Impact of medical imaging on the epigenome - low-dose exposure in the course of computed tomography does not induce detectable changes of DNA-methylation profiles in peripheral blood cells.

BACKGROUND: Computed tomography (CT) is a main contributor to artificial low-dose exposure. Understanding the biological effects induced by CT exposure and their dependency on the characteristics of photon spectra is essential for knowledge-driven risk assessment. In a previous gene expression study, we have identified upregulation of AEN, BAX, DDB2, EDA2R and FDXR after ex vivo exposure with single-energy CT and dual-energy CT (DECT). In this study, we focused on CT-induced changes of DNA methylation. This epigenetic modification of DNA is a central regulator of gene expression and instrumental in preserving genome integrity. Previous studies reported focal hypermethylation and global hypomethylation after exposure with doses above 100 mSv, however, the effect of low dose exposure on DNA methylation is hardly explored. MATERIALS AND METHODS: DNA was isolated from peripheral blood of three healthy individuals 6 h after ex vivo exposition to single-energy (80 kV and 150 kV) and DECT (80 kV/Sn150 kV) with a calculated effective dose of 7.0 ± 0.08 mSv. The experimental setting was identical to the one used in our previous gene expression study enabling a direct comparison of gene expression results with changes of DNA methylation identified in this study. DNA methylation was analyzed by high-throughput sequencing of bisulfite-treated DNA targeted methylation sequencing. RESULTS: Unsupervised hierarchical clustering based on DNA methylation profiles of all samples created three distinct clusters. Formation of these three clusters was solely determined by the origin of samples, indicating the absence of prominent irradiation-associated changes of DNA methylation. In line with this observation, inter-individual comparison of non-irradiated samples revealed 1163, 1224 and 4550 significant differentially methylated regions (DMRs), respectively, whereas the pairwise comparison of irradiated and non-irradiated samples failed to identify irradiation-induced DMRs in any of the three probands. This even applied to the genomic regions harboring AEN, BAX, DDB2, EDA2R and FDXR, the five genes known to be upregulated by CT exposure. CONCLUSIONS: CT exposure with various photon spectra did not result in detectable changes of DNA methylation. However, minor effects in a subpopulation of irradiated cells cannot be ruled out. Thus, future studies with extended observation intervals are needed to investigate DNA methylation changes that are induced by indirect effects at later points of time or become detectable by clonal expansion of affected cells. Moreover, our data suggest that DNA methylation analysis is less sensitive in detecting immediate effects of low-dose irradiation when compared to gene expression analysis.

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra.

Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79-18.26 mGy and dose length product (DLP) of 606.7-613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

Application of a Purified Protein From Natural Latex and the Influence of Suture Type on Achilles Tendon Repair in Rats.

BACKGROUND: The prolonged tendon-healing process, the high costs associated with treatment, the increase in the number of injuries over the past decades, and the lack of consensus on the optimal treatment of tendon injuries are a global problem. Restoring the normal tendon anatomy and decreasing the healing time are key factors for treatment advancement. HYPOTHESIS: Application of a purified protein from natural latex (PPNL) accelerates the healing process, increasing collagen synthesis and decreasing metalloproteinases. PPNL associated with a simpler suture technique should decrease the healing time. STUDY DESIGN: Controlled laboratory study. METHODS: Injury, surgery, and treatment with PPNL were conducted with male Sprague-Dawley rats. Two suture techniques were used: U-suture, a simpler and lesser traumatic technique, and Kessler-Tajima, to avoid strangulation of the microcirculation. Achilles tendons were completely sectioned, and 100 µL of 0.1% PPNL was applied on the tendon during surgery. Tendon morphology, distribution, and quantity of collagen types I and III, as well as expression of TIMP-1, TIMP-2, MMP-2, and MMP-9 and ultrastructural aspects of cells and collagen fibrils, were assessed after 2 and 4 weeks. RESULTS: PPNL treatment improved collagen type I synthesis and reduced MMP-2 expression. All groups showed a 6.8-times increase in tendon weight as compared with the control group after 2 weeks and a 5.2-times increase after 4 weeks. All groups showed an increase in diameter after 4 weeks, except for the ones treated with PPNL, which showed a slight reduction in diameter. The peak of concentration of collagen fibrils with a 80-nm diameter was 27.79% in the control group; all other experimental groups presented fibrils between 50 and 60 nm. However, the best results were observed with Kessler-Tajima suture associated with PPNL. CONCLUSION/CLINICAL RELEVANCE: There are no known medicines or substances capable of aiding the tendon healing process besides surgery. The discovery of a substance able to improve this process and decrease its duration represents an important advancement in orthopaedic medicine.

Morphological Characterization of the Myenteric Plexus of the Ileum and Distal colon of Dogs Affected by Muscular Dystrophy.

Duchenne-like muscular dystrophy (canine dystrophinopathy) is a hereditary degenerative disease characterized by muscle changes similar to those described for Duchenne muscular dystrophy (DMD) and by alterations in the smooth muscles of the gastrointestinal tract. Some authors have suggested that these abnormalities may be associated with intestinal motility. This study analyzed the nitrergic and cholinergic neurons and P2X7 receptor expression in the myenteric plexus of the ileum and distal colon of dogs with muscular dystrophy. Immunohistochemical techniques were used to detect nitric oxide synthase (NOS) and acetylcholine transferase (ChAT) expression and to label all HuC/D- and P2X7 receptor-immunoreactive (IR) neurons. Transmission electron microscopy and basic histology were performed for further analysis. The results showed that nitrergic neurons exhibited a Dogiel type I morphology in the ileum and distal colon. The neuronal profile results showed that there were fewer NOS-, ChAT-, and HuC/D-IR neurons in the ileum than in the distal colon in the dystrophic (DT) dogs. Additionally, there were more NOS-, ChAT- and HuC/D-IR neurons per ganglion in the distal colon than in the ileum. The P2X7 receptor-expressing neurons colocalized with nitrergic and cholinergic neurons. Transmission and light microscopy revealed collagen between the muscle fibers, between the circular and longitudinal muscle layers and within the myenteric ganglia of dogs with muscular dystrophy. These findings provide a morphological description of the myenteric neurons in the ileum and distal colon of these DT dogs and may contribute to a better understanding of the gastrointestinal disorders found in patients with DMD. Anat Rec, 301:673-685, 2018. © 2017 Wiley Periodicals, Inc.

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice (ob/ob).

AIM: To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice. METHODS: The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method. RESULTS: The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm(2)) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG. CONCLUSION: Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons.

Morphological observations of ampullae of lorenzini in Squatina guggenheim and S. occulta (Chondrichthyes, Elasmobranchii, Squatinidae).

We have conducted a morphological study of the ampullae of Lorenzini on two shark species from Squatina Genus. In both species, S. guggenheim and S. occulta, the ampullae were observed like small pores scattered in the head region similar to other species of the Chondrichthyes Class. However, differently of the other species a greatest density of ampullae of Lorenzini was observed along of the body surface. After fixation using 10% formaldehyde, the ampullae were removed and processed for light and scanning electron microscopy. Macroscopically, the two shark species differed by the presence of dorsal spines that appeared from the head to the first dorsal fin in S. guggenheim and were absent in S. occulta. Microscopically, there were no differences between the ampullae of Lorenzini channels in these two species. The wall of the ampulla was formed by a simple squamous epithelium. Bands of connective tissue, hyaline cartilage and collagen fibers were found between the ampulla and the skeletal striated muscle layer. Nerve branches responsible for conducting signal pulses to the central nervous system were visible between the muscle and connective tissue layers. Using scanning electron microscopy and histological analysis, we found that the channels were twisted and positioned parallel to the skin. The inside of the channels contained a large amount of a gelatinous secretion composed by polysaccharides. Therefore, we conclude that the morphological combination of extended distribution of the ampullae of Lorenzini and the body shape may represent an adaptation of these species to their way of life.

LOST MERISTEMS genes regulate cell differentiation of central zone descendants in Arabidopsis shoot meristems.

Meristems of seed plants continuously produce new cells for incorporation into maturing tissues. A tightly controlled balance between cell proliferation in the center and cell differentiation at the periphery of the shoot meristem maintains its integrity. Here, we describe the role of three GRAS genes, named LOST MERISTEMS genes, in shoot apical meristem maintenance and axillary meristem formation. Under short photoperiods, the lom1 lom2 and lom1 lom2 lom3 mutants have arrested meristems characterized by an over-proliferation of meristematic cells and loss of polar organization. They also show early arrest of axillary meristem development and formation of ectopic meristematic cell clusters within the stem. LOM1 and LOM2 transcripts accumulate in the peripheral and basal zones of the SAM and in vascular strands. We show that LOM1 and LOM2 promote cell differentiation at the periphery of shoot meristems and help to maintain their polar organization.

Killing of adherent oral microbes by a non-thermal atmospheric plasma jet.

Atmospheric plasma jets are being intensively studied with respect to potential applications in medicine. The aim of this in vitro study was to test a microwave-powered non-thermal atmospheric plasma jet for its antimicrobial efficacy against adherent oral micro-organisms. Agar plates and dentin slices were inoculated with 6 log(10) c.f.u. cm(-2) of Lactobacillus casei, Streptococcus mutans and Candida albicans, with Escherichia coli as a control. Areas of 1 cm(2) on the agar plates or the complete dentin slices were irradiated with a helium plasma jet for 0.3, 0.6 or 0.9 s mm(-2), respectively. The agar plates were incubated at 37 degrees C, and dentin slices were vortexed in liquid media and suspensions were placed on agar plates. The killing efficacy of the plasma jet was assessed by counting the number of c.f.u. on the irradiated areas of the agar plates, as well as by determination of the number of c.f.u. recovered from dentin slices. A microbe-killing effect was found on the irradiated parts of the agar plates for L. casei, S. mutans, C. albicans and E. coli. The plasma-jet treatment reduced the c.f.u. by 3-4 log(10) intervals on the dentin slices in comparison to recovery rates from untreated controls. The microbe-killing effect was correlated with increasing irradiation times. Thus, non-thermal atmospheric plasma jets could be used for the disinfection of dental surfaces.

Autologous stem cell transplant in 716 patients with multiple myeloma: low treatment-related mortality, feasibility of outpatient transplant, and effect of a multidisciplinary quality initiative.

We report on the feasibility of outpatient transplant in 716 patients undergoing autologous stem cell transplant for multiple myeloma at Mayo Clinic's site in Rochester, MN, from January 1, 2000, through October 31, 2007. We also report on the development and effect of a multidisciplinary quality initiative implemented by the Mayo Clinic Blood and Marrow Transplant Program involving physicians, nurses, pharmacists, dietitians, and financial specialists for outpatient management of patients undergoing stem cell transplant. This approach uses an electronic ordering system for diagnostic tests and chemotherapy to minimize medical errors. Analysis of hospitalization trends since inception of the program showed that 278 (39%) of the 716 patients treated completed the transplant procedure as outpatients. The median duration of hospitalization for all patients was 4 days; age and serum creatinine levels were predictive of the need for and duration of hospitalization. We also assessed recent treatment-related mortality rates during a 33-month period after implementation of the program (between January 1, 2005, and October 1, 2007). The 100-day survival rate was 99.5% for patients with low-risk myeloma (transplant during first plateau; n=201) and 97.2% for patients with high-risk myeloma (refractory, relapsing or second or greater plateau; n=71). The overall 100-day survival rate was 98.9%. Our experience shows that outpatient transplant is feasible for all patients with multiple myeloma and results in shorter hospital stays and low treatment-related mortality rates.

The kinesin I family member KIF5C is a novel substrate for protein kinase CK2.

Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic alpha- or alpha'-subunits and two regulatory beta-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2alpha' in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2alpha/CK2beta and CK2alpha'/CK2beta as well as by CK2alpha' alone but not by CK2alpha alone.