Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

Quantitative T2 Mapping of Acute Pancreatitis.

BACKGROUND: Quantification of the T2 signal by means of T2 mapping in acute pancreatitis (AP) has the potential to quantify the parenchymal edema. Quantitative T2 mapping may overcome the limitations of previously reported scoring systems for reliable assessment of AP. PURPOSE: To evaluate MR-derived pancreatic T2 mapping values in AP and correlate them with markers of disease severity. STUDY TYPE: Prospective single-center study. POPULATION: 76 adults with AP (20-91 years, females/males: 39/37). FIELD STRENGTH/SEQUENCE: Fat suppressed multiecho spin-echo prototype sequence to quantify T2 signal at 3T MRI. ASSESSMENT: The severity of AP was assessed clinically, biologically, and by contrast-enhanced CT (CECT) performed 48-72 hours after symptom onset. MRI was then performed ≤24 hours after CT. Two readers blinded to any clinical information independently evaluated the T2 values by placing three regions of interest inside the pancreatic head, body, and tail on the T2 mapping MR sequence. Results were compared with corresponding CECT images as the standard and clinical severity parameters, using the length of hospital stay as our primary endpoint. STATISTICAL TESTS: Continuous variables were compared using the Spearman's rank correlation coefficient, analysis of variance (ANOVA) or Student's t-test. RESULTS: T2 values significantly correlated with the length of hospital stay (rs (74) = 0.29), CT severity index (CTSI) (rs (73) = 0.61; CTSI 0-3: 72 ± 14 msec, CTSI 4-10: 88 ± 15), intensive care unit (ICU) admission (t(2.77) = -3.41) and presence of organ failure (t(6.72) = -3.42), whereas the CTSI and Ranson score were not significantly related with ICU admission (CTSI: P = 0.24; Ranson score: P = 0.24) and organ failure (CTSI: P = 0.11; Ranson score P = 0.11). CONCLUSION: T2 mapping correlates with AP severity parameters and is useful for assessing the severity of AP with higher sensitivity than the usual clinical and radiological scoring systems. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 2.

Novel high-dense microelectrode array based multimodal bioelectronic monitoring system for cardiac arrhythmia re-entry analysis.

In recent decades, significant progress has been made in the treatment of heart diseases, particularly in the field of personalized medicine. Despite the development of genetic tests, phenotyping and risk stratification are performed based on clinical findings and invasive in vivo techniques, such as stimulation conduction mapping techniques and programmed ventricular pacing. Consequently, label-free non-invasive in vitro functional analysis systems are urgently needed for more accurate and effective in vitro risk stratification, model-based therapy planning, and clinical safety profile evaluation of drugs. To overcome these limitations, a novel multilayer high-density microelectrode array (HD-MEA), with an optimized configuration of 512 sensing and 4 pacing electrodes on a sensor area of 100 mm2, was developed for the bioelectronic detection of re-entry arrhythmia patterns. Together with a co-developed front-end, we monitored label-free and in parallel cardiac electrophysiology based on field potential monitoring and mechanical contraction using impedance spectroscopy at the same microelectrode. In proof of principle experiments, human induced pluripotent stem cell (hiPS)-derived cardiomyocytes were cultured on HD-MEAs and used to demonstrate the sensitive quantification of contraction strength modulation by cardioactive drugs such as blebbistatin (IC50 = 4.2 µM), omecamtiv and levosimendan. Strikingly, arrhythmia-typical rotor patterns (re-entry) can be induced by optimized electrical stimulation sequences and detected with high spatial resolution. Therefore, we provide a novel cardiac re-entry analysis system as a promising reference point for diagnostic approaches based on in vitro assays using patient-specific hiPS-derived cardiomyocytes.

Sediment discharge from Greenland's marine-terminating glaciers is linked with surface melt.

Sediment discharged from the Greenland Ice Sheet delivers nutrients to marine ecosystems around Greenland and shapes seafloor habitats. Current estimates of the total sediment flux are constrained by observations from land-terminating glaciers only. Addressing this gap, our study presents a budget derived from observations at 30 marine-margin locations. Analyzing sediment cores from nine glaciated fjords, we assess spatial deposition since 1950. A significant correlation is established between mass accumulation rates, normalized by surface runoff, and distance down-fjord. This enables calculating annual sediment flux at any fjord point based on nearby marine-terminating outlet glacier melt data. Findings reveal a total annual sediment flux of 1.324 + /- 0.79 Gt yr-1 over the period 2010-2020 from all marine-terminating glaciers to the fjords. These estimates are valuable for studies aiming to understand the basal ice sheet conditions and for studies predicting ecosystem changes in Greenland's fjords and offshore areas as the ice sheet melts and sediment discharge increase.

Microcavity well-plate for automated parallel bioelectronic analysis of 3D cell cultures.

Three-dimensional (3D) in vitro cell culture models serve as valuable tools for accurately replicating cellular microenvironments found in vivo. While cell culture technologies are rapidly advancing, the availability of non-invasive, real-time, and label-free analysis methods for 3D cultures remains limited. To meet the demand for higher-throughput drug screening, there is a demanding need for analytical methods that can operate in parallel. Microelectrode systems in combination with microcavity arrays (MCAs), offer the capability of spatially resolved electrochemical impedance analysis and field potential monitoring of 3D cultures. However, the fabrication and handling of small-scale MCAs have been labour-intensive, limiting their broader application. To overcome this challenge, we have established a process for creating MCAs in a standard 96-well plate format using high-precision selective laser etching. In addition, to automate and ensure the accurate placement of 3D cultures on the MCA, we have designed and characterized a plug-in tool using SLA-3D-printing. To characterize our new 96-well plate MCA-based platform, we conducted parallel analyses of human melanoma 3D cultures and monitored the effect of cisplatin in real-time by impedance spectroscopy. In the following we demonstrate the capabilities of the MCA approach by analysing contraction rates of human pluripotent stem cell-derived cardiomyocyte aggregates in response to cardioactive compounds. In summary, our MCA system significantly expands the possibilities for label-free analysis of 3D cell and tissue cultures, offering an order of magnitude higher parallelization capacity than previous devices. This advancement greatly enhances its applicability in real-world settings, such as drug development or clinical diagnostics.

Performance of three predictive scores to avoid delayed diagnosis of significant blunt bowel and mesenteric injury: A 12-year retrospective cohort study.

BACKGROUND: Avoiding missed diagnosis and therapeutic delay for significant blunt bowel and mesenteric injuries (sBBMIs) after trauma is still challenging despite the widespread use of computed tomography (CT). Several scoring tools aiming at reducing this risk have been published. The purpose of the present work was to assess the incidence of delayed (>24 hours) diagnosis for sBBMI patients and to compare the predictive performance of three previously published scores using clinical, radiological, and laboratory findings: the Bowel Injury Prediction Score (BIPS) and the scores developed by Raharimanantsoa Score (RS) and by Faget Score (FS). METHODS: A population-based retrospective observational cohort study was conducted; it included adult trauma patients after road traffic crashes admitted to Lausanne University Hospital, Switzerland, between 2008 and 2019 (n = 1,258) with reliable information about sBBMI status (n = 1,164) and for whom all items for score calculation were available (n = 917). The three scores were retrospectively applied on all patients to assess their predictive performance. RESULTS: The incidence of sBBMI after road traffic crash was 3.3% (38 of 1,164), and in 18% (7 of 38), there was a diagnostic and treatment delay of more than 24 hours. The diagnostic performances of the FS, the RS, and the BIPS to predict sBBMI, expressed as the area under the receiver operating characteristic curve, were 95.3% (95% confidence interval [CI], 92.7-97.9%), 89.2% (95% CI, 83.2-95.3%), and 87.6% (95% CI, 81.8-93.3%) respectively. CONCLUSION: The present study confirms that diagnostic delays for sBBMI still occur despite the widespread use of abdominal CT. When CT findings during the initial assessment are negative or equivocal for sBBMI, using a score may be helpful to select patients for early diagnostic laparoscopy. The FS had the best individual diagnostic performance. However, the BIPS or the RS, relying on clinical and laboratory variables, may be helpful to select patients for early diagnostic laparoscopy when there are unspecific CT signs of bowel or mesenteric injury. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites.

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in reduced susceptibility to ART. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites.

Functional, biological, and radiological evaluation of the pancreaticojejunal anastomosis 1 year after pancreatoduodenectomy: a prospective study.

PURPOSE: This prospective study aimed to analyze the functional, biological, and radiological aspects of the pancreatic anastomosis 1 year after pancreatoduodenectomy (PD). METHODS: From 2016 to 2019, patients with PD indication were screened. Questionnaires about pancreas insufficiency, fecal elastase tests, and magnetic resonance imaging (MRI) were performed before and 1 year after PD. RESULTS: Twenty patients were prospectively included. The only difference between pre- and postoperative questionnaires was constipation (less frequent 1 year after PD). Median pre- and postoperative fecal elastase levels were 96 µg/g (IQR 15-196, normal value > 200) and 15 µg/g (IQR 15-26, p = 0.042). There were no significant differences in terms of main pancreatic duct (MPD) size (4, IQR 3-5 vs. 4 mm, IQR 3-5, p = 0.892), border regularity, stenosis, visibility, image improvement, and secondary pancreatic duct dilation before and after secretin injection. All patients but one (2 refused and 2 were lost to follow-up, 15/16, 94%) had a patent pancreaticojejunal anastomosis on 1-year MRI. CONCLUSION: Although median 1-year fecal elastase was significantly lower than preoperatively, suggesting that exocrine secretion was altered, the anatomical outcome as assessed by MRI was excellent showing high patency rate (15/16, 94%) at 1 year. This emphasizes the difference between anatomy and function.

Infection Studies with Airway Organoids from Carollia perspicillata Indicate That the Respiratory Epithelium Is Not a Barrier for Interspecies Transmission of Influenza Viruses.

Bats are a natural reservoir for many viruses and are considered to play an important role in the interspecies transmission of viruses. To analyze the susceptibility of bat airway cells to infection by viruses of other mammalian species, we developed an airway organoid culture model derived from airways of Carollia perspicillata. Application of specific antibodies for fluorescent staining indicated that the cell composition of organoids resembled those of bat trachea and lungs as determined by immunohistochemistry. Infection studies indicated that Carollia perspicillata bat airway organoids (AOs) from the trachea or the lung are highly susceptible to infection by two different porcine influenza A viruses. The bat AOs were also used to develop an air-liquid interface (ALI) culture system of filter-grown epithelial cells. Infection of these cells showed the same characteristics, including lower virulence and enhanced replication and release of the H1N1/2006 virus compared to infection with H3N2/2007. These observations agreed with the results obtained by infection of porcine ALI cultures with these two virus strains. Interestingly, lectin staining indicated that bat airway cells only contain a small amount of alpha 2,6-linked sialic acid, the preferred receptor determinant for mammalian influenza A viruses. In contrast, large amounts of alpha 2,3-linked sialic acid, the preferred receptor determinant for avian influenza viruses, are present in bat airway epithelial cells. Therefore, bat airway cells may be susceptible not only to mammalian but also to avian influenza viruses. Our culture models, which can be extended to other parts of the airways and to other species, provide a promising tool to analyze virus infectivity and the transmission of viruses both from bats to other species and from other species to bats. IMPORTANCE We developed an organoid culture system derived from the airways of the bat species Carollia perspicillata. Using this cell system, we showed that the airway epithelium of these bats is highly susceptible to infection by influenza viruses of other mammalian species and thus is not a barrier for interspecies transmission. These organoids provide an almost unlimited supply of airway epithelial cells that can be used to generate well-differentiated epithelial cells and perform infection studies. The establishment of the organoid model required only three animals, and can be extended to other epithelia (nose, intestine) as well as to other species (bat and other animal species). Therefore, organoids promise to be a valuable tool for future zoonosis research on the interspecies transmission of viruses (e.g., bat → intermediate host → human).

A non-antibiotic-disrupted gut microbiome is associated with clinical responses to CD19-CAR-T cell cancer immunotherapy.

Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell lymphoma patient cohort from five centers in Germany and the United States (Germany, n = 66; United States, n = 106; total, n = 172), we demonstrate that wide-spectrum antibiotics treatment ('high-risk antibiotics') prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely to be confounded by an increased pretreatment tumor burden and systemic inflammation in patients pretreated with high-risk antibiotics. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were noted between pre-CAR-T infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-month survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides, Ruminococcus, Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also being associated with pre-infusion peripheral T cell levels in these patients. Collectively, we identify conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy.

MDCT-findings in patients with non-occlusive mesenteric ischemia (NOMI): influence of vasoconstrictor agents.

OBJECTIVES: To evaluate the influence of vasoconstrictor agents (VCAs) on signs of vasoconstriction and bowel ischemia on MDCT detected in patients with non-occlusive mesenteric ischemia (NOMI). METHODS: This 8-year single-center retrospective study consecutively included all patients with histopathologically proven NOMI who underwent MDCT ≤ 48 h prior to surgical bowel resection. Two blinded radiologists jointly reviewed each examination for signs of bowel ischemia, abdominal organ infarct, mesenteric vessel size and regularity, and ancillary vascular findings. VCA administration (length and dosage), clinical and biochemical data, risk factors, and outcomes were retrieved from patients' medical records. Subgroup comparisons were performed. RESULTS: Ninety patients were included (59 males, mean age 65 years); 40 (44.4%) had received VCAs before MDCT. Overall mortality was 32% (n = 29), with no significant difference between the two groups. In patients treated with VCAs, the calibre of the superior mesenteric artery (SMA) was smaller (p = 0.032), and vasoconstriction of its branches tended to be more important (p = 0.096) than in patients not treated with VCAs. The presence and extent of bowel ischemia did not significantly correlate with VCA administration, but abdominal organ infarcts tended to be more frequent (p = 0.005) and involved more organs (p = 0.088). The VCA group had lower mean arterial pressure (p = 0.006) and lower hemoglobin levels (p < 0.001). Several biomarkers of organ failure and inflammation, differed significantly with VCA use, proving worse clinical condition. CONCLUSIONS: MDCT demonstrates more severe SMA vasoconstriction and tends to show increased abdominal organ infarcts after VCA administration in NOMI patients compared to NOMI patients not treated with VCAs. KEY POINTS: • In critically ill patients with NOMI, MDCT demonstrates VCA support via increased vasoconstriction of the main SMA and its branches. • VCA administration in NOMI patients tends to contribute to the development of organ infarcts, as shown on MDCT. • An important degree of vasoconstriction in NOMI patients may indicate insufficient resuscitation and, thus, help clinicians in further patient management.

[Further Development and Interoperability in Oncological Care Structures, Quality Control and Research]. / Weiterentwicklung und Vernetzung der onkologischen Versorgungsstrukturen, der Qualitätssicherung und der Forschung.

How can we improve the interoperability of medical guidelines and the implementation and measurement of outcomes in medical health care for cancer patients as well as for care providers? This is the aim of the working group "Quality and Cross-linking". The following publication gives an overview of the targets reached in the development of guidelines together with quality indicators and documentation in cancer registries.

Neuronal and glial cell co-culture organization and impedance spectroscopy on nanocolumnar TiN films for lab-on-a-chip devices.

Lab-on-a-chip devices, such as multielectrode arrays (MEAs), offer great advantages to study function and behavior of biological cells, such as neurons, outside the complex tissue structure. Nevertheless, in vitro systems can only succeed if they represent realistic conditions such as cell organization as similarly found in tissues. In our study, we employ a co-culture system of neuron-like (SH-SY5Y) and glial-like (U-87 MG) cells with various neuron-glial ratios to model different brain regions with different cellular compositions in vitro. We find that cell behavior in terms of cellular organization, as well as proliferation, depends on neuron-glial cell ratio, as well as the underlying substrate material. In fact, nanocolumnar titanium nitride (TiN nano), which exhibits improved electric properties for neural recording on MEA, shows improved biocompatible features compared to indium tin oxide (ITO). Moreover, electrochemical impedance spectroscopy experiments allow us to monitor cellular processes label-free in real-time over several days with multielectrode arrays. Additionally, electrochemical impedance experiments reveal superiority of TiN with nanocolumnar surface modification in comparison with ITO. TiN nano exhibits enhanced relative cell signals and improved signal-to-noise ratio, especially for smaller electrode sizes, which makes nanocolumnar TiN a promising candidate for research on neural recording and stimulation.