Tick-borne piroplasms and trypanosomes incidentally detected in eastern grey kangaroos (Macropus giganteus) during a mortality and morbidity event in southern New South Wales, Australia.

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo (Macropus giganteus) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms (Babesia and Theileria) and trypanosomes in affected animals. Blood (n = 89) and liver (n = 19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal's health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals' blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby (Notamacropus rufogriseus). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event.

Procedural safety of rotational atherectomy and modified balloon angioplasty: insights from a German national registry.

BACKGROUND: Modified balloons (MB) and rotational atherectomy (RA) are recommended tools for treatment of coronary plaques with superficial calcium. Knowledge about in-hospital safety is limited. METHODS: Patients with coronary artery disease who underwent coronary angiography with RA or MB angioplasty in Germany were identified via ICD and OPS codes from 2017 to 2020. Acute coronary syndromes were excluded. Since patients were not randomized toward MB or RA, potential confounding factors were taken into account using the propensity score methods. Thereby, inverse probability weighting was applied. RESULTS: Ten thousand.ninety-twopatients underwent RA with an increasing trend from 1817 in 2017 toward 3166 in 2020. MBs were used in 22,378 patients also with an increasing trend from 4771 in 2017 toward 6078 in 2020. Patients receiving RA were older (74.23 ± 8.68 vs. 71.86 ± 10.02, p < 0.001), had a higher Charlson Comorbidity Index (2.07 ± 1.75 vs. 1.99 ± 1.76, p = 0.001) and more frequently left main (17.96% vs. 12.91%, p < 0.001) or three vessel disease (66.25% vs. 58.10%, p < 0.001). Adjusted procedural risk of major adverse cardiac and cerebrovascular events (MACCE) was similar in both groups, while pericardial effusion (RR 2.69; 95% CI 1.88-3.86, p < 0.001), pericardial puncture/pericardiotomy/pericardial tamponade (RR 2.66; 95% CI 1.85-3.81, p < 0.001) and bleeding (RR 1.65; 95% CI 1.12-2.43, p < 0.011) occurred more frequently in patients receiving RA. Patients treated with RA at high volume centers were hospitalized shorter (p = 0.005) and had a lower rate of acute cerebrovascular events (p < 0.001). Rate of MACCE, bleeding and pericardial puncture were not influenced by the annual RA numbers per center. CONCLUSION: MBs had a lower risk of bleeding and pericardial puncture. Patients treated at centers with high annual RA procedure numbers had a lower risk of acute cerebrovascular events and were hospitalized shorter.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Reweighting and validation of the hospital frailty risk score using electronic health records in Germany: a retrospective observational study.

BACKGROUND: In the hospital setting, frailty is a significant risk factor, but difficult to measure in clinical practice. We propose a reweighting of an existing diagnoses-based frailty score using routine data from a tertiary care teaching hospital in southern Germany. METHODS: The dataset includes patient characteristics such as sex, age, primary and secondary diagnoses and in-hospital mortality. Based on this information, we recalculate the existing Hospital Frailty Risk Score. The cohort includes patients aged ≥ 75 and was divided into a development cohort (admission year 2011 to 2013, N = 30,525) and a validation cohort (2014, N = 11,202). A limited external validation is also conducted in a second validation cohort containing inpatient cases aged ≥ 75 in 2022 throughout Germany (N = 491,251). In the development cohort, LASSO regression analysis was used to select the most relevant variables and to generate a reweighted Frailty Score for the German setting. Discrimination is assessed using the area under the receiver operating characteristic curve (AUC). Visualization of calibration curves and decision curve analysis were carried out. Applicability of the reweighted Frailty Score in a non-elderly population was assessed using logistic regression models. RESULTS: Reweighting of the Frailty Score included only 53 out of the 109 frailty-related diagnoses and resulted in substantially better discrimination than the initial weighting of the score (AUC = 0.89 vs. AUC = 0.80, p < 0.001 in the validation cohort). Calibration curves show a good agreement between score-based predictions and actual observed mortality. Additional external validation using inpatient cases aged ≥ 75 in 2022 throughout Germany (N = 491,251) confirms the results regarding discrimination and calibration and underlines the geographic and temporal validity of the reweighted Frailty Score. Decision curve analysis indicates that the clinical usefulness of the reweighted score as a general decision support tool is superior to the initial version of the score. Assessment of the applicability of the reweighted Frailty Score in a non-elderly population (N = 198,819) shows that discrimination is superior to the initial version of the score (AUC = 0.92 vs. AUC = 0.87, p < 0.001). In addition, we observe a fairly age-stable influence of the reweighted Frailty Score on in-hospital mortality, which does not differ substantially for women and men. CONCLUSIONS: Our data indicate that the reweighted Frailty Score is superior to the original Frailty Score for identification of older, frail patients at risk for in-hospital mortality. Hence, we recommend using the reweighted Frailty Score in the German in-hospital setting.

Curiosities take the stage - role-play in parasitology teaching.

Careful observation of parasites, masters of camouflage, reveals an ingenious and fascinating world. However, students often perceive parasitology as impenetrable. What if a flamboyant flea circus director passionately introduced the multidimensional contexts of this discipline? Will role-play capture the imagination of students and guide them in their future learning?

Harnessing cholesterol uptake of malaria parasites for therapeutic applications.

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.

Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer.

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1's affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1's in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer's cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1's high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Uncovering atherosclerotic cardiovascular disease by PET imaging.

Assessing atherosclerosis severity is essential for precise patient stratification. Specifically, there is a need to identify patients with residual inflammation because these patients remain at high risk of cardiovascular events despite optimal management of cardiovascular risk factors. Molecular imaging techniques, such as PET, can have an essential role in this context. PET imaging can indicate tissue-based disease status, detect early molecular changes and provide whole-body information. Advances in molecular biology and bioinformatics continue to help to decipher the complex pathogenesis of atherosclerosis and inform the development of imaging tracers. Concomitant advances in tracer synthesis methods and PET imaging technology provide future possibilities for atherosclerosis imaging. In this Review, we summarize the latest developments in PET imaging techniques and technologies for assessment of atherosclerotic cardiovascular disease and discuss the relationship between imaging readouts and transcriptomics-based plaque phenotyping.

Association of rheumatological markers with neuronal antibodies, cerebrospinal fluid, electroencephalography, and magnetic resonance imaging findings in 224 patients with psychotic syndromes.

INTRODUCTION: Psychotic syndromes can have autoimmune-mediated causes in some patients. Thus, this retrospective work aims to investigate the role of rheumatological markers in the development of psychosis. PATIENTS AND METHODS: In total, 224 patients with psychotic syndromes receiving a "rheumatological laboratory screening" (including C-reactive protein [CRP], immunofixation, complement factors, rheumatoid factor [RF], antiphospholipid antibodies [APAs], antineutrophil cytoplasmic antibodies [ANCAs], and antinuclear antibodies [ANAs]) were analyzed. A further diagnostic work-up included investigations of neuronal antibodies and cerebrospinal fluid (CSF), as well as electroencephalography (EEG) and magnetic resonance imaging (MRI) of the brain. ANA testing was routinely performed in all patients using serum on human epithelioma-2 (Hep2) cells, and a subset of patients (N = 73) also underwent tissue-based assays from serum and CSF. The number of cases with autoimmune psychotic syndromes was descriptively collected, and ANA-positive and -negative patients were compared in detail. RESULTS: CRP was elevated in 9 % of patients, immunofixation identified alterations in 8 %, complement factor C3 was decreased in 14 %, RF was elevated in 1 %, APAs were elevated in 7 %, ANCAs were not clearly positive, and ANAs were positive in 19 % (extractable nuclear antigen [ENA] differentiation resulted in positive findings in 14 patients). From the 73 patient samples additionally investigated using tissue-based assays, there were 26 positive results for some kind of ANA (36 %), and overall using both methods, 54 patients (24 %) were considered positive for ANAs. A neuropsychiatric evaluation revealed a possible autoimmune psychotic syndrome in seven patients (3 %) and a probable autoimmune psychotic syndrome in two patients (1 %). ANA-positive patients were more frequently treated with antidepressants (p = 0.040) and had a higher number of somatic comorbidities (p < 0.001). In addition, (chronic) inflammatory MRI lesions (p = 0.008) and focal atrophies (p = 0.012) were found more frequently in ANA-positive than ANA-negative patients. DISCUSSION: Rheumatological screening led to suspicion of a possible or probable autoimmune psychotic syndrome in 4%. ANAs were associated with MRI pathologies. Therefore, rheumatological processes may contribute to the development of psychotic syndromes in rare cases.

Correction: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

[This corrects the article DOI: 10.1371/journal.ppat.1011517.].

Combination of high resolution MRI with 3D-printed needle guides for ex vivo myocardial biopsies.

Magnetic resonance imaging (MRI) provides a multitude of techniques to detect and characterize myocardial infarction. To correlate MRI findings with histology, in most cases terminal animal studies are performed; however, precise extraction and spatial correlation of myocardial tissue samples to MRI image data is difficult. In this proof of concept study, we present a 3D-printing technique to facilitate the extraction of tissue samples from myocardial regions. Initially, seven pig hearts embedded in formaldehyde were imaged on a clinical 3 T system to define biopsy targets on high resolution ex vivo images. Magnitude images and R2*-maps acquired with a 3D multi-echo gradient echo sequence and 0.58 mm isotropic resolution were used to create digital models of the cardiac anatomy. Biopsy guides were 3D-printed to steer the extraction of myocardial samples. In total, 61 tissue samples were extracted with an average offset of the tissue sample location from the target location of 0.59 ± 0.36 mm. This offset was not dependent on the distance of the target point to the epicardial surface. Myocardial tissue could be extracted from all samples. The presented method enables extraction of myocardial tissue samples that are selected by ex vivo MRI with submillimeter precision.

Cardioaortic dimensions in German landrace pigs derived from cardiac magnetic resonance imaging.

Pigs are frequently applied as animal models in cardiovascular research due to their anatomical and physiological similarity to humans. For study planning and refinement, precise knowledge of the cardioaortic dimensions is essential. In a retrospective single-center study, the cardioaortic dimensions and left ventricular function of German Landrace pigs were assessed using cardiac MRI. All parameters were compared between male and female pigs and analyzed for correlation with body weight. In total, 15 pigs were included (7 male and 8 female, weight 60.9 ± 7.0 kg). The left ventricle revealed an end-diastolic diameter of 50.5 ± 4.4 mm and an ejection fraction of 51.2 ± 9.8%. The diameters of the ascending and descending aorta were 21.3 ± 2.3 and 16.2 ± 1.4 mm, respectively. There were no significant differences between male and female pigs, except that males had a smaller end-diastolic left ventricular volume (p = 0.041). A moderate correlation was found between body weight and the aortic annulus diameter (R = 0.57, p = 0.027). In conclusion, cardiac MRI allows precise quantification of porcine cardioaortic dimensions. For medical device testing, size differences between pigs and humans should be considered.

A ubiquitous spectrolaminar motif of local field potential power across the primate cortex.

The mammalian cerebral cortex is anatomically organized into a six-layer motif. It is currently unknown whether a corresponding laminar motif of neuronal activity patterns exists across the cortex. Here we report such a motif in the power of local field potentials (LFPs). Using laminar probes, we recorded LFPs from 14 cortical areas across the cortical hierarchy in five macaque monkeys. The laminar locations of recordings were histologically identified by electrolytic lesions. Across all areas, we found a ubiquitous spectrolaminar pattern characterized by an increasing deep-to-superficial layer gradient of high-frequency power peaking in layers 2/3 and an increasing superficial-to-deep gradient of alpha-beta power peaking in layers 5/6. Laminar recordings from additional species showed that the spectrolaminar pattern is highly preserved among primates-macaque, marmoset and human-but more dissimilar in mouse. Our results suggest the existence of a canonical layer-based and frequency-based mechanism for cortical computation.

Comparing balloon-expandable and self-expanding transfemoral transcatheter aortic valve replacement based on subgroups in Germany 2019/2020.

BACKGROUND: Previously, overall comparable outcomes were seen for balloon-expandable (BE) or self-expanding (SE) transfemoral transcatheter aortic valve replacement (TAVR). However, subgroup analyses based on large case numbers are still needed. METHODS: German national data of all BE and SE transfemoral TAVR treating aortic valve stenosis in 2019 and 2020 were analysed. We then compared different outcomes and performed a subgroup analysis for the endpoint in-hospital mortality. RESULTS: Overall, 46,243 TAVR were analysed, 19,910 BE, and 26,333 SE. Patients in the SE group had a significantly higher logistic EuroSCORE (13.61 vs 12.66%, p < 0.001), age (81.55 vs 79.99a, p < 0.001), and proportion of women (54.82 vs 40.06%, p < 0.001). Both groups showed a similar in-hospital mortality with 2.37% in BE and 2.35% in SE (p = 0.916). In-hospital mortality also did not differ significantly after risk adjustment (OR = 0.98 [0.86, 1.13], p = 0.799). Patients in the SE group had a significantly lower risk of major bleeding (OR = 0.83 [0.73, 0.95], p = 0.006), but a significantly higher risk of stroke (OR = 1.38 [1.19, 1.59], p < 0.001), delirium (OR = 1.15 [1.06, 1.24], p = 0.001), and permanent pacemaker implantation (OR = 1.29 [1.21, 1.37], p < 0.001). In the subgroup analysis of in-hospital mortality, there were no significant differences in any of the observed subgroups (age < 75/75-79/80-84/ ≥ 85a, logistic EuroSCORE < 4/4- < 9/ ≥ 9, gender, NYHA III/IV, previous CABG, peripheral vascular disease, COPD, pulmonary hypertension, renal disease GFR < 30 ml/min, and diabetes mellitus). CONCLUSION: In the direct comparison of balloon-expandable and self-expanding TAVR, there are no differences for in-hospital mortality in subgroups.

Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays.

The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O2). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum, a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O2 consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O2 consumption in permeabilized asexual P. falciparum parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.

A role for ocular dominance in binocular integration.

Neurons in the primate primary visual cortex (V1) combine left- and right-eye information to form a binocular output. Controversy surrounds whether ocular dominance, the preference of these neurons for one eye over the other, is functionally relevant. Here, we demonstrate that ocular dominance impacts gain control during binocular combination. We recorded V1 spiking activity while monkeys passively viewed grating stimuli. Gratings were either presented to one eye (monocular), both eyes with the same contrasts (binocular balanced), or both eyes with different contrasts (binocular imbalanced). We found that contrast placed in a neuron's dominant eye was weighted more strongly than contrast placed in a neuron's non-dominant eye. This asymmetry covaried with neurons' ocular dominance. We then tested whether accounting for ocular dominance within divisive normalization improves the fit to neural data. We found that ocular dominance significantly improved model performance, with interocular normalization providing the best fits. These findings suggest that V1 ocular dominance is relevant for response normalization during binocular stimulation.

Catheter based left atrial appendage closure in-hospital outcomes in Germany from 2016 to 2020.

BACKGROUND: New and refined catheter based left atrial appendage (LAA) closure devices have been introduced in the past decade. The procedure can be performed using either an endocardial occlusion device or an epicardial loop stitch. We aimed to analyzed recent procedural safety. METHODS: Catheter based LAA closures were identified in a complete nationwide German dataset via ICD and OPS codes from 2016 to 2020. RESULTS: From 2016 to 2020, 28,039 endocardial and 213 epicardial occlusions were performed. Numbers of endocardial procedures increased from 5259 in 2016 to 5917 in 2020 (p = 0.020) in 387 centers with shifting of patients' characteristics towards older age (ß = 0.29, p < 0.001), more heart failure (ß = 1.01, p < 0.001) and renal disease (ß = 0.67, p = 0.001) and without a significant trend for in-hospital safety except more bleeding (ß = 0.12, p = 0.05). In-hospital major adverse cardiac and cerebrovascular events (MACCE) or pericardial puncture were independent on center procedure numbers. The loop stitch procedure was performed in 15 centers. Patients were younger (76.17 ± 8.16 vs. 73.16 ± 8.99, p < 0.001) and had a lower comorbidity index (2.29 ± 1.93 vs. 1.92 ± 1.64, p = 0.005). Adjusted risk difference for pericardial effusion (8.04%; 95% CI 3.01-13.08%; p = 0.002) and pericardial puncture (6.60%; 95% CI 3.85-9.35%; p < 0.001) was higher for the loop stitch procedure, while risk of bleeding (- 1.85%; 95% CI - 3.01 to - 0.69%; p = 0.002), intracerebral bleeding (- 0.37%; 95% CI - 0.59 to - 0.15%; p = 0.001) and shock (- 1.41%; 95% CI - 2.44 to - 0.39%; p = 0.007) was lower. No significant difference was observed for in-hospital MACCE. CONCLUSIONS: Endocardial occlusion was the major catheter based LAA closure procedure in Germany without improvements in in-hospital safety from 2016 to 2020. In-hospital MACCE was independent on endocardial LAAC center volumes. Conclusions on the comparison between the two procedure types must be made cautious as the LAA loop stitch occlusion was utilized limited in a minor number of centers. Catheter based left atrial appendage closure in-hospital outcomes in Germany from 2016 to 2020.