Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

The status of academic interventional radiologists in Germany with focus on gender disparity: how can we do better?

PURPOSE: The aim was to characterize the framework conditions in academic interventional radiology (IR) in Germany with focus on differences between genders. MATERIALS AND METHODS: After IRB approval, all members of The German Society for Interventional Radiology and Minimally Invasive Therapy (n = 1,632) were invited to an online survey on work and research. Statistical comparisons were undertaken with the Fisher's exact test, Wilcoxon rank sum test or Pearson's Chi-squared test. RESULTS: From 267 available questionnaires (general response rate 16.4%), 200 were fully completed. 40% of these (78/200) were involved in research (71% men vs. 29% women, p < 0.01) and eligible for further analysis. Of these, 6% worked part-time (2% vs. 17%, p < 0.05). 90% of the respondents spent less than 25% of their research during their paid working hours, and 41% performed more than 75% of their research during. leisure time. 28% received exemption for research. 88% were (rather) satisfied with their career. One in two participants successfully applied for funding, with higher success rates among male applicants (90% vs. 75%) and respondents with protected research time (93% vs. 80%). Compared to men, women rated their entrance in research as harder (p < 0.05), their research career as more important (p < 0.05), felt less noticed at congresses (93% vs. 53%, p < 0.01), less confident (98% vs. 71%, p < 0.01), and not well connected (77% vs. 36%, p < 0.01).  CONCLUSION: Women and men did research under the same circumstances; however, women were underrepresented. Future programs should generally focus on protected research time and gather female mentors to advance academic IR in Germany.

Influence of Mutations and N-Glycosylation Sites in the Receptor-Binding Domain (RBD) and the Membrane Protein of SARS-CoV-2 Variants of Concern on Antibody Binding in ELISA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI- cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI- cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.

Potential of a Bead-Based Multiplex Assay for SARS-CoV-2 Antibody Detection.

Serological assays for SARS-CoV-2 play a pivotal role in the definition of whether patients are infected, the understanding of viral epidemiology, the screening of convalescent sera for therapeutic and prophylactic purposes, and in obtaining a better understanding of the immune response towards the virus. The aim of this study was to investigate the performance of a bead-based multiplex assay. This assay allowed for the simultaneous testing of IgG antibodies against SARS-CoV-2 spike, S1, S2, RBD, and nucleocapsid moieties and S1 of seasonal coronaviruses hCoV-22E, hCoV-HKU1, hCoV-NL63, and hCoV-OC43, as well as MERS and SARS-CoV. We compared the bead-based multiplex assay with commercial ELISA tests. We tested the sera of 27 SARS-CoV-2 PCR-positive individuals who were previously tested with different ELISA assays. Additionally, we investigated the reproducibility of the results by means of multiple testing of the same sera. Finally, the results were correlated with neutralising assays. In summary, the concordance of the qualitative results ranged between 78% and 96% depending on the ELISA assay and the specific antigen. Repeated freezing-thawing cycles resulted in reduced mean fluorescence intensity, while the storage period had no influence in this respect. In our test cohort, we detected up to 36% of sera positive for the development of neutralising antibodies, which is in concordance with the bead-based multiplex and IgG ELISA.

Outcome of Endovascular and Open Treated Penetrating Aortic Ulcers.

PURPOSE: Penetrating aortic ulcer (PAU) is a rare etiology of acute aortic syndrome. Few studies exist regarding the perioperative outcome. The aim was to analyze clinical outcome and risk factors of mortality in this treatment population. METHODS: Retrospective, monocentric study from 2010 to 2021. Clinical data of endovascular or open treated PAU were analyzed. In-hospital mortality was selected as the primary study endpoint. Angio-morphologies were analyzed and risk factors for mortality were identified by using univariate analysis. RESULTS: Overall, 133 patients were identified. 29% (n=38) of patients presented symptomatically. In 64% (n=85), the PAU was localized in the thoracic aorta. On average, PAUs had a depth of 15.4±10.1 mm and a width of 17.9±9.6 mm. The patients had a median of 2 (95% confidence interval [CI]=2-3) high-risk features (HRF) as PAU depth >10 mm, PAU width >20 mm, aortic diameter >40 mm, symptomatic, intramural hematoma (IMH), pleural effusion. Significantly more HRF were observed in symptomatic patients (p=0.01). 53% (n=71) of patients were treated with thoracic endovascular aortic repair (TEVAR), 41% (n=54) by endovascular aortic repair (EVAR), and 6% (n=8) by open surgery. A hybrid procedure with cervical debranching was performed in 16% (n=21) and complex endovascular repair with fenestrated or branched endografts in 15% (n=20). Overall, complications greater than grade II according to the Clavien-Dindo classification occurred in 19% (n=25) and of the patients. In-hospital mortality manifested in 6% (n=8). Factors associated with increased mortality were the diameter of the aorta >40 mm (88% vs 39%, p=0.03), as well as symptomatic patients (63% vs 26%, p=0.04), coincident IMHs (38% vs 10%, p=0.05), and complex endovascular procedures (50% vs 50% p<0.01). Penetrating aortic ulcer width >20 mm tended to show higher mortality (75% vs 40%, p=0.06). Routine follow-up was available for 89% (n=117) for a median of 39 months (95% CI=25-42). One-year and 5-year survival were 83% and 60%, respectively, with 1 aortic pathology-related death. CONCLUSIONS: Treatment of PAU is associated with an acceptable perioperative morbidity and mortality. Risk factors associated with increased mortality are an elevated aortic diameter, the presence of IMHs, clinical symptomatology at presentation, and complex endovascular procedures.

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA. Methods: Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses. Results and discussion: For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

Streptococcal quorum sensing peptide CSP-7 contributes to muscle inflammation and wasting.

Muscle wasting diseases, such as cancer cachexia and age-associated sarcopenia, have a profound and detrimental impact on functional independence, quality of life, and survival. Our understanding of the underlying mechanisms is currently limited, which has significantly hindered the development of targeted therapies. In this study, we explored the possibility that the streptococcal quorum sensing peptide Competence Stimulating Peptide 7 (CSP-7) might be a previously unidentified contributor to clinical muscle wasting. We found that CSP-7 selectively triggers muscle cell inflammation in vitro, specifically the release of IL-6. Furthermore, we demonstrated that CSP-7 can traverse the gastrointestinal barrier in vitro and is present in the systemic circulation in humans in vivo. Importantly, CSP-7 was associated with a muscle wasting phenotype in mice in vivo. Overall, our findings provide new mechanistic insights into the pathophysiology of muscle inflammation and wasting.

Fusing Bismuth and Mercaptocarboranes: Design and Biological Evaluation of Low-Toxicity Antimicrobial Thiolato Complexes.

This study proposes an innovative strategy to enhance the pharmacophore model of antimicrobial bismuth thiolato complex drugs by substituting hydrocarbon ligand structures with boron clusters, particularly icosahedral closo-dicarbadodecaborane (C2 B10 H12 , carboranes). The hetero- and homoleptic mercaptocarborane complexes BiPh2 L (1) and BiL3 (2) (L=9-S-1,2-C2 B10 H11 ) were prepared from 9-mercaptocarborane (HL) and triphenylbismuth. Comprehensive characterization using NMR, IR, MS, and XRD techniques confirmed their successful synthesis. Evaluation of antimicrobial activity in a liquid broth microdilution assay demonstrated micromolar to submicromolar minimum inhibitory concentrations (MIC) suggesting high effectiveness against S. aureus and limited efficacy against E. coli. This study highlights the potential of boron-containing bismuth complexes as promising antimicrobial agents, especially targeting Gram-positive bacteria, thus contributing to the advancement of novel therapeutic approaches.

Comparative analysis of radiomics and deep-learning algorithms for survival prediction in hepatocellular carcinoma.

To examine the comparative robustness of computed tomography (CT)-based conventional radiomics and deep-learning convolutional neural networks (CNN) to predict overall survival (OS) in HCC patients. Retrospectively, 114 HCC patients with pretherapeutic CT of the liver were randomized into a development (n = 85) and a validation (n = 29) cohort, including patients of all tumor stages and several applied therapies. In addition to clinical parameters, image annotations of the liver parenchyma and of tumor findings on CT were available. Cox-regression based on radiomics features and CNN models were established and combined with clinical parameters to predict OS. Model performance was assessed using the concordance index (C-index). Log-rank tests were used to test model-based patient stratification into high/low-risk groups. The clinical Cox-regression model achieved the best validation performance for OS (C-index [95% confidence interval (CI)] 0.74 [0.57-0.86]) with a significant difference between the risk groups (p = 0.03). In image analysis, the CNN models (lowest C-index [CI] 0.63 [0.39-0.83]; highest C-index [CI] 0.71 [0.49-0.88]) were superior to the corresponding radiomics models (lowest C-index [CI] 0.51 [0.30-0.73]; highest C-index [CI] 0.66 [0.48-0.79]). A significant risk stratification was not possible (p > 0.05). Under clinical conditions, CNN-algorithms demonstrate superior prognostic potential to predict OS in HCC patients compared to conventional radiomics approaches and could therefore provide important information in the clinical setting, especially when clinical data is limited.

Avoiding adverse events in interventional radiology - a systematic review on the instruments.

BACKGROUND: Avoiding AEs is a pivotal fundament for high patient safety in an efficient interventional radiology (IR) department. Although IR procedures are considered to have a lower risk than their surgical alternatives, they account for one third of all radiological adverse events (AEs) and in general, the number of AEs is increasing. Thus, measures to prevent AEs in IR are of interest. METHODS: A systematic literature search was conducted via handsearch and Ovid. A structured data extraction was performed with all included studies and their quality of evidence was evaluated. Finally, data were aggregated for further statistical analysis. RESULTS: After screening 1,899 records, 25 full-text publications were screened for eligibility. Nine studies were included in the review. Of those, four studies investigated in simulator training, one in team training, three in checklists, and one in team time-out. Eight were monocenter studies, and five were conducted in a non-clinical context. Study quality was low. Aggregation and analysis of data was only possible for the studies about checklists with an overall reduction of the median error per procedure from 0.35 to 0.06, observed in a total of 20,399 and 58,963 procedures, respectively. CONCLUSION: The evidence on the instruments to avoid AEs in IR is low. Further research should be conducted to elaborate the most powerful safety tools to improve patient outcomes in IR by avoiding AEs.

Immunological Quantitation of the Glycation Site Lysine-414 in Serum Albumin in Human Plasma Samples by Indirect ELISA Using Highly Specific Monoclonal Antibodies.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance.

Outcome After Conservative and Endovascular Treatment of Stanford Type B Aortic Intramural Hematomas - A Single-Center Retrospective Study.

OBJECTIVES: Aortic intramural hematoma (IMH) is a rare disease. Thus far, only limited data is available and the indications for conservative and endovascular treatment are not well defined. The aim of this study was to investigate clinical presentation, course, CT imaging features and outcome of patients with type B aortic IMHs. METHODS: We included all patients with type B IMHs between 2012 and 2021 in this retrospective monocentric study. Clinical data, localization, thickness of IMHs and the presence of ulcer-like projections (ULPs) was evaluated before and after treatment. RESULTS: Thirty five patients (20 females; 70.3 y ± 11 y) were identified. Almost all IMHs (n = 34) were spontaneous and symptomatic with back pain (n = 34). At the time of diagnosis, TEVAR was deemed indicated in 9 patients, 26 patients were treated primarily conservatively. During the follow-up, in another 16 patients TEVAR was deemed indicated. Endovascularly and conservatively treated patients both showed decrease in thickness after treatment. Patients without ULPs showed more often complete resolution of the IMH than patients with ULPs (endovascularly treated 90.9% (10/11) vs 71.4% (5/7); conservatively treated 71.4% (10/14) vs 33.3% (1/3); P = .207). Complications after TEVAR occurred in 32% and more frequently in patients treated primarily conservatively (37.5% vs 22.2%). No in-hospital mortality was observed during follow-up. CONCLUSIONS: Prognosis of IMH seems favourable in both surgically as well as conservatively treated patients. However, it is essential to identify patients at high risk for complications under conservative treatment, who therefore should be treated by TEVAR. In our study, ULPs seem to be an adverse factor for remodeling.

cAMP-phosphodiesterase 4D7 (PDE4D7) forms a cAMP signalosome complex with DHX9 and is implicated in prostate cancer progression.

A robust body of work has demonstrated that a reduction in cAMP-specific 3',5'-cyclic phosphodiesterase 4D isoform 7 (PDE4D7) is linked with negative prostate cancer outcomes; however, the exact molecular mechanism that underpins this relationship is unknown. Epigenetic profiling has shown that the PDE4D gene can be hyper-methylated in transmembrane serine protease 2 (TMPRSS2)-ETS transcriptional regulator ERG (ERG) gene-fusion-positive prostate cancer (PCa) tumours, and this inhibits messenger RNA (mRNA) expression, leading to a paucity of cellular PDE4D7 protein. In an attempt to understand how the resulting aberrant cAMP signalling drives PCa growth, we immunopurified PDE4D7 and identified binding proteins by mass spectrometry. We used peptide array technology and proximity ligation assay to confirm binding between PDE4D7 and ATP-dependent RNA helicase A (DHX9), and in the design of a novel cell-permeable disruptor peptide that mimics the DHX9-binding region on PDE4D7. We discovered that PDE4D7 forms a signalling complex with the DExD/H-box RNA helicase DHX9. Importantly, disruption of the PDE4D7-DHX9 complex reduced proliferation of LNCaP cells, suggesting the complex is pro-tumorigenic. Additionally, we have identified a novel protein kinase A (PKA) phosphorylation site on DHX9 that is regulated by PDE4D7 association. In summary, we report the existence of a newly identified PDE4D7-DHX9 signalling complex that may be crucial in PCa pathogenesis and could represent a potential therapeutic target.

CT versus MR guidance for radiofrequency ablation in patients with colorectal liver metastases: a 10-year follow-up favors MR guidance.

OBJECTIVES: To compare the results of CT- vs MR-guided radiofrequency ablation (RFA) of liver metastases (LM) from colorectal cancer after 10 years of follow-up in an observational, retrospective, and multicentric study. METHODS: A total of 238 patients with 496 LM were treated with RFA either with CT (CT group) or magnetic resonance (MR group) guidance. Every ablated LM was assessed and followed up with diagnostic MRI. Technical success, technique efficacy, predictive factors, recurrence rates, and overall survival were assessed. RESULTS: The CT group comprised 143 patients and the MR group 77 patients. Eighteen patients underwent ablation with both modalities. Technical success per patient and per lesion was 88% and 93% for CT and 87% and 89.6% for MR, and technique efficacy was 97.1% and 98.6% for CT and 98.7% and 99.3% for MR respectively. Local recurrence following the first ablation (primary patency) occurred in 20.1% (CT) vs 4.6% (MR) (p < 0.001). Residual liver tumor, size of LM, and advanced N and M stage at initial diagnosis were independent predictors for overall survival in both groups. The median overall survival measured from first RFA treatment was 2.6 years. The 1-year, 5-year, and 10-year survival were 85.9%, 25.5%, and 19.1% respectively. CONCLUSIONS: The MR group had significantly better local control compared to the CT group. There was no significant difference in patient survival between the two groups. CLINICAL RELEVANCE STATEMENT: MR-guided radiofrequency ablation of colorectal liver metastases is safe and effective, and offers better local control than CT-guided ablation. KEY POINTS: • Imaging modality for radiofrequency ablation guidance is an independent predictor of local recurrence in colorectal liver metastases. • MR-guided radiofrequency ablation achieved better local control of liver metastases from colorectal cancer than CT-guided. • The number and size of liver metastases are, among others, independent predictors of survival. Radiofrequency ablation with MR guidance improved clinical outcome but does not affect survival.

Effect of Contrast Timing, Volume, and Flow Rate on Image Quality of Pulmonary Arteries in CTA.

PURPOSE: To evaluate the effect of contrast timing, contrast volume, and contrast flow rate on the image quality of pulmonary arteries in computed tomography angiography (CTA) and to assess if bolus-tracking region of interest (ROI) positioning in the left atrium, which is used for triple-rule-out CTA, allows for sufficient depiction of the pulmonary arteries. METHODS: In this retrospective single-center study, data were collected for patients who underwent thoracic CTA during a specific period. Two groups of 121 patients each were created based on bolus-tracking ROI positioning in the main pulmonary artery or left atrium using propensity score matching. Image quality of the pulmonary arteries was evaluated using quantitative and qualitative scores. Subgroups were formed to examine the influence of contrast volume and flow rate. Two radiologists determined if pulmonary embolism was present, if pulmonary embolism could be excluded with certainty, and from which level pulmonary embolism could be excluded with certainty. Interrater reliability also was evaluated. RESULTS: ROI positioning in the main pulmonary artery scored significantly higher compared with the left atrium. There was no significant difference in subgroups of patients who were examined with 60 mL or more contrast volume and less than 4 mL/s flow rate; scores were similar or better than in the overall study population. Pulmonary embolism was not able to be excluded with certainty for each 1 patient in these subgroups compared with a high percentage in the overall study population. DISCUSSION: ROI positioning in the left atrium in combination with the 60 mL or more contrast volume and less than 4 mL/s flow rate does not adversely affect depiction of the pulmonary arteries compared with conventional ROI positioning in the main pulmonary artery. CONCLUSION: When using 60 mL or more contrast volume and less than 4 mL/s flow rate, ROI positioning in the left atrium, which is used in triple-rule-out CTA, is sufficient for the assessment of pulmonary arteries.

Revisiting the roles of cAMP signalling in the progression of prostate cancer.

Prostate cancer is one of the most common cancers in men and one of the top causes of death in men worldwide. Development and function of both normal prostate cells and early-stage prostate cancer cells are dependent on the cross-talk between androgen signalling systems and a variety of other transduction pathways which drive differentiation of these cells towards castration-resistance. One such signalling pathway is the ubiquitous cAMP signalling axis which functions to activate spatially restricted pools of cAMP effectors such as protein kinase A (PKA). The importance of both PKA and cAMP in the development of prostate cancer, and their interactions with the androgen receptor, were the focus of a review by Merkle and Hoffmann in 2010. In this updated review, we revisit this topic with analysis of current PKA-related prostate cancer literature and introduce novel information on the relevance of another cAMP effector, the exchange protein directly activated by cAMP (EPAC).