The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane-proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel ß-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel ß-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent-in stark contrast to the primarily positive surface charge of the left-handed parallel ß-roll "type" protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left-handed parallel ß-roll structures may differ depending on their position in the structure.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Recent advances in the understanding of trimeric autotransporter adhesins.

Adhesion is the initial step in the infection process of gram-negative bacteria. It is usually followed by the formation of biofilms that serve as a hub for further spread of the infection. Type V secretion systems engage in this process by binding to components of the extracellular matrix, which is the first step in the infection process. At the same time they provide protection from the immune system by either binding components of the innate immune system or by establishing a physical layer against aggressors. Trimeric autotransporter adhesins (TAAs) are of particular interest in this family of proteins as they possess a unique structural composition which arises from constraints during translocation. The sequence of individual domains can vary dramatically while the overall structure can be very similar to one another. This patchwork approach allows researchers to draw conclusions of the underlying function of a specific domain in a structure-based approach which underscores the importance of solving structures of yet uncharacterized TAAs and their individual domains to estimate the full extent of functions of the protein a priori. Here, we describe recent advances in understanding the translocation process of TAAs and give an overview of structural motifs that are unique to this class of proteins. The role of BpaC in the infection process of Burkholderia pseudomallei is highlighted as an exceptional example of a TAA being at the centre of infection initiation.

The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance.

The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high-copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.

Magnetic resonance-imaging of the effect of targeted antiangiogenic gene delivery in a melanoma tumour model.

OBJECTIVES: We investigated the effect of targeted gene therapy to melanoma tumours (M21) by MR-imaging. METHODS: M21 and M21-L tumours were grown to a size of 850 mm(3). M21 and M21-L tumours were intravenously treated with an αvß3-integrin-ligand-coupled nanoparticle (RGDNP)/RAF(-) complex five times every 72 hours. MRI was performed at set time intervals 24h and 72h after the i.v. injection of the complex. The MRI protocol was T1-wt-SE±CM, T2-wt-FSE, DCE-MRI, Diffusion-wt-STEAM-sequence, T2-time obtained on a 1.5-T-GE-MRI device. RESULTS: The size of the treated M21 tumours kept nearly constant during the treatment phase (847.8±31.4 mm(3) versus 904.8±44.4 mm(3)). The SNR value (T2-weighted images) of the tumours was 36.7±0.6 and dropped down to 30.6±1.9 (p=0.004). At the beginning the SNR value (T1-weighted images) of the tumours after contrast medium application was 42.3±1.9 and dropped down to 28.5±3.0 (p<0.001). In the treatment group the diffusion coefficient increased significantly under therapy (0.54±0.01x10(-3) mm(2)/s versus 0.67±0.04x10(-3) mm(2)/s). The DCE-MRI showed a reduction of the slope and of the Akep of 67.8±4.3 % respectively 64.8±3.3 % compared to baseline. CONCLUSIONS: Targeted gene delivery therapy induces significant changes in MR-imaging. MRI showed a significant reduction of contrast medium uptake parameters and increase of the diffusion coefficient of the tumours. KEY POINT: • Treatment with targeted gene-delivery therapy can be monitored by MR imaging • DCE and diffusion-weighted imaging are appropriate methods for monitoring this therapy • Functional changes are significant prior to any morphological changes.

Segmentation-based partial volume correction for volume estimation of solid lesions in CT.

In oncological chemotherapy monitoring, the change of a tumor's size is an important criterion for assessing cancer therapeutics. Measuring the volume of a tumor requires its delineation in 3-D. This is called segmentation, which is an intensively studied problem in medical image processing. However, simply counting the voxels within a binary segmentation result can lead to significant differences in the volume, if the lesion has been segmented slightly differently by various segmentation procedures or in different scans, for example due to the limited spatial resolution of computed tomography (CT) or partial volume effects. This variability limits the sensitivity of size measurements and thus of therapy response assessments and it can even lead to misclassifications. We present a fast, generic algorithm for measuring the volume of solid, compact tumors in CT that considers partial volume effects at the border of a given segmentation result. The algorithm is an extension of the segmentation-based partial volume analysis proposed by Kuhnigk for the volumetry of solid lung lesions , such that it can be applied to inhomogeneous lesions and lesions with inhomogeneous surroundings. Our generalized segmentation-based partial volume correction is based on a spatial subdivision of the segmentation result, from which the fraction of tumor for each voxel is computed. It has been evaluated on phantom data, 1516 lesion segmentation pairs (lung nodules, liver metastases and lymph nodes) as well as 1851 lung nodules from the LIDC-IDRI database. The evaluations of our algorithm show a more accurate estimation of the real volume and its ability to reduce inter- and intra-observer variability significantly for each entity. Overall, the variability (interquartile range) for phantom data is reduced by 49% ( p ≪ 0.001) and the variability between different readers is reduced by 28% ( p ≪ 0.001). The average computation time is 0.2 s.

On the evaluation of segmentation editing tools.

Efficient segmentation editing tools are important components in the segmentation process, as no automatic methods exist that always generate sufficient results. Evaluating segmentation editing algorithms is challenging, because their quality depends on the user's subjective impression. So far, no established methods for an objective, comprehensive evaluation of such tools exist and, particularly, intermediate segmentation results are not taken into account. We discuss the evaluation of editing algorithms in the context of tumor segmentation in computed tomography. We propose a rating scheme to qualitatively measure the accuracy and efficiency of editing tools in user studies. In order to objectively summarize the overall quality, we propose two scores based on the subjective rating and the quantified segmentation quality over time. Finally, a simulation-based evaluation approach is discussed, which allows a more reproducible evaluation without the need for human input. This automated evaluation complements user studies, allowing a more convincing evaluation, particularly during development, where frequent user studies are not possible. The proposed methods have been used to evaluate two dedicated editing algorithms on 131 representative tumor segmentations. We show how the comparison of editing algorithms benefits from the proposed methods. Our results also show the correlation of the suggested quality score with the qualitative ratings.

Imaging of firefly luciferase activity under antiangiogenic therapy in a heat shock protein 70-transfected melanoma tumor model.

We investigated the effect of targeted gene therapy on heat shock protein 70 (Hsp70) expression in a melanoma tumor model (M21). M21 cells transfected with a plasmid containing the firefly luciferase reporter gene (ffluc), whose expression is driven by the hsp70 (hspa1b) or the cytomegalovirus (CMV) promoter, were grown to a size of 600 mm3. Five animals in each group were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein [RGD-NP/RAF(-)] complex. Bioluminescence imaging (BLI) (IVIS, Xenogen, Alameda, CA) was performed at set time intervals. Western blot analysis of the HSP70 protein was simultaneously performed. The size of the treated M21 tumors was nearly constant (637.8 ± 33.4 mm3 vs 674.8 ± 34.4 mm3). BLI showed that if transcription was controlled by the CMV promoter, firefly luciferase activity decreased to 51.1% ± 8.3%. When transcription was controlled by the hsp70 promoter, the highest firefly luciferase activity (4.4 ± 0.3-fold) was observed after 24 hours. In accordance with BLI, Western blot analysis showed an increase in the level of HSP70, with the maximum detection 24 hours after the injection of the RGD-NP/RAF(-) complex. Targeted antiangiogenic therapy can induce luciferase activity where transcription is controlled by an hsp70 promoter and HSP70 protein in melanoma tumors.

Painful muscle fibrosis following synthol injections in a bodybuilder: a case report.

INTRODUCTION: Synthol is a site enhancement oil used by bodybuilders to boost the cosmetic appearance of muscles. Here, we describe the case of a patient with severe side effects following repeated intramuscular injections of synthol in his right biceps muscle. CASE PRESENTATION: A 29-year-old Middle Eastern male bodybuilder, following intramuscular injections of synthol five years ago, presented with painful pressure in his right upper arm. On presentation to our clinic, his muscle appeared disfigured. Magnetic resonance imaging revealed scattered cystic fatty lesions in the muscle. The affected part was surgically removed and histopathology showed inflammatory changes with fibrosis and a so-called Swiss cheese pattern. CONCLUSION: Synthol injections that are used for the short-term enhancement of muscle appearance by bodybuilders bear the danger of long-term painful muscle fibrosis and disfigurement.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model.

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm(3). Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8 ± 103.4 mm(2) at the beginning versus 704.8 ± 94.4 mm(3) at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9% ± 4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5 ± 0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

Workflow-centred evaluation of an automatic lesion tracking software for chemotherapy monitoring by CT.

OBJECTIVES: In chemotherapy monitoring, an estimation of the change in tumour size is an important criterion for the assessment of treatment success. This requires a comparison between corresponding lesions in the baseline and follow-up computed tomography (CT) examinations. We evaluate the clinical benefits of an automatic lesion tracking tool that identifies the target lesions in the follow-up CT study and pre-computes the lesion volumes. METHODS: Four radiologists performed volumetric follow-up examinations for 52 patients with and without lesion tracking. In total, 139 lung nodules, liver metastases and lymph nodes were given as target lesions. We measured reading time, inter-reader variability in lesion identification and volume measurements, and the amount of manual adjustments of the segmentation results. RESULTS: With lesion tracking, target lesion assessment time decreased by 38 % or 22 s per lesion. Relative volume difference between readers was reduced from 0.171 to 0.1. Segmentation quality was comparable with and without lesion tracking. CONCLUSIONS: Our automatic lesion tracking tool can make interpretation of follow-up CT examinations quicker and provide results that are less reader-dependent. KEY POINTS: Computed tomography is widely used to follow-up lesions in oncological patients. Novel software automatically identifies and measures target lesions in oncological follow-up examinations. This enables a reduction of target lesion assessment. The automated measurements are less reader-dependent.

Use of in vivo bioluminescence and MRI to determine hyperthermia-induced changes in luciferase activity under the control of an hsp70 promoter.

We investigated the in vivo effect of hyperthermia on the expression of heat shock proteins and MRI changes in three tumor cell lines. Three tumor cell lines (SCCVII, NIH3T3, M21) were transfected with a plasmid containing the heat shock protein 70 gene (hsp70) promoter fragment and the luciferase reporter gene, and injected into mice. Tumors of 1100 mm³ in size were exposed to five different temperatures (38, 40, 42, 44 and 46 °C) in a water bath. Bioluminescence and MRI were performed at set time intervals. The MRI scan protocol was as follows: T1-weighted spin echo ± contrast medium, T2-weighted fast spin echo, dynamic contrast-enhanced MRI, diffusion-weighted stimulated echo acquisition mode sequence, T2 time obtained on a 1.5T General Electric MRI scanner. Immunoblotting was also performed. hsp70 transcription was strongly induced at 42 and 44 °C, reaching values as high as 8531.5 ± 432.1-fold above baseline in NIH3T3 tumors. At these temperatures, significant increases in the uptake of contrast medium, slope of initial enhancement, Ak(ep) values and apparent diffusion coefficient (ADC) were observed in the 8-h scan of the NIH3T3 cell line. In SCCVII tumors, ADC increased by about 23% (p = 0.010) in the scans performed at 8, 24, 48 and 96 h. At 46 °C, luciferase activity was reduced significantly in the three cell lines. In all tumor types, a significant increase in ADC was observed, which was highest in SCCVII tumors (33.8%; p < 0.01). In accordance with the bioluminescence results, significant Hsp70 protein production was shown by immunoblot analysis. The best correlation coefficient between luciferase activity and immunoblotting results was found for M21 tumors (r = 0.93, p < 0.0001). Different tissue types display distinct patterns of hsp70 transcription. MRI can be used, in combination with optical imaging, to provide information on hsp70 transcription and protein production. The major finding of the present study was that heat-related biochemical changes in tumor tissue can be determined by MRI.

Novel approach to complex pulmonary arteriovenous malformation embolization using detachable coils and Amplatzer vascular plugs.

INTRODUCTION: We evaluated the feasibility of a modified embolization technique of pulmonary arteriovenous malformations (PAVM) using venous sac embolization with detachable coils combined with the feeding artery embolization with the Amplatzer vascular plug (AVP). MATERIALS AND METHODS: We retrospectively studied technical and clinical success in the treatment of 11 complexe PAVMs. We recorded number and size of feeding arteries and draining vein, the last prior and post treatment in the follow up CT, size of PAVMs; and the number of devices needed to occlude each PAVM. RESULTS: 11 complexe PAVM were treated with detachable coils to venous sac embolization followed by AVP to embolize feeding arteries. In all but one case a complete occlusion of the PAVM was angiographically achieved. The mean number of feeding vessel was 2.64 ± 0.92 (2-5). The mean number of coils was 7.82 ± 5.09 (3-20 coils). CT-follow-up, that was possible in 8 patients, showed a significant reduction of the draining vein size. The mean diameter reduction of the draining vein was 62 ± 18% varying between 29% and 77%. In all but one case with the complexe angioarchitecture the reduction of draining vein size close to 70% was achieved. CONCLUSIONS: Our study implies that the venous sac embolization using the detachable coils followed by occlusion of the large feeding arteries using the AVP is a highly efficient method for the treatment of the complex PAVMs with large out-flow vessels and short feeding arteries.

Gene expression analysis of SCC tumor cells in muscle tissue.

The purpose of this study was to evaluate microarray technology of HNSCC cells in muscle tissue. 200 SCCVII tumor cells were injected intramuscularly into the right flank of ten C3H/Km mice each. One week later the animals were killed and the tissue taken out. Histology (H&E staining) and microarray of the tissue were performed. Histology showed a few tumor cells between the muscle fibers. Microarray technology showed different gene expression pattern of the muscle tissue with SCCVII cells in comparison with normal muscle tissue. Only those genes showing a fold change difference of 5 or higher were considered. Gene expression analysis revealed changes in the expression levels of SCCVII cells in muscle tissue in 220 genes. Significant gene expression differences between SCCVII cells in muscle tissue and pure muscle tissue could be seen.