Prevalence of Bacterial Contamination of Casting Material in a Pediatric Population.

Surgical site infection is a relatively common and devastating complication following pediatric orthopedic surgery. Many infections have been determined to be the result of settled airborne particles on surgical equipment and the sterile field. Fiberglass casts are commonly used orthopedic fixation devices before and after surgery; however, fiberglass casting material is expelled during the removal process and represents an uninvestigated area for the possibility of cast saw dust as a source of airborne bacterial contamination in an operating room setting. This study evaluates the prevalence and distribution of microbiota on 90 pediatric casts by collecting and culturing fiberglass cast material from 90 pediatric casts. Bacterial identification was performed using a Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry device. 81 out of 90 casts (90%) showed evidence of microbial contamination. Isolated species were very diverse and ranged from normal skin flora to opportunistic pathogens. The 5 most commonly isolated organisms were Acinetobacter pittii, Enterobacter cloacae, Micrococcus luteus, Staphylococcus epidermidis, and Staphylococcus hominis. Further investigation is required to determine if casting material is truly a cause of surgical site infection.

Use of Raman spectroscopy to decrease time for identifying the species of Candida growth in cultures.

BACKGROUND: The objective of this study is to establish Raman signatures from pure cultures of different Candida species using Raman Spectroscopy (RS) and use these signatures for rapid identification of unknown Candida species. METHODS: Pure cultures of five Candida species were evaluated using RS to build a limited signature library. 'Raman Processing' (RP) software was used for Principal Component Analysis (PCA) and Differential Functional Analysis (DFA). RESULTS: Eleven principal components described at least 95% variance in the spectra. Raman signatures from these known Candida species were able to identify the species of unknown Candida cultures with 100% accuracy. CONCLUSION: Raman spectroscopy can improve early identification of Candida species and may facilitate early optimal antifungal therapy.

Raman spectroscopy in the diagnosis of ulcerative colitis.

INTRODUCTION: At present, the diagnosis of ulcerative colitis (UC) requires the histologic demonstration of characteristic mucosal inflammatory changes. A rapid and noninvasive diagnosis would be of value, especially if it could be adapted to a simple rectal probe. Raman spectroscopy creates a molecular fingerprint of substances by detecting laser light scattered from asymmetric, vibrating, and chemical bonds. We hypothesize that Raman spectroscopy can distinguish UC from non-UC colon tissue rapidly and accurately. MATERIALS AND METHODS: Colon tissue specimens were obtained from patients operated at the Children's Hospital of Michigan, United States, including UC colon and non-UC colon. The samples were examined with a Renishaw inVia Raman microscope (Gloucestershire, United Kingdom) with a 785 nm laser. Principal component analysis and discriminant function analysis were used to classify groups. Final classification was evaluated against histologic diagnoses using leave-one-out cross-validation at a spectral level. RESULTS: We compared Raman spectroscopy examination of colon specimens from four patients with UC and four patients without UC. A total of 801 spectra were recorded from colon specimens. We evaluated 100 spectra each from the mucosal and serosal surfaces of patients with UC and 260 spectra from the mucosal surface and 341 spectra from the serosal surface of the patients who did not have UC. For samples from the mucosal surface, the Raman analysis had a sensitivity of 82% and a specificity of 89%. For samples from the serosal surface, Raman spectroscopy had a sensitivity of 87% and a specificity of 93%. When considering each tissue sample and deciding the diagnosis based on the majority of spectra from that sample, there were no errors in the diagnosis. CONCLUSIONS: Raman spectroscopy can distinguish UC from normal colon tissue rapidly and accurately. This technology offers the possibility of real-time diagnosis as well as the ability to study changes in UC-afflicted colon tissue that do not appear histologically.

Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression.

The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.

Vision 20/20: the role of Raman spectroscopy in early stage cancer detection and feasibility for application in radiation therapy response assessment.

Raman spectroscopy is an optical technique capable of identifying chemical constituents of a sample by their unique set of molecular vibrations. Research on the applicability of Raman spectroscopy in the differentiation of cancerous versus normal tissues has been ongoing for many years, and has yielded successful results in the context of prostate, breast, brain, skin, and head and neck cancers as well as pediatric tumors. Recently, much effort has been invested on developing noninvasive "Raman" probes to provide real-time diagnosis of potentially cancerous tumors. In this regard, it is feasible that the Raman technique might one day be used to provide rapid, minimally invasive real-time diagnosis of tumors in patients. Raman spectroscopy is relatively new to the field of radiation therapy. Recent work involving cell lines has shown that the Raman technique is able to identify proteins and other markers affected by radiation therapy. Although this work is preliminary, one could ask whether or not the Raman technique might be used to identify molecular markers that predict radiation response. This paper provides a brief review of Raman spectroscopic investigations in cancer detection, benefits and limitations of this method, advances in instrument development, and also preliminary studies related to the application of this technology in radiation therapy response assessment.

Differentiation of alloreactive versus CD3/CD28 stimulated T-lymphocytes using Raman spectroscopy: a greater specificity for noninvasive acute renal allograft rejection detection.

Acute rejection (AR) remains problematic in renal transplantation. As a marker, serum creatinine is limited, warranting a more effective screening tool. Raman spectroscopy (RS) can detect T-cell activation with high sensitivity. In this study we explore its specificity. Seventy-five inactivated, 40 alloantigen-activated, and 75 CD3/CD28-activated T cells were analyzed using RS. CD3/CD28-activated peak magnitudes (PM) were 4.3% to 23.9% lower than inactivated PM at positions: 903, 1031, 1069, 1093, 1155, 1326, and 1449 cm(-1), with a difference in peak ratio (PR) observed at the 1182:1195 cm(-1) position (0.91 +/- 0.06 vs. 1.2 +/- 0.01, respectively: P = 0.006). Differences in CD3/CD28- and alloantigen-activated PM were observed at: 903, 1031, 1093, 1155, 1326, and 1449 cm(-1), with no PR differences at the 1182:1195 cm(-1) position (0.91 +/- 0.06 vs. 0.86 +/- 0.09: P = 0.8). Spectral signature separation of CD3/CD28-and alloantigen-activated groups was 100% specific and sensitive. We conclude that RS can differentiate T cells activated by different stimuli with high sensitivity and specificity.

Raman spectroscopic differentiation of activated versus non-activated T lymphocytes: an in vitro study of an acute allograft rejection model.

Acute rejection (AR) remains a significant complication in renal transplant patients. Using serum creatinine for AR screening has proven problematic, and thus a noninvasive, highly sensitive and specific test is needed. T cells from human peripheral blood were analyzed using Raman spectroscopy. Fifty-one Mixed Lymphocyte Culture (MLC) activated T cells (ATC), 28 Mitomycin C inactivated T cells (ITC), and 35 resting T cells (RTC), were studied utilizing 785 and 514.5 nm wavelengths. Statistical analysis following subtraction of fluorescence used Student's t test to quantify peak ratio differences and discriminant function analysis (DFA), with three distinct sectors assigned for grouping purposes: Sector I, ITC; Sector II, ATC; Sector III, RTC. Differences between ATC and non-activated T cells (ITC and RTC) were found at 1182 and 1195 cm-1 peak positions for both wavelengths. Significant differences in peak ratios for 785 and 514.5 nm wavelengths existed between ATC and RTC (p=0.001 and p=0.006, respectively) and ATC and ITC (p=0.001 and p=0.001, respectively), with a trend in differences observed between ITC and RTC (p=0.07 and p=0.08, respectively). Analysis of the DFA-derived sector distribution for the 785 and 514.5 nm wavelengths revealed a sensitivity of 95.7% and 89.3%, respectively, and a specificity of 100% and 93.8%, respectively. This data suggests that Raman spectroscopy can detect significant differences between activated and nonactivated T cells based upon cell-surface receptor expression, thereby establishing unique signatures that can aid in the development of a noninvasive AR screening tool with high sensitivity and specificity.

Molecular organization in SAMs used for neuronal cell growth.

The attachment of cells onto solid supports is fundamental in the development of advanced biosensors or biochips. In this work, we characterize cortical neuron adhesion, growth, and distribution of an adhesive layer, depending on the molecular structure and composition . Neuronal networks are successfully grown on amino-terminated alkanethiol self-assembled monolayer (SAM) on a gold substrate without adhesion protein interfaces. Neuron adhesion efficiency was studied for amino-terminated, carboxy-terminated, and 1:1 mixed alkanethiol SAMs deposited on gold substrates. Atomic force microscopy and X-ray photoelectron spectroscopy were used to measure the roughness of gold substrate and thickness of SAM monolayers. Conformational ordering and ionic content of SAMs were characterized by vibrational sum frequency generation (VSFG) spectroscopy. Only pure amino-terminated SAMs provide efficient neuronal cell attachment. Ordering of the terminal amino groups does not affect efficiency of neuron adhesion. VSFG analysis shows that ordering of the terminal groups improves with decreasing surface roughness; however the number of gauche defects in alkane chains is independent of surface roughness. We monitor partial dissociation of carboxy groups in mixed SAMs that implies formation of NH3+ neighbors and appearance of catanionic structure. Such catanionic environment proved inefficient for neuron adhesion. Surface roughness of metal within the 0.7-2 nm range has little effect on the efficiency of neuron adhesion. This approach can be used to create new methods that help map structure-property relationships of biohybrid systems.

A neural cell culture study on thin film electrode materials.

Functional neural stimulation requires good interface between the neural cells and the electrode surfaces. In order to study the effect of electrode materials and surface structure on cell adhesion and biocompatibility, we cultured cortical neurons on thin films of platinum and iridium oxide. We used both flat, as-deposited and laser micro-structured films. The laser micro-structuring consisted of creating regular arrays of micro-bumps or holes with diameters of 4-5 mum and height of about 1.5 mum. The micro-bumps were fabricated onto platinum and iridium film surfaces deposited on borosilicate glass substrates, using mask-projection irradiation with single nano-second pulses from a KrF excimer laser (lambda = 248 nm). Amorphous and crystalline (deposited at 250 degrees C) IrO(2) films were deposited onto the laser micro-structured iridium films by pulsed-DC reactive sputtering to obtain micro-structured IrO(2) films. Cortical neurons isolated from rat embryo brain were cultured onto these film surfaces. Our results indicate that flat and micro-structured film surfaces are biocompatible and non-toxic for neural cell growth. The use of poly-D: -lysine as a mediator for cell adhesion onto the thin film surfaces is also discussed.

Culturing neuron cells on electrode with self-assembly monolayer.

The success of neuronal implantable microsystems relies on the quality of the interface with neuronal cells. Depending on the application, specifically engineered surfaces may either prevent or enhance cell/tissue growth with an appropriate host response. The surface chemistry and topography have major effects on the cell adherence and the interaction between the tissue and devices. We report on a simple technique to precisely explant cortical neurons in a serum-free medium on 2D electrode arrays and investigated the pad size effect on neuron cell culture and immobilization. We produced gold patterns on glass substrates using microfabrication processes. 11-Amino-1-undecanethiol self-assembled monolayer was coated only on the gold surface. Cortical neurons were cultured on the arrays to examine the dependence of neuron growth and cells distribution on pad size. We found that the terminal functional groups of the highly oriented 11-amino-1-undecanethiol thin film are essential for generating cell-adhesive areas for the rat cortical neurons. A 50 microm x 50 microm SAM pad size was found to be suitable for single cortical neuron immobilization, while the larger pads provide excellent neuron coverage. This technology may enable precise and localized neuron stimulation and surveillance for both biological research and medical applications.

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro.

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests.

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro.

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.

Genotoxicity of cyanobacterial extracts containing microcystins from Polish water reservoirs as determined by SOS chromotest and comet assay.

Toxicity of cyanobacterial blooms, an increasing problem around the world, is connected to the increase in bloom samples containing microcystins, caused by excessive eutrophication of drinking- and recreational water reservoirs. Microcystins are the most common group of cyanobacterial hepatotoxins. In Poland they are produced mainly by the Microcystis genus. The toxicity of microcystins has been well documented, but investigation into their genotoxicity has been insufficient relative to the study of their overall toxicity. Therefore, the aim of this study was the estimation and comparison of the genotoxicity of cyanobacterial extracts with microcystins (CEMs) using the SOS chromotest (bacterial test) with Escherichia coli PQ37 and the comet assay with human lymphocytes. Cyanobacterial bloom samples were collected in the summer months from two Polish water reservoirs, one at Sulejów and one at Jeziorsko. The SOS chromotest, which used prokaryotic cells (without metabolic activation), and the comet assay, which used eukaryotic cells, both indicated the potential genotoxic effect of CEMs. Cyanobacterial extracts caused DNA damage in human lymphocytes in vitro. The maximum level of DNA damage was observed after 12 h incubation with CEMs. The bacterial test indicated a dependence of the degree of CEM genotoxicity, the composition, and the concentration of microcystins in each bloom sample examined with the time of exposure. Differences between the genotoxicity of cyanobacterial extract and the standard microcystin-LR were noticeable. This was probably caused by the interaction of different microcystin variants. The results showed that CEMs from Polish water reservoirs were genotoxic, which was reflected by the stimulation of the SOS repair system in bacterial cells (SOS chromotest) and by the damage induced in DNA in human lymphocytes (comet assay).

Radiation-induced DNA damage and its repair in lymphocytes of patients with head and neck cancer and healthy donors.

BACKGROUND: DNA repair capacity may be an important factor in determining both individual susceptibility to cancer and the response to cancer therapy. The aim of this work was to compare DNA damage and the repair process in cells originating from healthy donors and cancer patients. MATERIALS AND METHODS: Using the micronucleus and comet assays, we compared the induction of DNA damage and its repair in lymphocytes isolated from blood samples of 14 healthy donors and 24 patients with head and neck tumours. Gamma-rays at the dose of 2 or 4 Gy were used as the damaging factor. The micronucleus test was performed according to Fenech (1) and the comet assay according to Green et al. (2). RESULTS AND CONCLUSION: Lymphocytes of both healthy donors and tumour patients showed great diversification in reaction to the same dose of gamma irradiation as well as differences in the kinetics of DNA repair. The patient group contained significantly more individuals whose lymphocytes were characterized by higher background DNA damage and higher damage inducibility. Blood cells of donors showing high damage inducibility also showed increased levels of micronuclei induced by ionizing radiation. Micronuclei induction did not correlate with a high level of unrepaired DNA damage.

Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement.

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells.

Lipid peroxidation, DNA damage, and cellular morphology of R1 Rhabdomyosarcoma cell line irradiated in vitro by gamma-rays with different dose rates.

The study examines the relationship between lipid peroxidation, DNA damage, and cell morphology after the exposure of R1 Rhabdomyosarcoma cells to two different dose-rates of gamma rays. Exponential cultures of R1 cells were irradiated with single dose of 5 Gy at high dose rate (0.833 Gy/min) and low dose rate (0.0707 Gy/min). The concentration of two aldehydes, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), were determined. DNA damage induction and repair were measured by using the alkaline version of the comet assay. Cellular alteration was also estimated microscopically as was the frequency of cells with micronuclei and proportion of apoptosis and necrosis. These parameters were evaluated immediately (time 0) and after different times up to 48 h of incubation in 37 degrees C, after irradiation. Results indicate that a low dose rate in comparison to high dose rate caused a significantly higher increase of aldehydes concentration observed at 12 h, followed by obviously higher DNA damage at 48 h and altered cellular morphology. The inverse dose-rate effect estimated for the gamma rays Co-60 source was found to be related to the measured biochemical and morphological parameters.