[State of the art: Simulation in US]. / State of the art: Simulation in der Ultraschallausbildung.

Technical simulation of diagnostic and therapeutic procedures is of growing relevance for student education and advanced medical training and has already been introduced in the field of ultrasound. This review gives a broad overview on different levels of simulation for ultrasound diagnostics and highlights the technical background of the methodology. A critical review of the literature reveals recommendations for implementing simulation techniques in medical studies and professional ultrasound training. An analysis of strengths and weaknesses shows the advantages of simulation especially in the context of individual learning situations and COVID-19-related restrictions for personal interaction. However, simulation techniques cannot replace the experiences of complex clinical examinations with direct interaction to real patients. Therefore, future applications may focus on repetition and assessment of achieved competencies by using standardized feedback mechanisms in order to preserve the limited resources for practical medical training.

Mechanistic modeling of cation exchange chromatography scale-up considering packing inhomogeneities.

In process development and characterization, the scale-up of chromatographic steps is a crucial part and brings a number of challenges. Usually, scale-down models are used to represent the process step, and constant column properties are assumed. The scaling is then typically based on the concept of linear scale-up. In this work, a mechanistic model describing an anti-Langmuirian to Langmuirian elution behavior of a polypeptide, calibrated with a pre-packed 1 ml column, is applied to demonstrate the scalability to larger column volumes up to 28.2 ml. Using individual column parameters for each column size, scaling to similar eluting salt concentrations, peak heights, and shapes is experimentally demonstrated by considering the model's relationship between the normalized gradient slope and the eluting salt concentration. Further scale-up simulations show improved model predictions when radial inhomogeneities in packing quality are considered.

Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling.

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of LNJ in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

Ultrasound Elastography for Characterization of Focal Liver Lesions.

Focal liver lesions (FLL) are typically detected by conventional ultrasound or other imaging modalities. After the detection of FLL, further characterization is essential, and this can be done by contrast-enhanced imaging techniques, e.g., contrast-enhanced ultrasound (CEUS) and magnetic resonance imaging (MRI) or by means of biopsy with histological evaluation. Elastographic techniques are nowadays integrated into high-end ultrasound systems and their value for the detection of severe liver fibrosis and cirrhosis has been shown in studies and meta-analyses. The use of an ultrasound elastographic technique for the differentiation of malignant and benign liver tumors is less well-established. This review summarizes the current data on utility and performance of ultrasound elastography for the characterization of FLL.

Mechanistic modeling and simulation of a complex low and high loading elution behavior of a polypeptide in cation exchange chromatography.

The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled. Model calibration in the linear range of the isotherm is done by applying counterion-induced linear gradient elution experiments between pH 3.3 and 4.3. Inverse fitting with column loads up to 25 mg/mLCV is performed for parameter estimation in the nonlinear range. The polypeptide elution peak shows an anti-Langmuirian behavior with fronting under low loading conditions and a switch to a Langmuirian behavior with increasing load. This unusual elution behavior could be described with an extended version of the sigmoidal Self-Association isotherm including two activity coefficients for the polypeptide and counterion in solution. The activity coefficient of the solute polypeptide shows a strong influence on the model parameters and is crucial in the linear and nonlinear range of the isotherm. The modeling procedure results in a unique and robust model parameter set that is sufficient to describe the complex elution behavior and allows modeling over the full isotherm range.

Conventional ultrasound for diagnosis of hepatic steatosis is better than believed.

BACKGROUND: Hepatic steatosis is a condition frequently encountered in clinical practice, with potential progression towards fibrosis, cirrhosis, and hepatocellular carcinoma. Detection and staging of hepatic steatosis are of most importance in nonalcoholic fatty liver disease (NAFLD), a disease with a high prevalence of more than 1 billion individuals affected. Ultrasound (US) is one of the most used noninvasive imaging techniques used in the diagnosis of hepatic steatosis. Detection of hepatic steatosis with US relies on several conventional US parameters, which will be described. US is the first-choice imaging in adults at risk for hepatic steatosis. The use of some scoring systems may add additional accuracy especially in assessing the severity of hepatic steatosis. SUMMARY: In the presented paper, we discuss screening and risk stratification, ultrasound features for diagnosing hepatic steatosis, B-mode criteria, focal fatty patterns and Doppler features of the hepatic vessels, and the value of the different US signs for the diagnosis of liver steatosis including classifying the severity of steatosis using different US scores. Limitations of conventional B-mode and Doppler features in the evaluation of hepatic steatosis are also discussed, including those in grading and assessing the complications of steatosis, namely fibrosis and nonalcoholic steatohepatitis. KEY MESSAGES: Ultrasound is the first-line imaging examination for the screening and follow-up of patients with liver steatosis. The use of some scoring systems may add additional accuracy in assessing the severity of steatosis. Conventional B-mode and Doppler ultrasound have limitations in grading and assessing the complications of steatosis.

Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders.

Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.

Unusual Diversity of Myoglobin Genes in the Lungfish.

Myoglobin is a respiratory protein that serves as a model system in a variety of biological fields. Its main function is to deliver and store O2 in the heart and skeletal muscles, but myoglobin is also instrumental in homeostasis of nitric oxide (NO) and detoxification of reactive oxygen species (ROS). Almost every vertebrate harbors a single myoglobin gene; only some cyprinid fishes have two recently duplicated myoglobin genes. Here we show that the West African lungfish Protopterus annectens has at least seven distinct myoglobin genes (PanMb1-7), which diverged early in the evolution of lungfish and showed an enhanced evolutionary rate. These myoglobins are lungfish specific, and no other globin gene was found amplified. The myoglobins are differentially expressed in various lungfish tissues, and the brain is the main site of myoglobin expression. The typical myoglobin-containing tissues, the skeletal muscle and the heart, have much lower myoglobin mRNA levels. Muscle and heart express distinct myoglobins (PanMb1 and PanMb3, respectively). In cell culture, lungfish myoglobins improved cellular survival under hypoxia albeit with different efficiencies and reduced the production of reactive oxygen species. Only Mb2 and Mb6 enhanced the energy status of the cells. The unexpected diversity of myoglobin hints to a functional diversification of this gene: some myoglobins may have adapted to the O2 requirements of the specific tissue and help the lungfish to survive hypoxic periods; other myoglobins may have taken over the roles of neuroglobin and cytoglobin, which appear to be missing in the West African lungfish.

Membrane-bound globin X protects the cell from reactive oxygen species.

Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role.

E2F1-associated purine synthesis pathway is a major component of the MET-DNA damage response network.

Various lines of investigation support a signaling interphase shared by receptor tyrosine kinases and the DNA damage response. However, the underlying network nodes and their contribution to the maintenance of DNA integrity remain unknown. We explored MET-related metabolic pathways whose interruption compromises proper resolution of DNA damage. Discovery metabolomics combined with transcriptomics identified changes in pathways relevant to DNA repair following MET inhibition (METi). METi by tepotinib was associated with formation of γH2AX foci and with significant alterations in major metabolic circuits such as glycolysis, gluconeogenesis, and purine, pyrimidine, amino acids, and lipids metabolism. 5'-Phosphoribosyl-N-formylglycinamide (FGAR), a de novo purine synthesis pathway metabolite, was consistently decreased in in vitro and in vivo MET-dependent models, and a METi-related depletion of dNTPs was observed. METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase (GART) which catalyzes FGAR synthesis. Genes encoding these enzymes are regulated through E2F1, whose levels decrease upon METi in MET-driven cells and xenografts. Transient E2F1 overexpression prevented dNTPs depletion and the concomitant METi-associated DNA damage in MET-driven cells. We conclude that DNA damage following METi results from dNTPs reduction via downregulation of E2F1 and a consequent decline of de novo purine synthesis.

Effect of salt modulators on the elution behavior of insulin and the separation of product-related impurities in reversed-phase chromatography.

In this work, the effect of the salt modulators potassium chloride, ammonium chloride, ammonium sulfate, and potassium sulfate on the elution behavior of insulin in reversed-phase chromatography with ethanol as the organic modifier was investigated. Without the addition of salt modulators, insulin shows the formation of multiple peaks under non-linear loading conditions, presumably due to an aggregate formation equilibrium. Flow rate and temperature did not influence the appearance of multiple peaks. The addition of chloride and sulfate salt modulators changed the monomer-multimer equilibrium, and multi-peak formation no longer occurred. Chloride salts induce a Langmuirian elution behavior, whereas sulfate salts induce additional insulin-insulin interactions resulting in an anti-Langmuirian elution behavior. The elution behavior can be influenced by the combination of both chloride and sulfate salts and by varying the concentration ratio. The separation with respect to two product-related impurities also showed significant differences under Langmuirian and anti-Langmuirian elution conditions and the purification of insulin could be optimized. Induced anti-Langmuirian elution by lowering the chloride/sulfate ratio suppresses an observed tag-along effect of one variant resulting in a slightly smaller pool volume with increased insulin concentration and a significantly increased insulin recovery.

A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response.

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.

HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

Reinvestigating the clinical relevance of the m6A writer METTL3 in urothelial carcinoma of the bladder.

METTL3 is the major writer of N6-Methyladenosine (m6A) and has been associated with controversial roles in cancer. This is best illustrated in urothelial carcinoma of the bladder (UCB), where METTL3 was described to have both oncogenic and tumor-suppressive functions. Here, we reinvestigated the role of METTL3 in UCB. METTL3 knockout reduced the oncogenic phenotype and m6A levels of UCB cell lines. However, complete depletion of METTL3/m6A was not achieved due to selection of cells expressing alternative METTL3 isoforms. Systematic vulnerability and inhibitor response analyses suggested that uroepithelial cells depend on METTL3 for viability. Furthermore, expression and survival analyses of clinical data revealed a complex role for METTL3 in UCB, with decreased m6A mRNA levels in UCB tumors. Our results suggest that METTL3 expression may be a suitable diagnostic UCB biomarker, as the enzyme promotes UCB formation. However, the suitability of the enzyme as a therapeutic target should be evaluated carefully.

The ultrasound use of simulators, current view, and perspectives: Requirements and technical aspects (WFUMB state of the art paper).

Simulation has been shown to improve clinical learning outcomes, speed up the learning process and improve learner confidence, whilst initially taking pressure off busy clinical lists. The World Federation for Ultrasound in Medicine and Biology (WFUMB) state of the art paper on the use of simulators in ultrasound education introduces ultrasound simulation, its advantages and challenges. It describes different simulator types, including low and high-fidelity simulators, the requirements and technical aspects of simulators, followed by the clinical applications of ultrasound simulation. The paper discusses the role of ultrasound simulation in ultrasound clinical training, referencing established literature. Requirements for successful ultrasound simulation acceptance into educational structures are explored. Despite being in its infancy, ultrasound simulation already offers a wide range of training opportunities and likely holds the key to a broader point of care ultrasound education for medical students, practicing doctors, and other health care professionals. Despite the drawbacks of simulation, there are also many advantages, which are expanding rapidly as the technology evolves.

The use of simulation in medical ultrasound: Current perspectives on applications and practical implementation (WFUMB state-of-the-art paper).

Simulation has been shown to improve clinical learning outcomes, speed up the learning process, and improve trainee confidence, while taking the pressure off initial face-to-face patient clinical areas. The second part of The World Federation for Ultrasound in Medicine and Biology state-of-the-art paper on the use of simulators provides a general approach on the practical implementation. The importance of needs assessment before developing a simulation-based training program is outlined. We describe the current practical implementation and critically analyze how simulators can be integrated into complex task scenarios to train small or large groups. A wide range of simulation equipment is available especially for those seeking interventional ultrasound training, ranging from animal tissue models, simple synthetic phantoms, to sophisticated high-fidelity simulation platforms using virtual reality. Virtual reality simulators provide feedback and thereby allow trainees to not only to practice their motor skills and hand eye coordination but also to interact with the simulator. Future developments will integrate more elements of automated assessment and artificial intelligence, thereby enabling enhanced realistic training experience and improving skill transfer into clinical practice.

Early detection of pancreatic tumors by advanced EUS imaging.

The early detection of pancreatic ductal adenocarcinoma (PDAC) dramatically improves outcome. All available state-of-the-art imaging methods allow early detection with EUS being the best technique for exclusion of PDAC and detection of very early PDAC. Etiological differentiation of small SPL is important to guide individually tailored patients' management including radical surgery in resectable PDAC, medical (neoadjuvant or palliative intended) treatment in patients with non-resectable malignancy, pancreatic parenchyma saving strategies in some non-PDAC, and follow-up in particular in low-grade PanNEN or other small benign lesions. Multimodality EUS imaging including B-Mode assessment, elastography, contrast-enhancement and EUS-guided sampling is the most appropriate technique for diagnosis and risk assessment of small SPL. We present a review discussing modern (endoscopic) ultrasound imaging techniques including contrast enhanced ultrasound and elastography for the early detection and characterization of solid pancreatic lesions.

VueBox® for quantitative analysis of contrast-enhanced ultrasound in liver tumors1.

Dynamic contrast-enhanced ultrasound (DCE-US) enables quantification of tumor perfusion. VueBox is a platform independent external software using DICOM cine loops which objectively provides various DCE-US parameters of tumor vascularity. This review summaries its use for diagnosis and treatment monitoring of liver tumors. The existing literature provides evidence on the successful application of Vuebox based DCE-US for characterization and differential diagnosis of focal liver lesions, as well as on its use for monitoring of local ablative therapies and of modern systemic treatment in oncology.

Discovery of Inhibitors of DNA Methyltransferase 2, an Epitranscriptomic Modulator and Potential Target for Cancer Treatment.

Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.

General principles of image optimization in EUS.

With the development of modern EUS, multiple imaging functions, transducer settings, and examination modes have become available for clinical settings. While the major determinants of the ultrasound beam are still comprised of the signal wavelength, its frequency range, and its amplitude, other modifications and calculations have gained more interest for advanced users, such as tissue harmonic imaging (THI), spatial and frequency compounding, certain versions of speckle reduction, and various Doppler/duplex settings. The goal of such techniques is a better, perhaps more realistic image, with reduced artifacts (such as speckle), better image contrast, and an improved signal-to-noise ratio. In addition, "add-ons" such as THI, which is based on the phenomenon of nonlinear distortion of acoustic signals as they travel through tissues, provide greater contrast and an enhanced spatial resolution than conventional EUS. Finally, optimization of spectral and color Doppler imaging in EUS requires experience and knowledge about the basic principles of Doppler/duplex phenomena. For these purposes, factors such as adjustment of Doppler controls, Doppler angle, color gain, spectral wall filters, and others require special attention during EUS examinations. Incorporating these advanced techniques in EUS examinations may be time-consuming and cumbersome. Hence, practical guidelines enabling endosonographers to steer safely through the large quantity of technological properties and settings (knobology) are appreciated. This review provides an overview of the role of important imaging features to be adjusted before, during, and after EUS procedures.