Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.     

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1)

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.     

Molecular simulations of intermediate and long alkanes adsorbed on graphite: Tuning of non-bond interactions

The interplay between the torsional potential energy and the scaling of the 1-4 van der Waals and Coulomb interactions determines the stiffness of flexible molecules. In this paper we demonstrate for the first time that the precise value of the nonbond scaling factor (SF)—often a value assumed without justification—has a significant effect on the critical properties and mechanisms of systems undergoing a phase transition, and that, for accurate simulations, this scaling factor is highly dependent on the system under consideration. In particular, by analyzing the melting of n-alkanes (hexane C6, dodecane C12, tetracosane C24) on graphite, we show that the SF is not constant over varying alkane chain lengths when the structural correlated transformations are concerned. Instead, monotonic decrease of SF with the molecular length drives a cross-over between two distinct mechanisms for melting in such systems. In a broad sense we show that the choice for SF in any simulation containing adsorbed or correlated long molecules needs to be carefully considered.     

Identification of Treponema denticola in subgingival samples by PCR technology and its correlation with clinical diagnosis

Treponema denticola has been associated with gingivitis and chronic periodontitis. The aim of this study was to identify Treponema denticola in subgingival samples using PCR technology and to correlate it with clinical diagnosis of subjects. The study was carried out on seventy patients (20-84 years of age; mean age, 45.06 +/- 12.58) of which 22 individuals with no detectable gingivitis or periodontitis, 4 subjects with chronic gingivitis and 44 subjects with chronic periodontitis. Subgingival plaque samples were collected from five sites in each patient. DNA was extracted from the samples using QIAamp DNA Mini Kit (QIAGEN). Treponema denticola and other four periodontopathogens were found using multiplex polymerase chain reaction followed by a reverse hybridization. The relationship between clinical diagnoses and detection of Treponema denticola was determined with Fisher exact test. The results showed significant differences between diagnostic groups regarding subject proportion. Treponema denticola was detected in 2 out of 22 subjects with no detectable gingivitis or periodontitis, 2 out of 4 subjects with chronic gingivitis, and 40 out of 44 subjects with chronic periodontitis. Our findings suggest that Treponema denticola is closely connected to the initiation and progression of periodontal disease.     

Hepatitis B, C and D coinfection in HIV-infected patients: prevalence and progress

The HAART therapy has improved life expectancy enabling long latency conditions caused by the hepatitis viruses that became the leading cause of death in HIV infected patients. In this study a group of 300 patients aged from 18 to 63 years were selected in order to assess the prevalence and consequences of HIV and the hepatitis B (HBV), C (HCV) and D (HDV) viruses coinfections. Study groups were designed for each coinfection. These groups were in turn divided in case groups formed of coinfected participants and control groups consisting of mono-infected participants. This classification was obtained by testing the participants for the presence of specific infection markers using the ELISA technique. As a result, in regard to the HIV/HBV coinfection the study group consisted of 16 coinfected participants and 114 HBV-infected participants resulting in a prevalence of the coinfection of 14%. In the case of the HIV/HDV coinfection the study group consisted of 5 coinfected participants and 45 HDV-infected participants. The prevalence of the HIV/HCV coinfection was 25% out of the 170 HCV-infected participants. The effect of the coinfections on the expression and levels of the infection markers was analyzed in constrast to those encountered in the case of the mono-infection. The observed changes in the expression of the specific hepatitis markers indicate the impact of the coinfection with HIV on the progression of the hepatitis infections. In addition, the inadequate immune response towards the hepatitis viruses in the case of the coinfected participants leads to the development of cirrhosis and end stage liver disease.     

miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response

Although there is evidence that redox regulation has an essential role in malignancies, its impact on tumor prognosis remains unclear. Here we show crosstalk between oxidative stress and the miR-200 family of microRNAs that affects tumorigenesis and chemosensitivity. miR-141 and miR-200a target p38α and modulate the oxidative stress response. Enhanced expression of these microRNAs mimics p38α deficiency and increases tumor growth in mouse models, but it also improves the response to chemotherapeutic agents. High-grade human ovarian adenocarcinomas that accumulate miR-200a have low concentrations of p38α and an associated oxidative stress signature. The miR200a-dependent stress signature correlates with improved survival of patients in response to treatment. Therefore, the role of miR-200a in stress could be a predictive marker for clinical outcome in ovarian cancer. In addition, although oxidative stress promotes tumor growth, it also sensitizes tumors to treatment, which could account for the limited success of antioxidants in clinical trials.     

Habitat use and dispersal characteristic by <Emphasis Type="Italic">Stethophyma grossum</Emphasis>: the role of habitat isolation and stable habitat conditions towards low dispersal

Species in a highly fragmented environment, such as the intensively used agricultural landscapes of Europe, are expected to be in danger of extinction. We hypothesize according to Kisdi’s theory (Am Nat 159:579–596, 2002) that species in fragmented landscapes with isolated habitats in general tend to possess low dispersal. In order to verify this hypothesis we studied the movement patterns of Stethophyma grossum, a hygrophilous species of wetlands, by mark–release–recapture techniques in a landscape with scattered suitable habitats over 3 years. The study focused on the major population in this landscape (site #1) as dispersal behaviour was assumed to be greatest. Actually, marked individuals of S. grossum were never found in any further suitable habitats in close vicinity to site #1. Despite that the peatland meadow of study site #1 was all over covered with homogenous vegetation only 6% (1.8 ha) of the whole area (30 ha) were occupied by S. grossum. The mean recapture rate over 3 years amounted to 39% with no significant differences between males and females. Both covered little distances within their mean range size of 1.8 ha; the median distances were 36.91 m for males and 26.65 m for females. We confirm the hypothesis that sub-populations of species in longstanding naturally isolated habitats, which habitat conditions have been stable; evolved low dispersal with little movements which are routine movements to find mating partners or food.     

New approaches to disease mapping in admixed populations

Admixed populations such as African Americans and Hispanic Americans are often medically underserved and bear a disproportionately high burden of disease. Owing to the diversity of their genomes, these populations have both advantages and disadvantages for genetic studies of complex phenotypes. Advances in statistical methodologies that can infer genetic contributions from ancestral populations may yield new insights into the aetiology of disease and may contribute to the applicability of genomic medicine to these admixed population groups.     

Hypoxic alterations of cortisol circadian rhythm in man after simulation of a long duration flight

Fatigue is often reported after long duration flights. Mild hypobaric hypoxia caused by pressurisation may be involved in this effect through disruption of circadian rhythms, independently of the number of time zones crossed. In this controlled crossover study, we assessed the effects of two levels of hypoxia equivalent to 8000 and 12,000 ft on the circadian rhythm of plasma cortisol, a marker of the circadian time structure. Sixteen healthy young male volunteers (23-39 years) were exposed in a hypobaric chamber for 8 h (08:00-16:00 h) to 8000 ft, followed 4 weeks later to 12,000 ft. Plasma cortisol was assayed during two 24-h cycles (control and hypoxic exposure) every 2h in all subjects. We found a significant change in the pattern of cortisol secretion during both hypoxic exposures, with an initial fall in cortisol followed by a transient rebound, whereas the phase and the 24-h mean level remained unchanged. The change in cortisol pattern followed the alterations in autonomic balance assessed by heart rate variability (HRV) spectral analysis. The normalised high frequencies and the low-to-high frequencies ratio showed a significant shift toward sympathetic dominance with some differences in time course for both altitudes studied. HRV analysis improved the interpretation of cortisol 24-h profiles. Our data, which strongly suggest that prolonged mild hypoxia alters the expression of cortisol circadian rhythm, should be taken into account to interpret secretory rhythm changes after transmeridian flights.     

A bacterial model for studying effects of human mutations in vivo: Escherichia coli strains mimicking a common polymorphism in the human MTHFR gene

A simple bacterial model for studying effects of human mutations in vivo, when homologous genes exist in bacterial and human cells, is presented. We have constructed Escherichia coli strains bearing different alleles of the metF gene, an ortologue of human MTHFR gene, coding for 5,10-methylenetetrahydrofolate reductase. These strains bear a null mutation in the chromosomal metF gene and different metF alleles on plasmid(s), and thus there are merozygotes mimicking wild-type homozygotes, heterozygotes and recessive mutant homozygotes. The A177V mutantion in metF corresponds to one of the most common MTHFR polymorphism, A222V, which has been shown to be associated with increased levels of homocysteine in plasma that, in turn, causes many serious medical problems. Results of relatively simple and quick experiments with these strains are compatible with previously published reports on effects of the A222V substitution in the product of MTHFR gene. In addition, these results suggest either impairment of formation of heterodimers and/or heterotetramers by wild-type and A177V metF variants or dominance of the wild-type polypepides in such structures. Moreover, positive effects of folic acid and vitamins B2 and B12 on physiology of the mutant cells, suggested on the basis of clinical studies, is confirmed. Therefore, we conclude that the bacterial model described in this report may be a useful tool in studies on human mutations.