Simulation of Radiation-Induced DNA Damage and Protection by Histones Using the Code RITRACKS.

(1) Background: DNA damage is of great importance in the understanding of the effects of ionizing radiation. Various types of DNA damage can result from exposure to ionizing radiation, with clustered types considered the most important for radiobiological effects. (2) Methods: The code RITRACKS (Relativistic Ion Tracks), a program that simulates stochastic radiation track structures, was used to simulate DNA damage by photons and ions spanning a broad range of linear energy transfer (LET) values. To perform these simulations, the transport code was modified to include cross sections for the interactions of ions or electrons with DNA and amino acids for ionizations, dissociative electron attachment, and elastic collisions. The radiochemistry simulations were performed using a step-by-step algorithm that follows the evolution of all particles in time, including reactions between radicals and DNA structures and amino acids. Furthermore, detailed DNA damage events, such as base pair positions, DNA fragment lengths, and fragment yields, were recorded. (3) Results: We report simulation results using photons and the ions 1H+, 4He2+, 12C6+, 16O8+, and 56Fe26+ at various energies, covering LET values from 0.3 to 164 keV/µm, and performed a comparison with other codes and experimental results. The results show evidence of DNA protection from damage at its points of contacts with histone proteins. (4) Conclusions: RITRACKS can provide a framework for studying DNA damage from a variety of ionizing radiation sources with detailed representations of DNA at the atomic scale, DNA-associated proteins, and resulting DNA damage events and statistics, enabling a broader range of future comparisons with experiments such as those based on DNA sequencing.

Longitudinal intravital imaging of the bone marrow for analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Evidence of bisphosphonate-conjugated sitafloxacin eradication of established methicillin-resistant S. aureus infection with osseointegration in murine models of implant-associated osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Evidence of Bisphosphonate-Conjugated Sitafloxacin Eradication of Established Methicillin-Resistant S. aureus Infection with Osseointegration in Murine Models of Implant-Associated Osteomyelitis.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve "target-and-release" drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (bisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy, while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Epidemiological updates of post-traumatic related limb osteomyelitis in china: a 10 years multicentre cohort study.

BACKGROUND: Post-traumatic related limb osteomyelitis (PTRLO) is a complex bone infection. Currently, there are no available microbial data on a national scale that can guide appropriate antibiotic selection, and explore the dynamic changes in dominant pathogens over time. This study aimed to conduct a comprehensive epidemiological analysis of PTRLO in China. METHODS: The study was approved by the Institutional Research Board (IRB), and 3526 PTRLO patients were identified from 212 394 traumatic limb fracture patients at 21 hospitals between 1 January 2008 and 31 December 2017. A retrospective analysis was conducted to investigate the epidemiology of PTRLO, including changes in infection rate (IR), pathogens, infection risk factors and antibiotic resistance and sensitivity. RESULTS: The IR of PTRLO increased gradually from 0.93 to 2.16% (Z=14.392, P <0.001). Monomicrobial infection (82.6%) was significantly higher than polymicrobial infection (17.4%) ( P <0.001). The IR of Gram-positive (GP) and Gram-negative (GN) pathogens showed a significant increase from the lowest 0.41% to the highest 1.15% (GP) or 1.62% (GN), respectively. However, the longitudinal trend of GP vs. GN's composition did not show any significance (Z=±1.1918, P >0.05). The most prevalent GP strains were Methicillin-sensitive Staphylococcus aureus (MSSA) (17.03%), Methicillin-resistant Staphylococcus aureus (MRSA) (10.46%), E. faecalis (5.19%) and S. epidermidis (4.87%). In contrast, the dominant strains GN strains were Pseudomonas Aeruginosa (10.92%), E. cloacae (10.34%), E. coli (9.47%), Acinetobacter Baumannii (7.92%) and Klebsiella Pneumoniae (3.33%). In general, the high-risk factors for polymicrobial infection include opened-fracture (odds ratio, 2.223), hypoproteinemia (odds ratio, 2.328), and multiple fractures (odds ratio, 1.465). It is important to note that the antibiotics resistance and sensitivity analysis of the pathogens may be influenced by complications or comorbidities. CONCLUSIONS: This study provides the latest data of PTRLO in China and offers trustworthy guidelines for clinical practice. (China Clinical Trials.gov number, ChiCTR1800017597).

Reduced angiogenesis and delayed endochondral ossification in CD163-/- mice highlights a role of M2 macrophages during bone fracture repair.

While recent studies showed that macrophages are critical for bone fracture healing, and lack of M2 macrophages have been implicated in models of delayed union, functional roles for specific M2 receptors have yet to be defined. Moreover, the M2 scavenger receptor CD163 has been identified as a target to inhibit sepsis following implant-associated osteomyelitis, but potential adverse effects on bone healing during blockage therapy have yet to be explored. Thus, we investigated fracture healing in C57BL/6 versus CD163-/- mice using a well-established closed, stabilized, mid-diaphyseal femur fracture model. While gross fracture healing in CD163-/- mice was similar to that of C57BL/6, plain radiographs revealed persistent fracture gaps in the mutant mice on Day 14, which resolved by Day 21. Consistently, 3D vascular micro-CT demonstrated delayed union on Day 21, with reduced bone volume (74%, 61%, and 49%) and vasculature (40%, 40%, and 18%) compared to C57BL/6 on Days 10, 14, and 21 postfracture, respectively (p < 0.01). Histology confirmed large amounts of persistent cartilage in CD163-/- versus C57BL/6 fracture callus on Days 7 and 10 that resolves over time, and immunohistochemistry demonstrated deficiencies in CD206+ M2 macrophages. Torsion testing of the fractures confirmed the delayed early union in CD163-/- femurs, which display decreased yield torque on Day 21, and a decreased rigidity with a commensurate increase in rotation at yield on Day 28 (p < 0.01). Collectively, these results demonstrate that CD163 is required for normal angiogenesis, callus formation, and bone remodeling during fracture healing, and raise potential concerns about CD163 blockade therapy.

Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.

Influence of dimethoate on acetylcholinesterase activity and locomotor function in terrestrial isopods.

Locomotor behavior in terrestrial organisms is crucial for burrowing, avoiding predators, food seeking, migration, and reproduction; therefore, it is a parameter with ecological relevance. Acetylcholinesterase (AChE) is a nervous system enzyme inhibited by several compounds and widely used as an exposure biomarker in several organisms. Moreover, changes in energy reserves also may indicate an exposure to a stress situation. The aim of this study is to link biomarkers of different levels of biological organization in isopods exposed to increasing doses of dimethoate in semifield conditions. Locomotor parameters, AChE activity, and energy reserves (lipid, glycogen, and protein contents) were evaluated in the isopod Porcellio dilatatus after 48-h and 10-d exposure to dimethoate-contaminated soil. Results showed a clear impairment of both locomotor and AChE activity during the entire study, although effects were more pronounced after 48 h. Most locomotor parameters and AChE activity showed a clear dose-response relationship. By contrast, no clear trend was observed on energetic components. A positive and significant relationship was found between AChE activity and those locomotor parameters indicating activity, and the opposite was observed with those locomotor parameters indicating confusion and disorientation. The results obtained in this study enhance the importance of linking biochemical responses to parameters with ecological relevance at individual level, the value of locomotor behavior as an important marker to assess effects of toxicants, and also the usefulness and the acquisition of ecological relevance by AChE as a biomarker, by linking it with ecologically relevant behavioral parameters.

Toxicological, cellular and gene expression responses in earthworms exposed to copper and cadmium.

This study correlates sub-organismal changes with toxicological effects in earthworms (Lumbricus rubellus) exposed to copper and cadmium. Both metals reduced survival and reproduction at the highest concentration (LC50 5.11 microM Cu g(-1) and 4.04 microM Cd g(-1); cocoon production EC50s 5.17 microM Cu g(-1) and 1.86 microM Cd g(-1), all values as dry mass soil). Cadmium significantly reduced lysosomal membrane stability (at 1.86 microM Cd g(-1) and higher), upregulated metallothionein gene expression (at least sevenfold in all treatments) and reduced lysosome-associated-glycoprotein gene expression. Copper did not lower lysosomal membrane stability, but did upregulate metallothionein gene expression (at 2.5 microM Cu g(-1)), reduce lysosome-associated-glycoprotein gene expression and gave a nonlinear pattern for mitochondrial ribosomal subunit transcript expression (reduced at 0.35 and 0.811 microM Cu g(-1); higher at 2.5 microM Cu g(-1)). Correlation of metal body residue concentrations and cellular and molecular genetic responses with juvenile production rate confirmed a relationship for metallothionein expression, lysosomal membrane stability and cadmium tissue concentration in cadmium-exposed worms. Relationships between responses were also found for both metals. These suggested mechanisms for the interaction of cadmium and copper with specific gene products and with organelle (mitochondrial, lysosomal) functioning.

Pedological characterisation of sites along a transect from a primary cadmium/lead/zinc smelting works.

A pedological characterisation of seven sites along a transect from a smelter at Avonmouth, UK, was undertaken. Site locations comprised a mixture of both grassland (5 sites) and oak tree dominated (2 sites) areas. Geographically, sites were either low lying or on adjacent elevated ground. Across the transect, a severe gradient of metal concentrations was found with highest values for organic soil horizons from close to the factory. Comparisons with quality standards indicate that these metal levels are likely to be a major ecological stressor. In addition to the strong metal gradient, a number of other between site differences were also observed. These were firstly water regime, which at the low lying sites close to the smelter showed influence by both, groundwater and stagnant water, while at more distant sites indicated susceptibility to stagnant water only, and secondly soil pH, which showed large between site variations, although no consistent trend along the transect. Humus forms at sites more than 1.5 km from the smelter were characteristic for the soil conditions and land-use present, while those at sites within 1.5 km showed disturbed profiles. Indeed, the humus types present at these locations suggest that the activity of soil invertebrates may be inhibited. This is almost certainly due primarily to the high concentrations of metals that were present in mineral soil and organic horizons at the sites. However, the potential influences of water regime and pH could also be relevant factors influencing the results of any further studies conducted at these study sites.

Quantifying copper and cadmium impacts on intrinsic rate of population increase in the terrestrial oligochaete Lumbricus rubellus.

Demographic methods can translate toxicant effects on individuals into consequences for populations. To date few such studies have been conducted with longer-lived invertebrates. This is because full life-cycle experiments are difficult with such species. Here we report the effects of copper and cadmium on the key demographic parameter intrinsic rate of population increase (r) in a long-lived invertebrate (an earthworm). The approach used to derive r was based on robust measurement of effects on life-cycle traits in three specifically designed toxicity tests and integration of this data within a demographic model. The three laboratory tests used to generate values for specific life-cycle parameters under copper and cadmium exposure were suited to the task. Significant effects on a range of separate adult and juvenile life-cycle parameters were seen. Integration of parameter values within the demographic equation indicated that for copper, r was reduced only at a concentration that also caused adult mortality. For cadmium, a more graded exposure-dependent effect on fitness was seen, with r reduced at sublethal concentrations. The concentration response patterns for r found for the two metals suggest significantly different consequences for earthworm populations exposed to sublethal levels of copper and cadmium.

Interspecific comparisons of relative assimilation efficiencies for zinc and cadmium in an ecological series of talitrid amphipods (Crustacea).

A dual-labelling radioisotope technique has enabled the relative assimilation efficiencies of an essential (Zn) and a non-essential trace metal (Cd) to be determined in an ecological series of talitrid amphipods from low-shore to fully terrestrial habitats. All species exhibited high assimilation efficiencies for zinc and cadmium (within the range 66.7-96.4%). Interspecific differences in assimilation rates showed no trend with increasing terrestriality of the habitat. Elevated dietary zinc concentrations failed to enhance zinc assimilation efficiency. Incorporation of antibiotics into the diet to eliminate gut microflora did not affect zinc assimilation efficiencies. The implications of having high trace-metal assimilation efficiencies are discussed in terms of land invasion by talitrids.