Testing Laser-Structured Antimicrobial Surfaces Under Space Conditions: The Design of the ISS Experiment BIOFILMS

Maintaining crew health and safety are essential goals for long-term human missions to space. Attaining these goals requires the development of methods and materials for sustaining the crew’s health and safety. Paramount is microbiological monitoring and contamination reduction. Microbial biofilms are of special concern, because they can cause damage to spaceflight equipment and are difficult to eliminate due to their increased resistance to antibiotics and disinfectants. The introduction of antimicrobial surfaces for medical, pharmaceutical and industrial purposes has shown a unique potential for reducing and preventing biofilm formation. This article describes the development process of ESA’s BIOFILMS experiment, that will evaluate biofilm formation on various antimicrobial surfaces under spaceflight conditions. These surfaces will be composed of different metals with and without specified surface texture modifications. Staphylococcus capitis subsp. capitis, Cupriavidus metallidurans and Acinetobacter radioresistens are biofilm forming organisms that have been chosen as model organisms. The BIOFILMS experiment will study the biofilm formation potential of these organisms in microgravity on the International Space Station on inert surfaces (stainless steel AISI 304) as well as antimicrobial active copper (Cu) based metals that have undergone specific surface modification by Ultrashort Pulsed Direct Laser Interference Patterning (USP-DLIP). Data collected in 1 x g has shown that these surface modifications enhance the antimicrobial activity of Cu based metals. In the scope of this, the interaction between the surfaces and bacteria, which is highly determined by topography and surface chemistry, will be investigated. The data generated will be indispensable for the future selection of antimicrobial materials in support of human- and robotic-associated activities in space exploration

The transcriptomic landscape of cupriavidus metallidurans CH34 acutely exposed to copper

Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5′ end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance

Environmental Conditions Modulate the Transcriptomic Response of Both <i>Caulobacter crescentus</i> Morphotypes to Cu Stress

Bacteria encounter elevated copper (Cu) concentrations in multiple environments, varying from mining wastes to antimicrobial applications of copper. As the role of the environment in the bacterial response to Cu ion exposure remains elusive, we used a tagRNA-seq approach to elucidate the disparate responses of two morphotypes of Caulobacter crescentus NA1000 to moderate Cu stress in a complex rich (PYE) medium and a defined poor (M2G) medium. The transcriptome was more responsive in M2G, where we observed an extensive oxidative stress response and reconfiguration of the proteome, as well as the induction of metal resistance clusters. In PYE, little evidence was found for an oxidative stress response, but several transport systems were differentially expressed, and an increased need for histidine was apparent. These results show that the Cu stress response is strongly dependent on the cellular environment. In addition, induction of the extracytoplasmic function sigma factor SigF and its regulon was shared by the Cu stress responses in both media, and its central role was confirmed by the phenotypic screening of a sigF::Tn5 mutant. In both media, stalked cells were more responsive to Cu stress than swarmer cells, and a stronger basal expression of several cell protection systems was noted, indicating that the swarmer cell is inherently more Cu resistant. Our approach also allowed for detecting several new transcription start sites, putatively indicating small regulatory RNAs, and additional levels of Cu-responsive regulation

Genomic and Transcriptomic Changes that Mediate Increased Platinum Resistance in Cupriavidus metallidurans

The extensive anthropogenic use of platinum, a rare element found in low natural abundance in the Earth’s continental crust and one of the critical raw materials in the EU innovation partnership framework, has resulted in increased concentrations in surface environments. To minimize its spread and increase its recovery from the environment, biological recovery via different microbial systems is explored. In contrast, studies focusing on the effects of prolonged exposure to Pt are limited. In this study, we used the metal-resistant Cupriavidus metallidurans NA4 strain to explore the adaptation of environmental bacteria to platinum exposure. We used a combined Nanopore–Illumina sequencing approach to fully resolve all six replicons of the C. metallidurans NA4 genome, and compared them with the C. metallidurans CH34 genome, revealing an important role in metal resistance for its chromid rather than its megaplasmids. In addition, we identified the genomic and transcriptomic changes in a laboratory-evolved strain, displaying resistance to 160 µM Pt4+. The latter carried 20 mutations, including a large 69.9 kb deletion in its plasmid pNA4_D (89.6 kb in size), and 226 differentially-expressed genes compared to its parental strain. Many membrane-related processes were affected, including up-regulation of cytochrome c and a lytic transglycosylase, down-regulation of flagellar and pili-related genes, and loss of the pNA4_D conjugative machinery, pointing towards a significant role in the adaptation to platinum

Feasibility and accuracy of quantitative imaging on a 1.5 T MR-linear accelerator

PURPOSE: Systems for magnetic resonance (MR-) guided radiotherapy enable daily MR imaging of cancer patients during treatment, which is of interest for treatment response monitoring and biomarker discovery using quantitative MRI (qMRI). Here, the performance of a 1.5 T MR-linac regarding qMRI was assessed on phantoms. Additionally, we show the feasibility of qMRI in a prostate cancer patient on this system for the first time. MATERIALS AND METHODS: Four 1.5 T MR-linac systems from four institutes were included in this study. T1 and T2 relaxation times, and apparent diffusion coefficient (ADC) maps, as well as dynamic contrast enhanced (DCE) images were acquired. Bland-Altman statistics were used, and accuracy, repeatability, and reproducibility were determined. RESULTS: Median accuracy for T1 ranged over the four systems from 2.7 to 14.3%, for T2 from 10.4 to 14.1%, and for ADC from 1.9 to 2.7%. For DCE images, the accuracy ranged from 12.8 to 35.8% for a gadolinium concentration of 0.5 mM and deteriorated for higher concentrations. Median short-term repeatability for T1 ranged from 0.6 to 5.1%, for T2 from 0.4 to 1.2%, and for ADC from 1.3 to 2.2%. DCE acquisitions showed a coefficient of variation of 0.1-0.6% in the signal intensity. Long-term repeatability was 1.8% for T1, 1.4% for T2, 1.7% for ADC, and 17.9% for DCE. Reproducibility was 11.2% for T1, 2.9% for T2, 2.2% for ADC, and 18.4% for DCE. CONCLUSION: These results indicate that qMRI on the Unity MR-linac is feasible, accurate, and repeatable which is promising for treatment response monitoring and treatment plan adaptation based on daily qMRI

Feasibility and accuracy of quantitative imaging on a 1.5 T MR-linear accelerator

PURPOSE: Systems for magnetic resonance (MR-) guided radiotherapy enable daily MR imaging of cancer patients during treatment, which is of interest for treatment response monitoring and biomarker discovery using quantitative MRI (qMRI). Here, the performance of a 1.5 T MR-linac regarding qMRI was assessed on phantoms. Additionally, we show the feasibility of qMRI in a prostate cancer patient on this system for the first time. MATERIALS AND METHODS: Four 1.5 T MR-linac systems from four institutes were included in this study. T1 and T2 relaxation times, and apparent diffusion coefficient (ADC) maps, as well as dynamic contrast enhanced (DCE) images were acquired. Bland-Altman statistics were used, and accuracy, repeatability, and reproducibility were determined. RESULTS: Median accuracy for T1 ranged over the four systems from 2.7 to 14.3%, for T2 from 10.4 to 14.1%, and for ADC from 1.9 to 2.7%. For DCE images, the accuracy ranged from 12.8 to 35.8% for a gadolinium concentration of 0.5 mM and deteriorated for higher concentrations. Median short-term repeatability for T1 ranged from 0.6 to 5.1%, for T2 from 0.4 to 1.2%, and for ADC from 1.3 to 2.2%. DCE acquisitions showed a coefficient of variation of 0.1-0.6% in the signal intensity. Long-term repeatability was 1.8% for T1, 1.4% for T2, 1.7% for ADC, and 17.9% for DCE. Reproducibility was 11.2% for T1, 2.9% for T2, 2.2% for ADC, and 18.4% for DCE. CONCLUSION: These results indicate that qMRI on the Unity MR-linac is feasible, accurate, and repeatable which is promising for treatment response monitoring and treatment plan adaptation based on daily qMRI

Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8(+) lymphocytes in primary sarcomas is subtype dependent

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma (n = 46), Ewing sarcoma (n = 32), alveolar rhabdomyosarcoma (n = 20), embryonal rhabdomyosarcoma (n = 77), synovial sarcoma (n = 22) and desmoplastic small round cell tumors (DSRCT) (n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was describe