Cone-Beam CT-Guided Transarterial Tagging of Endophytic Renal Tumors with Indocyanine Green for Robot-Assisted Partial Nephrectomy.

PURPOSE: To evaluate the safety, efficacy, and clinical impact of preoperative cone-beam computed tomography (CT)-guided selective embolization of endophytic renal tumors with the fluorescent dye indocyanine green (ICG) and ethiodized oil in patients undergoing robot-assisted partial nephrectomy (RAPN) using near-infrared fluorescence imaging (NIR-FI). MATERIALS AND METHODS: Patients with renal endophytic tumors eligible for RAPN and transarterial embolization with ICG and ethiodized oil were prospectively enrolled. Technical success was defined as the completion of the embolization procedure. Radiographic success, defined as ethiodized oil accumulation in the nodule, was classified as poor, moderate, good, or optimal on the basis of postembolization cone-beam CT. Surgical visibility of the tumors during RAPN with the use of NIR-FI was classified as follows: (a) not visible, (b) visible with poorly defined margins, and (c) visible with well-defined margins. RESULTS: Forty-one patients underwent preoperative selective embolization. Technical success was 100%. Ethiodized oil accumulation on cone-beam CT was poor in 2 (4.9%), moderate in 6 (14.6%), good in 25 (61.0%), and optimal in 8 (19.5%) of 41 patients. During RAPN with NIR-FI, tumors were visible with well-defined margins in 26 (63.4%), visible with blurred margins in 14 (34.1%), and not visible in 1 (2.4%) of 41 cases. There were no adverse events following endovascular embolization. CONCLUSIONS: Preoperative transarterial superselective embolization of endophytic renal tumors with ICG and ethodized oil in patients undergoing RAPN is safe and effective, allowing accurate intraoperative visualization and resection of endophytic tumors.

Balloon occluded TACE (B-TACE) vs DEM-TACE for HCC: a single center retrospective case control study.

BACKGROUND: To compare oncological results and safety profile of balloon micro-catheter trans-arterial chemoembolization (b-TACE) and drug-eluting-microsphere (DEM-TACE) in patients with hepatocellular-carcinoma (HCC). METHODS: This is a case-control, retrospective, single-center study. Between January-2015/March-2019, 149 patients (131 males [87.9%]) with 226 HCC were treated, 22 patients (35 HCC; 19 [86.4%] males) with b-TACE and 127 with DEM-TACE (191 HCC, 112 [88.2%] males). Embolization protocol was standardized (sequential 100 ± 25 and 200 ± 25 µm microspheres). Results were evaluated by modified-response-evaluation-criteria-in-solid-tumor [mRECIST] at 1, 3-6 and 9-12 months and time to recurrence after complete response [TTR] at 1 years. Cox's regression weighted with tumor dimensions was performed. Adverse events (AEs) were recorded. RESULTS: mRECIST oncological response at all time points (1, 3-6 and 9-12 months) for both treatments were similar, with the exception of Objective response rate at 9-12 months. Objective response at 1 and 3-6 months between b-TACE vs DEM-TACE [23/35 (65.7%) vs 119/191 (62.3%), 21/29 (72.4%) vs 78/136 (57.4%) (p > 0.05), respectively]. On the contrary, at 9-12 months, it was significantly higher in b-TACE subgroup than DEM-TACE (15/19 [78.9%] vs 48/89 [53.9%], p = 0.05). TTR for complete response at 1 year had a better trend for b-TACE vs DEM-TACE (278.0 days [196.0-342.0] vs 219.0 days [161.0-238.0], OR 0.68 [0.4-1.0], p = 0.10). The use of balloon micro-catheter reduced the relative risk of the event of recurrence by 0.63 [CI95% 0.38-1.04]; p = 0.07). No significant differences were found in AEs rate. CONCLUSION: b-TACE showed a trend of better oncological response over DEM-TACE with and longer TTR with a similar adverse events rate, in patients presenting with larger tumors.

Sequential dual-phase cone-beam CT is able to intra-procedurally predict the one-month treatment outcome of multi-focal HCC, in course of degradable starch microsphere TACE.

OBJECTIVE: To evaluate the prognostic value of sequential dual-phase CBCT (DP-CBCT) imaging performed during degradable starch microsphere TACE (DSM-TACE) session in predicting the HCC's response to treatment, evaluate with modify response evaluation criteria in solid tumours (mRECIST) at 1-month multi-detector CT (MDCT) follow-up. MATERIALS AND METHODS: Between January and May 2018, 24 patients (68.5 ± 8.5 year [45-85]) with HCC lesions (n = 96 [average 4/patient]) were prospectively enrolled. Imaging assessment included: pre-procedural MDCT, intra-procedural DP-CBCT performed before first and second DSM-TACEs and 1-month follow-up MDCT. Lesions' attenuation/pseudo-attenuation was defined as average value measured on ROIs (HU for MDCT; arbitrary unit called HU* for CBCT). Lesions' attenuation modification was correlated with the post-procedural mRECIST criteria at 1-month MDCT. RESULTS: Eighty-two DSM-TACEs were performed. Lesion's attenuation values were: pre-procedural MDCT arterial phase (AP) 107.00 HU (CI 95% 100.00-115.49), venous phase (VP) 85.00 HU (CI 95% 81.13-91.74); and lesion's pseudo-attenuation were: first CBCT-AP 305.00 HU* (CI 95% 259.77-354.04), CBCT-VP 155.00 HU* (CI 95% 135.00-163.34). For second CBCT were: -AP 210.00 HU* (CI 95% 179.47-228.58), -VP 141.00 HU* (CI 95% 125.47-158.11); and for post-procedural MDCT were: -AP 95.00 HU (CI 95% 81.35-102.00), -VP 83.00 HU (CI 95% 78.00-88.00). ROC curve analysis showed that a higher difference pseudo-attenuation between first and second DP-CBCTs is related to treatment response. The optimal cut-off value of the difference between first and second CBCT-APs to predict complete response, objective response (complete + partial response) and overall disease control (objective response + stable disease) were > 206 HU* (sensitivity 80.0%, specificity 81.7%), > 72 HU* (sensitivity 79.5%, specificity 83.0%) and > - 7 HU* (sensitivity 91.6%, specificity 65.4%), respectively. CONCLUSIONS: DP-CBCT can predict intra-procedurally, by assessing lesion pseudo-attenuation modification, the DSM-TACE 1-month treatment outcome.

Urinary Proteomics Profiles Are Useful for Detection of Cancer Biomarkers and Changes Induced by Therapeutic Procedures.

Boron neutron capture therapy (BNCT) is a binary cancer treatment modality where two different agents (10B and thermal neutrons) have to be present to produce an effect. A dedicated trial design is necessary for early clinical trials. The concentration of 10B in tissues is an accepted surrogate to predict BNCT effects on tissues. Tissue, blood, and urines were sampled after infusion of two different boron carriers, namely BSH and BPA in the frame of the European Organisation for Research and Treatment of Cancer (EORTC) trial 11001. In this study, urine samples were used to identify protein profiles prior and after drug infusion during surgery. Here, an approach that is based on the mass spectrometry (MS)-based proteomic analysis of urine samples from head and neck squamous cell carcinoma (HNSCC) and thyroid cancer patients is presented. This method allowed the identification of several inflammation- and cancer-related proteins, which could serve as tumor biomarkers. In addition, changes in the urinary proteome during and after therapeutic interventions were detected. In particular, a reduction of three proteins that were involved in inflammation has been observed: Galectin-3 Binding Protein, CD44, and osteopontin. The present work represents a proof of principle to follow proteasome changes during complex treatments based on urine samples.

Pictorial guide for variants of Covid-19: CT imaging and interpretation.

Typical radiologic images of Covid-19 pneumonia consists in a wide spectrum of chest manifestations, which range from peripheral predominant ground-glass opacities to an organizing pneumonia pattern, with additional features including crazy-paving, consolidations, fibrotic streaks and linear opacities. With variants imaging profile of Covid-19 evolves, producing relatively atypical/indeterminate CT pattern of pulmonary involvement, which overlap with imaging features of a variety of other respiratory diseases, including infections, drug reaction and hypersensitivity pneumonia. Our knowledge of these radiological findings is incomplete and there is a need to strengthen the recognition of the many faces of Covid-19 pneumonia.

Intra-procedural dual phase cone beam computed tomography has a better diagnostic accuracy over pre-procedural MRI and MDCT in detection and characterization of HCC in cirrhotic patients undergoing TACE procedure.

PURPOSE: This study was directed to compare diagnostic accuracy of dual-phase cone beam computed tomography (DP-CBCT) vs pre-procedural second line imaging modality (SLIM [multidetector computed tomography and magnetic resonance imaging]) to detect and characterize hepatocellular carcinoma (HCC) in cirrhotic patients with indication for trans-arterial chemoembolization (TACE). METHODS: This is a single centre, retrospective, and observational study. Exclusion criteria were not-assisted DP-CBCT TACE, and unavailable follow-up SLIM. We evaluated 280 consecutive patients (January/2015-Febraury/2019). Seventy-two patients were eligible. Three radiologists in consensus reviewed: pre-procedural SLIM, DP-CBCT, and SLIM at follow-up, with 4 months of interval between each reading. Hyper-vascular foci (HVF) were detected and characterized. Diameter was recorded. Radiological behaviour, according to LI-RADS criteria, of HFV throughout follow-up time was the reference standard. Diagnostic accuracy was calculated for pre-procedural SLIM and DP-CBCT and evaluated through receiver operating characteristic curve. HVF only visible on DP-CBCT (defined as occult) were analysed. Tumour diameters were compared. RESULTS: Median time between pre-procedural SLIM and DP-CBCT and between DP-CBCT and definitive radiological diagnosis of HVF were 46.0 days (95%CI 36.5-55.0) and 30.5 days (95%CI 29.0-33.0), respectively. DP-CBCT had a better diagnostic performance than pre-examination SLIM (sensitivity 99%vs78%; specificity 89%vs85%; PPV 99%vs99%; NPV 92%vs30%; and accuracy 94%vs79%). DP-CBCT diagnosed 63 occult HVF. Occult HCC were 54/243 (22.2%). Six were occult angiomas. Three were false positive. Mean diameter was significantly higher in DP-CBCT vs pre-procedural SLIM (+7.5% [95%CI 3.7-11.3], p < 0.05). CONCLUSIONS: DP-CBCT has a better diagnostic accuracy and NPV than pre-procedural SLIM in cirrhotic patients with indication for TACE.

Balloon-Occluded Transcatheter Arterial Chemoembolization (b-TACE) for Hepatocellular Carcinoma Performed with Polyethylene-Glycol Epirubicin-Loaded Drug-Eluting Embolics: Safety and Preliminary Results.

PURPOSE: To report technical success, safety profile and oncological results of balloon-occluded transcatheter arterial chemoembolization using a balloon micro-catheter and epirubicin-loaded polyethylene-glycol (PEG) microsphere (100 ± 25 µm and 200 ± 50 µm) in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This is a single-centre, single-arm, retrospective study with 6-month follow-up. Twenty-two patients (Child-Pugh A 68% [15/22], B in 32% [7/22]; age 67.05 ± 14 years) with 29 HCC were treated in 24 procedures. Technical success is defined: ability to place the balloon micro-catheter within the required vascular segment, balloon-occluded arterial stump pressure drops and assessment of microsphere deposition. Laboratory assessment pre/post-procedural and complications were analysed, respectively, according to Common Terminology Criteria for Adverse Events (CTCAEv5) and CIRSE system. Postembolization syndrome (PES) was defined as fever and/or nausea and/or pain onset. Oncological results were evaluated using m-RECIST criteria with CT/MRI imaging at 1 and 3-6 months. In partial responder patients, pre/post-procedural tumour volume was compared. RESULTS: Pre-planned feeder was reached in all cases. Pressure drop average was 51.1 ± 21.6 mmHg. Exclusive target embolization was achieved in 14/24 procedures (58.3%). Laboratory test modifications were all grade 1. 4/24 adverse events occurred (17%): pseudo-aneurysm of the feeder (grade 3), liver abscess (grade 2) and 2 asymptomatic segmentary biliary tree dilatations (grade 2). PES occurred in 8/24 (33%). The complete response at 1 and 3-6 months was 44.8% (13/29) and 52.9% (9/17), respectively. The partial response at 1 and 3-6 months was 55% (16/29) and 4/17 (23.5%), respectively. Among partial responder patients, the average percentage of tumour volume reduction was 64.9 ± 27.3%. CONCLUSION: Epirubicin-loaded PEG microsphere b-TACE is technically feasible, safe and effective procedure for HCC treatment.

A combined approach of mass spectrometry, molecular modeling, and site-directed mutagenesis highlights key structural features responsible for the thermostability of Sulfolobus solfataricus carboxypeptidase.

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme, consisting of four identical subunits with a M(r) of 43,000. In a previous paper (Occhipinti et al., Biophys J 2003; 85:1165-1175), we developed a structure of the enzyme by molecular modeling and validated it by site-directed mutagenesis and small angle X-ray scattering. Here, we report investigations aimed at further validating the model, as well as at identifying molecular determinants responsible for thermostability. To this end, we took advantage of mass spectrometry techniques, notably LC-MS/MS. The structure was confirmed by such approaches, in that they lead to the identification of a disulfide bridge formed by Cys286 and Cys293, whose location in the model is well suited for giving rise to the crosslink. More notably, we also identified a protease-resistant core consisting of the N- and C-terminal antiparallel alpha-helices, which in the model are predicted to interact with each other via hydrophobic quadrants. On the basis of the model, we also tentatively identified the most tightly interacting residues as Leu7, Ala380, and Leu376. Although the replacement of Ala380 by serine did not detectably impair protein stability, a dramatic drop in thermostability was observed when the two leucines were replaced by either aspartate (L7D; L376D) or asparagine (L7N; L376N). We then investigated the kinetic thermal stability of the wild type and the mutants by determining the thermodynamic activation parameters, DeltaG++, DeltaH++, and DeltaS++. Besides highlighting the key role of the hydrophobic core in thermostability, these results suggest clearly different mechanisms of destabilization by the single mutations, depending on whether the leucines are replaced by asparagines or aspartates.

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach.

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.

LC-MS characterization of terpene lactones in plasma of experimental animals treated with Ginkgo biloba extracts Correlation with pharmacological activity.

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS) was applied to determine the concentration of terpene lactone in plasma of guinea pigs after chronic administration of Ginkgo biloba extract enriched in ginkgoterpenes in free form (IDN 5380) or complexed with soy phosphlipids (IDN 5381). Oral treatment of the animals with ginkgoterpenes resulted to inhibit the bronchoconstriction (ITP) and the concomitant increase of the levels of thromboxane B2 (TXB2) in the circulation caused by histamine (HIST) and platelet activating factor (PAF) in normal guinea pigs or by ovalbumin (OA) in actively sensitized guinea pigs. To compare the protective activities of G. biloba forms (IDN 5380 and IDN 5381), ED50 and dose ratio (DR) values for both parameters (ITP and TXB2) were evaluated. The phytosomic form (IDN 5381) significantly reduced (two- to four-fold as compared to free form, P < 0.001) the HIST, PAF or OA-induced airway changes and TXB2 release. In addition it has been observed that the absence of ginkgolide C (GC) in plasma samples (in human and animals) was due to its rapid methylation.

Proteomic analysis of urine exosomes reveals renal tubule response to leptospiral colonization in experimentally infected rats.

BACKGROUND: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. CONCLUSIONS: We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.

New approach for the detection of BSH and its metabolites using capillary electrophoresis and electrospray ionization mass spectrometry.

Boron neutron capture therapy is a promising binary treatment for cancer. It is based on the nuclear fission that occurs when non-radioactive 10B absorbs thermal neutrons. One of the two boron compounds currently used in clinical trials for this therapy is BSH. To ensure differentiated retention in the tumour versus normal tissue prior to treatment, routine analytical methods to determine pharmacokinetics must be available. For this purpose we have developed a new, easy and time saving approach, in which the separation of boron derivatives is performed by means of capillary electrophoresis (CE). The CE method allows analyses to be performed in short times (less than 18 min), sensitively (LOD 8 pg loaded on the capillary) quantitatively (LOQ 5 microg/ml) and with a high efficiency of separation. Moreover it is simpler than HPLC and more reproducible (intra- and inter-day values were +/-1% and +/-3%, respectively), and does not require a specific column of derivatization. Mass spectrometry analysis of boron derivatives in different samples was also performed to ensure correct attribution of the CE peaks.

Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Metabolism of borono-phenylalanine-fructose complex (BPA-fr) and borocaptate sodium (BSH) in cancer patients--results from EORTC trial 11001.

Within the clinical trial EORTC 11001, patients were infused with (10)B-enriched borono-phenylalanine-fructose complex (BPA-fr), or borocaptate sodium (BSH) solutions, which are used as boron carriers for boron neutron capture therapy. Urine samples were periodically collected and analyzed by (10)B NMR spectroscopy. The results revealed time-dependent metabolic changes of the administered compounds. BPA-fr dissociated to the constituents BPA and fructose, and the borate group was partly cleaved from BPA. BSH was partly aggregated to a dimer form, BSSB. These observations were previously reported for cultured cells and animal models, and are confirmed here in human cancer patients.

Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach.

For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of bilobalide in plasma and brain of rats after oral administration of its phospholipidic complex.

Standardized extracts of Ginkgo biloba L. leaves are widely used in clinical practice for the symptomatic treatment of mild to moderate dementia syndromes, cerebral insufficiency and for the enhancement of cognitive function. The main active components present in G. biloba extracts are flavonol-glycosides and terpene-lactones. In recent investigations, the sesquiterpene trilactone bilobalide has been described to exert an interesting neuroprotective effect when administered systemically to experimental animals. Oral administration of terpene-lactones either as standardized extracts or purified products is characterized by a low bioavailability. While preparing phospholipidic complex of G. biloba extracts or bilobalide, plasma levels of terpenes and sesquiterpene increase. In the present study, phospholipidic complex of bilobalide (IDN 5604) has been administered orally to rats and bilobalide levels have been determined in plasma and brain by means of a validated method based on liquid chromatography coupled to atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS). Due to its sensitivity (about 3pmol/ml) and specificity, LC/APCI-ITMS method proved to be a very powerful tool for pharmacokinetic studies of Ginkgo terpene-lactones. The results of the present study clearly confirm the improvement of oral bioavailability of bilobalide administered as phospholipidic complex and, for the first time, demonstrate the detection of significative amounts of bilobalide in brain. This last finding agrees with the neuroprotective activity observed for bilobalide.