The results obtained confirm that colposcopic evaluation is unable to distinguish between immature metaplastic transformation of the epithelium and metaplastic epithelium with initial neoplastic transformation.

The emergency department gynaecologist and emergency postcoital contraception.

The emergency department gynaecologist is often faced with requests for emergency postcoital contraception. The physician on duty is usually very busy and does not always have enough time to perform a complete evaluation of the woman's state of health. The emergency gynaecologist who prescribes postcoital contraception also has a number of other problems to cope with in regard to the pharmacological preparations on the market, the efficacy of various methods, the monitoring of side-effects which are not, always, tolerated by all patients and the outcome of his therapeutic prescription. All these aspects should be emphasized in the "first aid" counselling offered to the patients. In conclusion, we consider that any women who decides to use postcoital contraception should have the right to receive assistance of guaranteed quality throughout the period that elapses between taking the drug and the subsequent menstrual cycle. This is not strictly guaranteed by an emergency gynaecological service.

Gynaecological and obstetric audit at a G&O emergency department.

The present study takes into account only the patients that are sent home because their conditions do not require emergency hospitalization in order to try to understand the reasons why a woman seeks emergency treatment at a G&O emergency service.

Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo.

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.