Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion.

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.

Computer-aided characterization of early cancer in Barrett's esophagus on i-scan magnification imaging: a multicenter international study.

BACKGROUND AND AIMS: We aimed to develop a computer-aided characterization system that could support the diagnosis of dysplasia in Barrett's esophagus (BE) on magnification endoscopy. METHODS: Videos were collected in high-definition magnification white-light and virtual chromoendoscopy with i-scan (Pentax Hoya, Japan) imaging in patients with dysplastic and nondysplastic BE (NDBE) from 4 centers. We trained a neural network with a Resnet101 architecture to classify frames as dysplastic or nondysplastic. The network was tested on 3 different scenarios: high-quality still images, all available video frames, and a selected sequence within each video. RESULTS: Fifty-seven patients, each with videos of magnification areas of BE (34 dysplasia, 23 NDBE), were included. Performance was evaluated by a leave-1-patient-out cross-validation method. In all, 60,174 (39,347 dysplasia, 20,827 NDBE) magnification video frames were used to train the network. The testing set included 49,726 i-scan-3/optical enhancement magnification frames. On 350 high-quality still images, the network achieved a sensitivity of 94%, specificity of 86%, and area under the receiver operator curve (AUROC) of 96%. On all 49,726 available video frames, the network achieved a sensitivity of 92%, specificity of 82%, and AUROC of 95%. On a selected sequence of frames per case (total of 11,471 frames), we used an exponentially weighted moving average of classifications on consecutive frames to characterize dysplasia. The network achieved a sensitivity of 92%, specificity of 84%, and AUROC of 96%. The mean assessment speed per frame was 0.0135 seconds (SD ± 0.006). CONCLUSION: Our network can characterize BE dysplasia with high accuracy and speed on high-quality magnification images and sequence of video frames, moving it toward real-time automated diagnosis.

Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity.

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity-and not inverse similarity-between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.

Identifying key mechanisms leading to visual recognition errors for missed colorectal polyps using eye-tracking technology.

BACKGROUND AND AIM: Lack of visual recognition of colorectal polyps may lead to interval cancers. The mechanisms contributing to perceptual variation, particularly for subtle and advanced colorectal neoplasia, have scarcely been investigated. We aimed to evaluate visual recognition errors and provide novel mechanistic insights. METHODS: Eleven participants (seven trainees and four medical students) evaluated images from the UCL polyp perception dataset, containing 25 polyps, using eye-tracking equipment. Gaze errors were defined as those where the lesion was not observed according to eye-tracking technology. Cognitive errors occurred when lesions were observed but not recognized as polyps by participants. A video study was also performed including 39 subtle polyps, where polyp recognition performance was compared with a convolutional neural network. RESULTS: Cognitive errors occurred more frequently than gaze errors overall (65.6%), with a significantly higher proportion in trainees (P = 0.0264). In the video validation, the convolutional neural network detected significantly more polyps than trainees and medical students, with per-polyp sensitivities of 79.5%, 30.0%, and 15.4%, respectively. CONCLUSIONS: Cognitive errors were the most common reason for visual recognition errors. The impact of interventions such as artificial intelligence, particularly on different types of perceptual errors, needs further investigation including potential effects on learning curves. To facilitate future research, a publicly accessible visual perception colonoscopy polyp database was created.

Polyp characterization using deep learning and a publicly accessible polyp video database.

OBJECTIVES: Convolutional neural networks (CNN) for computer-aided diagnosis of polyps are often trained using high-quality still images in a single chromoendoscopy imaging modality with sessile serrated lesions (SSLs) often excluded. This study developed a CNN from videos to classify polyps as adenomatous or nonadenomatous using standard narrow-band imaging (NBI) and NBI-near focus (NBI-NF) and created a publicly accessible polyp video database. METHODS: We trained a CNN with 16,832 high and moderate quality frames from 229 polyp videos (56 SSLs). It was evaluated with 222 polyp videos (36 SSLs) across two test-sets. Test-set I consists of 14,320 frames (157 polyps, 111 diminutive). Test-set II, which is publicly accessible, 3317 video frames (65 polyps, 41 diminutive), which was benchmarked with three expert and three nonexpert endoscopists. RESULTS: Sensitivity for adenoma characterization was 91.6% in test-set I and 89.7% in test-set II. Specificity was 91.9% and 88.5%. Sensitivity for diminutive polyps was 89.9% and 87.5%; specificity 90.5% and 88.2%. In NBI-NF, sensitivity was 89.4% and 89.5%, with a specificity of 94.7% and 83.3%. In NBI, sensitivity was 85.3% and 91.7%, with a specificity of 87.5% and 90.0%, respectively. The CNN achieved preservation and incorporation of valuable endoscopic innovations (PIVI)-1 and PIVI-2 thresholds for each test-set. In the benchmarking of test-set II, the CNN was significantly more accurate than nonexperts (13.8% difference [95% confidence interval 3.2-23.6], P = 0.01) with no significant difference with experts. CONCLUSIONS: A single CNN can differentiate adenomas from SSLs and hyperplastic polyps in both NBI and NBI-NF. A publicly accessible NBI polyp video database was created and benchmarked.

Role of Conformational Dynamics of Sulfotransferases SULT1A1 and SULT1A3 in Substrate Specificity.

Sulfotransferases (SULTs) are phase II metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3'-Phosphoadenosine 5'-Phosphosulfate (PAPS) to a wide variety of endogenous compounds, drugs and natural products. Although SULT1A1 and SULT1A3 share 93% identity, SULT1A1, the most abundant SULT isoform in humans, exhibits a broad substrate range with specificity for small phenolic compounds, while SULT1A3 displays a high affinity toward monoamine neurotransmitters like dopamine. To elucidate the factors determining the substrate specificity of the SULT1 isoenzymes, we studied the dynamic behavior and structural specificities of SULT1A1 and SULT1A3 by using molecular dynamics (MD) simulations and ensemble docking of common and specific substrates of the two isoforms. Our results demonstrated that while SULT1A1 exhibits a relatively rigid structure by showing lower conformational flexibility except for the lip (loop L1), the loop L2 and the cap (L3) of SULT1A3 are extremely flexible. We identified protein residues strongly involved in the recognition of different substrates for the two isoforms. Our analyses indicated that being more specific and highly flexible, the structure of SULT1A3 has particularities in the binding site, which are crucial for its substrate selectivity.

Performance of artificial intelligence for detection of subtle and advanced colorectal neoplasia.

OBJECTIVES: There is uncertainty regarding the efficacy of artificial intelligence (AI) software to detect advanced subtle neoplasia, particularly flat lesions and sessile serrated lesions (SSLs), due to low prevalence in testing datasets and prospective trials. This has been highlighted as a top research priority for the field. METHODS: An AI algorithm was evaluated on four video test datasets containing 173 polyps (35,114 polyp-positive frames and 634,988 polyp-negative frames) specifically enriched with flat lesions and SSLs, including a challenging dataset containing subtle advanced neoplasia. The challenging dataset was also evaluated by eight endoscopists (four independent, four trainees, according to the Joint Advisory Group on gastrointestinal endoscopy [JAG] standards in the UK). RESULTS: In the first two video datasets, the algorithm achieved per-polyp sensitivities of 100% and 98.9%. Per-frame sensitivities were 84.1% and 85.2%. In the subtle dataset, the algorithm detected a significantly higher number of polyps (P < 0.0001), compared to JAG-independent and trainee endoscopists, achieving per-polyp sensitivities of 79.5%, 37.2% and 11.5%, respectively. Furthermore, when considering subtle polyps detected by both the algorithm and at least one endoscopist, the AI detected polyps significantly faster on average. CONCLUSIONS: The AI based algorithm achieved high per-polyp sensitivities for advanced colorectal neoplasia, including flat lesions and SSLs, outperforming both JAG independent and trainees on a very challenging dataset containing subtle lesions that could have been overlooked easily and contribute to interval colorectal cancer. Further prospective trials should evaluate AI to detect subtle advanced neoplasia in higher risk populations for colorectal cancer.

Feasibility of intraprocedural integration of cardiac CT to guide left ventricular lead implantation for CRT upgrades.

BACKGROUND: Optimal positioning of the left ventricular (LV) lead is an important determinant of cardiac resynchronization therapy (CRT) response. OBJECTIVE: Evaluate the feasibility of intraprocedural integration of cardiac computed tomography (CT) to guide LV lead implantation for CRT upgrades. METHODS: Patients undergoing LV lead upgrade underwent ECG-gated cardiac CT dyssynchrony and LV scar assessment. Target American Heart Association segment selection was determined using latest non-scarred mechanically activating segments overlaid onto real-time fluoroscopy with image co-registration to guide optimal LV lead implantation. Hemodynamic validation was performed using a pressure wire in the LV cavity (dP/dtmax) ). RESULTS: 18 patients (male 94%, 55.6% ischemic cardiomyopathy) with RV pacing burden 60.0 ± 43.7% and mean QRS duration 154 ± 30 ms underwent cardiac CT. 10/10 ischemic patients had CT evidence of scar and these segments were excluded as targets. Seventeen out of 18 (94%) patients underwent successful LV lead implantation with delivery to the CT target segment in 15 out of 18 (83%) of patients. Acute hemodynamic response (dP/dtmax ≥ 10%) was superior with LV stimulation in CT target versus nontarget segments (83.3% vs. 25.0%; p = .012). Reverse remodeling at 6 months (LV end-systolic volume improvement ≥15%) occurred in 60% of subjects (4/8 [50.0%] ischemic cardiomyopathy vs. 5/7 [71.4%] nonischemic cardiomyopathy, p = .608). CONCLUSION: Intraprocedural integration of cardiac CT to guide optimal LV lead placement is feasible with superior hemodynamics when pacing in CT target segments and favorable volumetric response rates, despite a high proportion of patients with ischemic cardiomyopathy. Multicentre, randomized controlled studies are needed to evaluate whether intraprocedural integration of cardiac CT is superior to standard care.

Total area of spontaneous portosystemic shunts independently predicts hepatic encephalopathy and mortality in liver cirrhosis.

BACKGROUND & AIMS: Spontaneous portosystemic shunts (SPSS) frequently develop in liver cirrhosis. Recent data suggested that the presence of a single large SPSS is associated with complications, especially overt hepatic encephalopathy (oHE). However, the presence of >1 SPSS is common. This study evaluates the impact of total cross-sectional SPSS area (TSA) on outcomes in patients with liver cirrhosis. METHODS: In this retrospective international multicentric study, CT scans of 908 cirrhotic patients with SPSS were evaluated for TSA. Clinical and laboratory data were recorded. Each detected SPSS radius was measured and TSA calculated. One-year survival was the primary endpoint and acute decompensation (oHE, variceal bleeding, ascites) was the secondary endpoint. RESULTS: A total of 301 patients (169 male) were included in the training cohort. Thirty percent of all patients presented with >1 SPSS. A TSA cut-off of 83 mm2 was used to classify patients with small or large TSA (S-/L-TSA). Patients with L-TSA presented with higher model for end-stage liver disease score (11 vs. 14) and more commonly had a history of oHE (12% vs. 21%, p <0.05). During follow-up, patients with L-TSA experienced more oHE episodes (33% vs. 47%, p <0.05) and had lower 1-year survival than those with S-TSA (84% vs. 69%, p <0.001). Multivariate analysis identified L-TSA (hazard ratio 1.66; 95% CI 1.02-2.70, p <0.05) as an independent predictor of mortality. An independent multicentric validation cohort of 607 patients confirmed that patients with L-TSA had lower 1-year survival (77% vs. 64%, p <0.001) and more oHE development (35% vs. 49%, p <0.001) than those with S-TSA. CONCLUSION: This study suggests that TSA >83 mm2 increases the risk for oHE and mortality in patients with cirrhosis. Our results support the clinical use of TSA/SPSS for risk stratification and decision-making in the management of patients with cirrhosis. LAY SUMMARY: The prevalence of spontaneous portosystemic shunts (SPSS) is higher in patients with more advanced chronic liver disease. The presence of more than 1 SPSS is common in advanced chronic liver disease and is associated with the development of hepatic encephalopathy. This study shows that total cross-sectional SPSS area (rather than diameter of the single largest SPSS) predicts survival in patients with advanced chronic liver disease. Our results support the clinical use of total cross-sectional SPSS area for risk stratification and decision-making in the management of SPSS.

Protocol analysis of dual-energy CT for optimization of kidney stone detection in virtual non-contrast reconstructions.

OBJECTIVES: Previous studies have shown that split-bolus protocols in virtual non-contrast (VNC) reconstructions of dual-energy computed tomography (DE-CT) significantly decrease radiation dose in patients with urinary stone disease. To evaluate the impact on kidney stone detection rate of stone composition, size, tube voltage, and iodine concentration for VNC reconstructions of DE-CT. METHODS: In this prospective study, 16 kidney stones of different sizes (1.2-4.5 mm) and compositions (struvite, cystine, whewellite, brushite) were placed within a kidney phantom. Seventy-two scans with nine different iodine contrast agents/saline solutions with increasing attenuation (0-1400 HU) and different kilovoltage settings (70 kV/150 kV; 80 kV/150 kV; 90 kV/150 kV; 100 kV/150 kV) were performed. Two experienced radiologists independently rated the images for the presence and absence of stones. Multivariate classification tree analysis and descriptive statistics were used to evaluate the diagnostic performance. RESULTS: Classification tree analysis revealed a higher detection rate of renal calculi > 2 mm in size compared with that of renal calculi < 2 mm (84.7%; 12.7%; p < 0.001). For stones with a diameter > 2 mm, the best results were found at 70 kV/Sn 150 kV and 80 kV/Sn 150 kV in scans with contrast media attenuation of 600 HU or less, with sensitivity of 99.6% and 96.0%, respectively. A higher luminal attenuation (> 600 HU) resulted in a significantly decreased detection rate (91.8%, 0-600 HU; 70.7%, 900-1400 HU; p < 0.001). In our study setup, the detection rates were best for cystine stones. CONCLUSION: Scan protocols in DE-CT with lower tube current and lower contrast medium attenuation show excellent results in VNC for stones larger than 2 mm but have limitations for small stones. KEY POINTS: • The detection rate of virtual non-contrast reconstructions is highly dependent on the surrounding contrast medium attenuation at the renal pelvis and should be kept as low as possible, as at an attenuation higher than 600 HU the VNC reconstructions are susceptible to masking ureteral stones. • Protocols with lower tube voltages (70 kV/Sn 150 kV and 80 kV/Sn 150 kV) improve the detection rate of kidney stones in VNC reconstructions. • The visibility of renal stones in virtual non-contrast of dual-energy CT is highly associated with the size, and results in a significantly lower detection rate in stones below 2 mm.

Association Between Portosystemic Shunts and Increased Complications and Mortality in Patients With Cirrhosis.

BACKGROUND & AIMS: Spontaneous portosystemic shunts (SPSS) have been associated with hepatic encephalopathy (HE). Little is known about their prevalence among patients with cirrhosis or clinical effects. We investigated the prevalence and characteristics of SPSS in patients with cirrhosis and their outcomes. METHODS: We performed a retrospective study of 1729 patients with cirrhosis who underwent abdominal computed tomography or magnetic resonance imaging analysis from 2010 through 2015 at 14 centers in Canada and Europe. We collected data on demographic features, etiology of liver disease, comorbidities, complications, treatments, laboratory and clinical parameters, Model for End-Stage Liver Disease (MELD) score, and endoscopy findings. Abdominal images were reviewed by a radiologist (or a hepatologist trained by a radiologist) and searched for the presence of SPSS, defined as spontaneous communications between the portal venous system or splanchnic veins and the systemic venous system, excluding gastroesophageal varices. Patients were assigned to groups with large SPSS (L-SPSS, ≥8 mm), small SPSS (S-SPSS, <8 mm), or without SPSS (W-SPSS). The main outcomes were the incidence of complications of cirrhosis and mortality according to the presence of SPSS. Secondary measurements were the prevalence of SPSS in patients with cirrhosis and their radiologic features. RESULTS: L-SPSS were identified in 488 (28%) patients, S-SPSS in 548 (32%) patients, and no shunt (W-SPSS) in 693 (40%) patients. The most common L-SPSS was splenorenal (46% of L-SPSS). The presence and size of SPSS increased with liver dysfunction: among patients with MELD scores of 6-9, 14% had L-SPSS and 28% had S-SPSS; among patients with MELD scores of 10-13, 30% had L-SPSS and 34% had S-SPSS; among patients with MELD scores of 14 or higher, 40% had L-SPSS and 32% had S-SPSS (P < .001 for multiple comparison among MELD groups). HE was reported in 48% of patients with L-SPSS, 34% of patients with S-SPSS, and 20% of patients W-SPSS (P < .001 for multiple comparison among SPSS groups). Recurrent or persistent HE was reported in 52% of patients with L-SPSS, 44% of patients with S-SPSS, and 37% of patients W-SPSS (P = .007 for multiple comparison among SPSS groups). Patients with SPSS also had a larger number of portal hypertension-related complications (bleeding or ascites) than those W-SPSS. Quality of life and transplantation-free survival were lower in patients with SPSS vs without. SPSS were an independent factor associated with death or liver transplantation (hazard ratio, 1.26; 95% confidence interval, 1.06-1.49) (P = .008) in multivariate analysis. When patients were stratified by MELD score, SPSS were associated with HE independently of liver function: among patients with MELD scores of 6-9, HE was reported in 23% with L-SPSS, 12% with S-SPSS, and 5% with W-SPSS (P < .001 for multiple comparison among SPSS groups); among those with MELD scores of 10-13, HE was reported in 48% with L-SPSS, 33% with S-SPSS, and 23% with W-SPSS (P < .001 for multiple comparison among SPSS groups); among patients with MELD scores of 14 or more, HE was reported in 59% with L-SPSS, 57% with S-SPSS, and 48% with W-SPSS (P = .043 for multiple comparison among SPSS groups). Patients with SPSS and MELD scores of 6-9 were at higher risk for ascites (40.5% vs 23%; P < .001) and bleeding (15% vs 9%; P = .038) than patients W-SPSS and had lower odds of transplant-free survival (hazard ratio 1.71; 95% confidence interval, 1.16-2.51) (P = .006). CONCLUSIONS: In a retrospective analysis of almost 2000 patients, we found 60% to have SPSS; prevalence increases with deterioration of liver function. SPSS increase risk for HE and with a chronic course. In patients with preserved liver function, SPSS increase risk for complications and death. ClinicalTrials.gov ID NCT02692430.

Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions.

Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism.

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

The ribs unfolded - a CT visualization algorithm for fast detection of rib fractures: effect on sensitivity and specificity in trauma patients.

OBJECTIVE: To assess a radiologist's detection rate of rib fractures in trauma CT when reading curved planar reformats (CPRs) of the ribs compared to reading standard MPRs. METHODS: Two hundred and twenty trauma CTs (146 males, 74 females) were retrospectively subjected to a software algorithm to generate CPRs of the ribs. Patients were split into two equal groups. Sixteen patients were excluded due to insufficient segmentation, leaving 107 patients in group A and 97 patients in group B. Two radiologists independently evaluated group A using CPRs and group B using standard MPRs. Two different radiologists reviewed both groups with the inverse methods setting. Results were compared to a standard of reference created by two senior radiologists. RESULTS: The reference standard identified 361 rib fractures in 61 patients. Reading CPRs showed a significantly higher overall sensitivity (P < 0.001) for fracture detection than reading standard MPRs, with 80.9% (584/722) and 71.5% (516/722), respectively. Mean reading time was significantly shorter for CPRs (31.3 s) compared to standard MPRs (60.7 s; P < 0.001). CONCLUSION: Using CPRs for the detection of rib fractures accelerates the reading of trauma patient chest CTs, while offering an increased overall sensitivity compared to conventional standard MPRs. KEY POINTS: • In major blunt trauma, rib fractures are diagnosed with Computed Tomography. • Image processing can unfold all ribs into a single plane. • Unfolded ribs can be read twice as fast as axial images. • Unfolding the ribs allows a more accurate diagnosis of rib fractures.

Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor.

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or ß(2) adrenergic (ß2AR) receptors and fluorescently tagged proteins (ß-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P(2) in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P(2). Here we show that ligand-induced interaction of AT1, 5HT2C and ß(2)A receptors with ß-arrestin-2 was unaffected by PtdIns(4,5)P(2) depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P(2). Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P(2) depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P(2), these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Rapid detection of bone metastasis at thoracoabdominal CT: accuracy and efficiency of a new visualization algorithm.

PURPOSE: To retrospectively assess the use of a combination of cancellous bone reconstructions (CBR) and multiplanar reconstructions (MPRs) for the detection of bone metastases at thoracoabdominal computed tomography (CT) compared with the use of MPRs alone. MATERIALS AND METHODS: The study was approved by the local institutional review board. Included were 156 consecutive patients with confirmed cancer who underwent a whole-body positron emission tomography (PET)/CT examination for clinical purposes (93 male and 63 female patients; mean age ± standard deviation, 59.8 years ± 14.9; range, 11-85 years). Only the CT images were processed with the CBR algorithm, which segments the bones and removes the cortical layer from the images. The PET images served as part of the reference standard. Images from 15 patients were used as a training set. Four radiologists independently evaluated images of half of the remaining 141 patients by using CBRs and MPRs together, and the other half by using MPRs only. Radiologists were blinded to patient names, and patient order was randomized. Results for detection rates and reporting time were recorded and compared with a standard of reference for each patient that was created by one senior radiologist and one nuclear medicine specialist by using all available CT and PET data, CBRs, and follow-up examinations. General estimation equations were used for statistical analysis. RESULTS: There were 349 lesions found in 103 patients, with 203 classified as malignant. Each patient was assessed by two readers per method, leading to a total of 698 lesions. The detection rate for all bone lesions was 35% (247 of 698) for MPRs and 74% (520 of 698) when CBRs and MPRs were used together, which was significantly higher (P < .001). The average reading time decreased from 85 to 43 seconds (P < .001) when both reconstructions were used. CONCLUSION: Advanced visualization of cancellous bone significantly increased the detection of bone metastases and reduced the time for interpretation.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

Dynamic Perviousness Predicts Revascularization Success in Acute Ischemic Stroke.

BACKGROUND: The predictive value of thrombus perviousness in acute ischemic stroke (AIS), as measured by computed tomography (CT), has been intensively studied with conflicting results. In this study, we investigate the predictive potential of the novel concept of dynamic perviousness using three-dimensional (3D) volumetric evaluation of occlusive thrombi. METHODS: The full thrombus volume in 65 patients with a hyperdense artery sign on non-contrast CT (NCCT), who underwent mechanical thrombectomy (MT), was segmented. Perviousness maps were computed voxel-wise for the entire thrombus volume as thrombus attenuation increase (TAI) between NCCT and CT angiography (CTA) as well as between CTA and late venous phase CT (CTV). Perviousness was analyzed for its association with NIHSS at admission, Thrombolysis In Cerebral Infarction (TICI) score, and number of MT passes. RESULTS: The mean late-uptake TAI of thrombi with NIHSS scores greater than 21 at admission was approximately 100% higher than for lower scored NIHSS (p between 0.05 and 0.005). Concerning revascularization results, thrombi requiring less than four MT passes had ca. 80% higher group mean late-uptake TAI than clots requiring four or more passes (p = 0.03), and thrombi with TICI score III had ca. 95% higher group mean late-uptake TAI than thrombi with TICI II (p = 0.03). Standard perviousness showed no significant correlation with MT results. CONCLUSION: Standard thrombus perviousness of 3D clot volume is not associated with revascularization results in AIS. In contrast, dynamic perviousness assessed with a voxel-wise characterization of 3D thrombi volume may be a better predictor of MT outcomes than standard perviousness.

Dynamic Perviousness: A Novel Imaging Marker for Predicting Mechanical Thrombectomy Outcomes in Acute Ischemic Stroke.

BACKGROUND: The predictive value of thrombus standard perviousness (SP) in acute ischemic stroke (AIS) for the technical success rates of mechanical thrombectomy (MT) or functional outcomes is not yet conclusive. We investigated the relationship between dynamic perviousness (DP) and revascularization results using time-dependent enhancement curve types determined with computed tomography (CT). METHODS: A retrospective analysis of 137 AIS patients was performed. DP was calculated as the thrombus attenuation increase (TAI) using three time points and categorized into four groups: (1) no enhancement (CNE); (2) late enhancement (CLE); (3) early enhancement with washout (CW); (4) early enhancement without washout (CNW). Associations with the technical success rate and functional outcomes were assessed. RESULTS: Late enhancement (CLE) had approximately two times higher odds for successful MT as compared to clots with other enhancement dynamics. The odds ratios (logistic regression model with CNW as the reference) for the TICI III scores were 4.04 (p = 0.067), 1.82 (p = 0.3), and 1.69 (p = 0.4) for CLE, CW, and CNE, respectively. The NIHSS scores at discharge and mRS scores at three months showed regression coefficients (linear regression model with CNW as reference) of -3.05 (p = 0.10), -1.17 (p = 0.51), and -1.24 (p = 0.47); and -1.30 (p = 0.097), -0.85 (p = 0.25), and -0.15 (p = 0.83) for CLE, CW, and CNE, respectively. CONCLUSIONS: Thrombi with late enhancement patterns showed a higher revascularization rate and better outcomes as compared to clots with early uptake or no washout.

First Results on the Presence of Mycotoxins in the Liver of Pregnant Fallow Deer (Dama dama) Hinds and Fetuses.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.