A droplet-chip/mass spectrometry approach to study organic synthesis at nanoliter scale.

A droplet-based microfluidic device with seamless hyphenation to electrospray mass spectrometry was developed to rapidly investigate organic reactions in segmented flow providing a versatile tool for drug development. A chip-MS interface with an integrated counterelectrode allowed for a flexible positioning of the chip-emitter in front of the MS orifice as well as an independent adjustment of the electrospray potentials. This was necessary to avoid contamination of the mass spectrometer as well as sample overloading due to the high analyte concentrations. The device was exemplarily applied to study the scope of an amino-catalyzed domino reaction with low picomole amount of catalyst in individual nanoliter sized droplets.

Circulating serum CK level vs. muscle impairment for in situ monitoring burden of disease in Mdx-mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) consists of a lack in the expression of the subsarcolemmal protein dystrophin causing progressive muscle dysfunction. Among the widely applied animal models in DMD research is the C57BL/1010ScSn-Dmdmdx mouse, commonly referred to as the "mdx mouse". The potential benefit of novel interventions in this model is often assessed by variables such as functional improvement, histological changes, and creatine kinase (CK) serum levels as an indicator for the extent of in situ muscle damage. OBJECTIVE: Our objective was to determine to what extent the serum CK-level serves a surrogate for muscle dysfunction. METHODS: In this trial mdx mice were subjected to a four-limb wire-hanging test (WHT) to assess the physical performance as a reference for muscle function. As CK is a component of the muscle fiber cytosol, its serum activity is supposed to positively correlate with progressing muscle damage. Hence serum CK levels were measured to detect the degree of muscle impairment. The functional tests and the serum CK levels were analyzed for their specific correlation. RESULTS: Although physical performance decreased during the course of the experiment, latency to fall times in the WHT did not correlate with the CK level in mdx mice. CONCLUSION: Our data suggests that the serum CK activity might be a critical parameter to monitor the progression of muscle impairment in mdx mice. Further this study emphasizes the complexity of the DMD phenotype in the mdx mouse, and the care with which isolated parameters in this model should be interpreted.

Sonoelastography can be used to monitor the restoration of Achilles tendon elasticity after injury.

PURPOSE: The aim of the current study was to evaluate an ultrasound approach for depicting elastic recovery after stem cell application on injured Achilles tendons. MATERIALS AND METHODS: A rabbit Achilles tendon injury model was used and randomized hind limbs received an extracellular matrix either with autologous mesenchymal stem cells (group 2, n = 6) or without (group 3, n = 6). The cells were harvested from the rabbits' nuchal fat body. Untreated Achilles tendons (group 1, n = 6) served as controls. Specimens were harvested after 8 weeks and analyzed longitudinally for elasticity using a high resolution 6-15 MHz matrix linear probe. For each tendon, real-time color-coded sonoelastography sequences were recorded for 20 seconds and 10 color histogram frames were obtained. Defined regions of interest (ROIs) were placed on the injury (n = 3) and on the adjacent uninjured tendon tissue (n = 3). In total, 180 measurements were obtained for semi-quantitative analysis. RESULTS: Repeated measures ANOVA demonstrated a higher elasticity for the stem cell-seeded matrix (group 2) in comparison to the unseeded matrix (group 3) (p < 0.001). No significant difference was found between the injured tendon tissue treated with stem cell-seeded matrix (group 2) and the uninjured Achilles tendons (group 1) (p > 0.05). Moreover, no differences were found between the measurements at different points in time (p > 0.05). CONCLUSION: Our results indicate that autologous mesenchymal stem cell application successfully restores the mechanical properties of injured tendon tissue. Furthermore, sonoelastography makes it possible to monitor the elasticity of injured Achilles tendons.

Loss of PTEN is associated with elevated EGFR and HER2 expression and worse prognosis in salivary gland cancer.

BACKGROUND: Activity of the tumour-suppressor gene PTEN is reduced in different types of cancer and implicates non-responsiveness to targeted therapy. This study evaluates the gene and protein status of PTEN in salivary gland carcinomas. METHODS: A total of 287 carcinomas of the major and minor salivary glands were investigated for phosphatase and tensin homologue located on chromosome 10 (PTEN) deletion and loss of PTEN expression using fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Results were correlated to clinicopathological parameters, long-term survival, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (IHC and FISH) status of the tumours. RESULTS: Hemizygous deletions of PTEN were found in 35 out of 232 (15.1%) carcinomas, while homozygous deletions were observed in 17 out of 232 (7.3%) tumours. Phosphatase and tensin homologue located on chromosome 10 deletion was common in certain histological subtypes and especially homozygous deletion was associated with high-grade malignancy, lymph node metastases and unfavourable long-term prognosis (P<0.001). Loss of PTEN expression was present in 59 out of 273 (21.6%) carcinomas and was significantly correlated to genomic PTEN deletion, high-grade malignancy (P<0.001), increased tumour size (P=0.036), lymph node metastases (P=0.007) and worse disease-specific survival (P=0.002). Genomic PTEN deletion, in particular homogenous deletion (P<0.001) predominantly occurred in tumours with increased gene copy number of EGFR (60.0%) and/or amplification of HER2 (63.6%). Loss of PTEN expression was frequently found in tumours overexpressing EGFR (28.6%) and/or HER2 (52.6%). CONCLUSION: PTEN function is reduced in different types of salivary gland cancer indicating unfavourable prognosis. Its association with EGFR and HER2 signalling might affect targeted therapy.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines.

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133(+) cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Long-term efficacy and safety of chronic globus pallidus internus stimulation in different types of primary dystonia.

BACKGROUND: Deep brain stimulation (DBS) of the globus pallidus internus (GPi) offers a very promising therapy for medically intractable dystonia. However, little is known about the long-term benefit and safety of this procedure. We therefore performed a retrospective long-term analysis of 18 patients (age 12-78 years) suffering from primary generalized (9), segmental (6) or focal (3) dystonia (minimum follow-up: 36 months). METHODS: Outcome was assessed using the Burke-Fahn-Marsden (BFM) scores (generalized dystonia) and the Tsui score (focal/segmental dystonia). Follow-up ranged between 37 and 90 months (mean 60 months). RESULTS: Patients with generalized dystonia showed a mean improvement in the BFM movement score of 39.4% (range 0-68.8%), 42.5% (range -16.0 to 81.3%) and 46.8% (range -2.7 to 83.1%) at the 3- and 12-month, and long-term follow-up, respectively. In focal/segmental dystonia, the mean reduction in the Tsui score was 36.8% (range 0-100%), 65.1% (range 16.7-100%) and 59.8% (range 16.7-100%) at the 3- and 12-month, and long-term follow-up, respectively. Local infections were noted in 2 patients and hardware problems (electrode dislocation and breakage of the extension cable) in 1 patient. CONCLUSION: Our data showed Gpi-DBS to offer a very effective and safe therapy for different kinds of primary dystonia, with a significant long-term benefit in the majority of cases.

N-heterocyclic carbenes: organocatalysts displaying diverse modes of action.

Within the context of Lewis base catalysis N-heterocyclic carbenes represent an extremely versatile class of organocatalyst that allows for a great variety of different transformations. Starting from the early investigations on benzoin, and later Stetter reactions, the mechanistic diversity of N-heterocyclic carbenes, depending on their properties, has led to the development of several unprecedented catalytic reactions. This article will provide an overview of the versatile reactivity of N-heterocyclic carbenes.

A novel one-pot pyrrole synthesis via a coupling-isomerization-Stetter-Paal-Knorr sequence.

[reaction: see text]. 1,2,3,5-tetrasubstituted pyrroles can be synthesized in good yields in a one-pot, three-step, four-component process by a coupling-isomerization-Stetter reaction-Paal-Knorr sequence of an electron-poor (hetero)aryl halide, a terminal propargyl alcohol, an aldehyde, and a primary amine. The structures of the 1,4-diketone 4f and the pyrrole 6b were additionally supported by X-ray structure analyses.

Synthesis of functionalized ethynylphenothiazine fluorophores.

Alkynylated and butadiynyl-bridged phenothiazines with variable functionalization can be synthesized in good yields by cross-coupling and condensation approaches. In addition, the structure of the diethynylated phenothiazine (7a) has been corroborated by an X-ray structure analysis. These oligofunctional heterocycles are fluorescent with modest quantum yields (Phi(f) = 20-35%) and represent suitable building blocks for novel photoexcitable molecular wires.

A novel 1,5-benzoheteroazepine synthesis via a one-Pot coupling-isomerization-cyclocondensation sequence

2,4-Di(hetero)aryl substituted 2,3-dihydro 1,5-benzoheteroazepines (hetero = NH, O, S) can be readily sythesized in a one-pot process initiated by a coupling-isomerization sequence of an electron poor (hetero)aryl halide and a terminal propargyl alcohol subsequently followed by a cyclocondensation with 2-amino, 2-hydroxy, or 2-mercapto anilines. In addition, the structures were established unambiguously by an X-ray structure analysis of 7a.

[Mumps epidemic in the health area of Toledo with immunization intervention. Estimation of vaccine effectiveness]. / Epidemia de parotiditis en el área sanitaria de Toledo con intervención mediante inmunización. Estimación de la efectividad de la vacuna.

BACKGROUND: The aim of this study was to describe a mumps epidemic occurred among school children in Toledo, Spain, between 1993 and 1994 were vaccination was undertaken as control measure, and to estimate vaccine efficacy under field conditions. MATERIAL AND METHODS: Attack rates for vaccinated (at 15 months of age) and unvaccinated population were calculated. A retrospective cohort study was designed to estimate vaccine efficacy. RESULTS: A total of 19 outbreaks in different schools were declared. 216 cases and 4,059 contacts, between 10-17 years old, were investigated. The attack rate for the school population was 5.3%. By age, the highest attack rate was for those in the 12-13 age group (7.9%). Estimated vaccine efficacy was 76% (95% confidence interval 66-87%) for the whole study population: For children among 10-13 years old it was 87% (95% confidence interval 76-93%), and 46% (95% confidence interval 20-70%) for those aged 14-17 years. CONCLUSIONS: Vaccination is a good control measure in mumps outbreaks (the attack rate for the study population was 5.1%). Vaccine efficacy was high for the 10-13 age group and in agreement with the results of seroepidemiological studies.

[Study of 2 food poisoning outbreaks in Mora (Toledo) with the same source of infection]. / Investigación de dos brotes de toxiinfección alimentaria en Mora (Toledo) con una fuente de infección común.

BACKGROUND: The occurrence of an outbreak needs an immediate investigation, because on it depends the necessary measures to control the spread of the epidemic, and it is also the way of knowing the causal factor. We present the results of the investigation of two food poisoning outbreaks (outbreak A and outbreak B) occurred in Mora (Toledo). Both outbreaks were reported at the same time, the source of infection was the same and the total number of exposed people was 620. METHODS: A case-control study was carried out. A standardised questionnaire for food poisoning outbreaks was used to collect the data. Data were analysed using classic methods and also logistic regression models. RESULTS: A total of 236 individuals were interviewed (119 for outbreak A and 117 for outbreak B), 146 of them were cases (73 in each outbreak). The mean incubation period was of 26.18 (+/- 12.35) and 25.2 (+/- 19.9) hours respectively. The main symptoms were diarrhoea, fever and abdominal pain. The mean age of the cases was 43.42 (+/- 17.03) years old for outbreak A and 42.4 (+/- 19.9) for outbreak B. The statistical analysis showed a significant association between the intake of cake and the disease (adjusted odds ratio = 25.00; 95% confidence interval = 6,4996.15 for outbreak A and adjusted odds ratio = 64.62; 95% confidence interval = 8.10-515.3 for outbreak B). We also obtained a laboratory confirmation showing the presence of Salmonella Entérica I, Infantis 6.7:r:1.5 in samples of cases, cake and samples of the person who prepared the cake. CONCLUSIONS: The logistic regression model, used for the analysis was useful to detect and summarise data in a more efficient manner than simple stratified analysis. The collaboration of the Primary Health Care professionals contributed to the success of the investigation.

Chronic hepatitis in patients with hemophilia A: histologic studies in patients with intermittently abnormal liver function tests.

Recent studies in multiply transfused patients with hemophilia A and persistent liver function abnormalities have shown a high incidence of chronic active hepatitis. The purpose of the present study was to determine the severity of liver disease in multiply transfused patients with intermittent liver enzyme abnormalities. Fifteen patients with elevated enzymes on two or three out of four determinations at 6-mo intervals were studied. None had signs or symptoms of chronic liver disease. Thirteen had serologic evidence of prior exposure to the hepatitis B virus. Liver biopsy performed on these patients after replacement therapy with factor VIII showed chronic persistent hepatitis or other mild forms of liver disease in 14 of the 15 patients. Patients with chronic persistent hepatitis had significantly higher mean liver enzymes at time of biopsy than patients with milder forms of hepatic inflammation, but there was no relationship between liver histology and hepatitis B serology or the amount of factor VIII used in the 6 mo preceding biopsy. These findings support the continued use of factor VIII concentrates in patients with hemophilia.

Amyloidosis and factor X deficiency.

The association of amyloidosis and factor X deficiency has been reported 18 times. We have added a 19th case and compiled a summary of all previously reported cases. The pathogenesis of this association is discussed and therapeutic recommendations are offered based on the accumulated experience.