Engineering custom-designed osteochondral tissue grafts

Tissue engineering is expected to help us outlive the failure of our organs by enabling the creation of tissue substitutes capable of fully restoring the original tissue function. Degenerative joint disease, which affects one-fifth of the US population and is the country's leading cause of disability, drives current research of actively growing, functional tissue grafts for joint repair. Toward this goal, living cells are used in conjunction with biomaterial scaffolds (serving as instructive templates for tissue development) and bioreactors (providing environmental control and molecular and physical regulatory signals). In this review, we discuss the requirements for engineering customized, anatomically-shaped, stratified grafts for joint repair and the challenges of designing these grafts to provide immediate functionality (load bearing, structural support) and long-term regeneration (maturation, integration, remodeling).     

The impact of long-term past exposure to elemental mercury on antioxidative capacity and lipid peroxidation in mercury miners

Limited information is available on the effects of chronic mercury exposure in relation to the risk of cardiovascular disease (CVD). It is known from in vitro and in vivo studies that Hg can promote lipid peroxidation through promotion of free radical generation, and interaction with antioxidative enzymes and reduction of bioavailable selenium. The objective of the study was to test the hypothesis that long-term past occupational exposure to elemental Hg (Hg⁰) can modify antioxidative capacity and promote lipid peroxidation in miners.

The study population comprised 54 mercury miners and 58 workers as the control group. The miners were examined in the post-exposure period. We evaluated their previous exposure to Hg⁰, the putative appearance of certain nonspecific symptoms and signs of micromercurialism, as well as the main behavioural and biological risk factors for CVD, and determined: 1) Hg and Se levels in blood and urine, 2) antioxidative enzymes, Cu/Zn superoxide dismutase (CuZn-SOD), catalase (CAT), and selenoenzyme glutathione peroxidase (GSH-Px) activity in erythrocytes as indirect indices of free radical activity, 3) pineal hormone melationin (MEL) in blood and urine, and 4) lipid hydroperoxides (LOOHs) and malondialdehyde (MDA) as lipid peroxidation products.

The mercury miners were intermittently exposed to Hg⁰ for periods of 7 to 31 years. The total number of exposure periods varied from 13 to 119. The cumulative U-Hg peak level varied from 794-11,365 μg/L. The current blood and urine Hg concentrations were practically on the same level in miners and controls. Miners showed some neurotoxic and nephrotoxic sequels of micromercurialism. No significant differences in behavioural and biological risk factors for CVD were found between miners and controls. A weak correlation (r = 0.36, p < 0.01) between systolic blood pressure and average past exposure U-Hg level was found. The mean P-Se in miners (71.4 μg/L) was significantly lower (p < 0.05) than in the controls (77.3 μg/L), while the mean U-Se tended to be higher (p < 0.05) in miners (16.5 μg/g creatinine) than in the controls (14.0 μg/g creatinine). Among antioxidative enzyme activities, only CAT in erythrocytes was significantly higher (p < 0.01) in miners (3.14 MU/g Hb) than in the controls (2.65 MU/g Hb). The mean concentration of B-MEL in miners (44.3 ng/L) was significantly higher (p < 0.01) than in the controls (14.9 ng/L). The mean value of U-MEL sulphate (31.8 μg/L) in miners was significantly lower (p < 0.01) than in the control group (46.9 μg/L). Among the observed lipid peroxidative products, the mean concentration of U-MDA was statistically higher (p < 0.01) in miners (0.21 μmol/mmol creatinine) than in the controls (0.17 μmol/mmol creatinine).

In the group of miners with high mercury accumulation and the presence of some nonspecific symptoms and signs of micromercurialism, the results of our study partly support the assumption that long-term occupational exposure to Hg⁰ enhances the formation of free radicals even several years after termination of occupational exposure. Therefore, long-term occupational exposure to Hg⁰ could be one of the risk factors for increased lipid peroxidation and increased mortality due to ischaemic heart disease (ICH) found among the mercury miners of the Idrija Mine.     

Reactivity of pyrimidine nucleosides under the conditions of the pyrimidine sequencing reactions of Maxam and Gilbert

Several pyrimidine nucleosides and polydeoxyribonucleotides have been examined with respect to their ultraviolet absorption spectra and the kinetics of their decomposition under the conditions used in the C- and (C + T)-specific treatments of the sequencing procedure of Maxam and Gilbert. In hydrazine-water (56:44, v/v), at 20 degrees C, thymidine was found to be virtually fully ionized. Under these conditions, the susceptibility to hydrazinolysis decreased in the order N3-methylthymidine greater than uridine approximately equal to deoxyuridine greater than deoxycytidine approximately equal to polydeoxycytidylate greater than polythymidylate much greater than thymidine greater than 5-methyldeoxycytidine. Addition of sodium chloride to 1.5 M slightly accelerated hydrazinolysis of those nucleosides bearing nonionizable heterocyclic moieties and retarded hydrazinolysis severalfold for those nucleosides containing an ionizable aglycone (thymidine, deoxyuridine, uridine). It is concluded that the nucleosides of this latter class react with hydrazine largely or exclusively in their nonionized form and that the main effect of added salt is increased ionization of the heterocycles, resulting in a decreased population of nonionized nucleosides.     

Extracellular Vesicle-Mediated Transfer of Genetic Information between the Hematopoietic System and the Brain in Response to Inflammation

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.     

The Influence of Very Low Illumination on the Postural Sway of Young and Elderly Adults

The purpose of the present study was to evaluate the influence of very low ambient illumination and complete darkness on the postural sway of young and elderly adults. Eighteen healthy young participants aged 23.8±1.5 years and 26 community-dwelling elderly aged 69.8±5.6 years were studied. Each participant performed four tests while standing on a force platform in the following conditions: in normal light (215 lx) with open eyes and with closed eyes, in very low illumination (0.25 lx) with open eyes, and in complete darkness with open eyes. The sequences of the tests in the altered visual conditions were determined by random blocs. Postural sway was assessed by means of the force platform measurements. The centre of pressure variables: the medio-lateral and antero-posterior path lengths, mean velocities, sway areas, and fractal dimensions were analysed. Very low illumination resulted in a statistically significant increase in postural sway in both the young and elderly groups compared to normal light, although the increase was significantly smaller than those observed in the eyes closed and complete darkness condition, and no significant effects of illumination on fractal dimensions were detected. The gains of the sways in the very low or no illumination conditions relative to the normal light condition were significantly larger in the group of young participants than in the group of elderly participants (up to 50% and 25%, respectively). However, the response patterns to changes in illumination were similar in the young and elderly participants, with the exception of the short-range fractal dimension of the medio-lateral sway. In conclusion, very low illumination resulted in increased postural sway compared to normal illumination; however, in the closed eye and complete darkness conditions, postural sway was significantly higher than in the very low illumination condition regardless of the age of the participants.     

New cytotoxic steroids from the soft coral Clavularia viridis

Ten new cytotoxic steroids, stoloniferones H-Q (1-10) were isolated from the methylene chloride solubles of the soft coral Clavularia viridis. The structures of the metabolites were elucidated on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analysis and their cytotoxicity against selected cancer cells was measured in vitro.     

Heterogeneity in expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> colicin K activity gene <Emphasis Type="Italic">cka</Emphasis> is controlled by the SOS system and stochastic factors

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.     

Correlation of Culture Positivity,PCR Positivity,and Burden of Borrelia burgdorferi Sensu Lato in Skin Samples of Erythema Migrans Patients with Clinical Findings

Background

Limited data are available regarding the relationship of Borrelia burden in skin of patients with erythema migrans (EM) and the disease course and post-treatment outcome.

Methods

We studied 121 adult patients with EM in whom skin biopsy specimens were cultured and analyzed by quantitative PCR for the presence of Borreliae. Evaluation of clinical and microbiological findings were conducted at the baseline visit, and 14 days, 2, 6, and 12 months after treatment with either amoxicillin or cefuroxime axetil.

Results

In 94/121 (77.7%) patients Borrelia was detected in skin samples by PCR testing and 65/118 (55.1%) patients had positive skin culture result (96.8% B. afzelii, 3.2% B. garinii). Borrelia culture and PCR results correlated significantly with the presence of central clearing and EM size, while Borrelia burden correlated significantly with central clearing, EM size, and presence of newly developed or worsened symptoms since EM onset, with no other known medical explanation (new or increased symptoms, NOIS). In addition, the logistic regression model for repeated measurements adjusted for time from inclusion, indicated higher Borrelia burden was a risk factor for incomplete response (defined as NOIS and/or persistence of EM beyond 14 days and/or occurrence of new objective signs of Lyme borreliosis). The estimated association between PCR positivity and unfavorable outcome was large but not statistically significant, while no corresponding relationship was observed for culture positivity.

Conclusions

Higher Borrelia burden in EM skin samples was associated with more frequent central clearing and larger EM lesions at presentation, and with a higher chance of incomplete response.