A streamlined mass spectrometry–based proteomics workflow for large-scale FFPE tissue analysis

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)–based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The Kety-Schmidt technique for repeated measurements of global cerebral blood flow and metabolism in the conscious rat.

Cerebral activation will increase cerebral blood flow (CBF) and cerebral glucose uptake (CMRglc) more than it increases cerebral uptake of oxygen (CMR(O2)). To study this phenomenon, we present an application of the Kety-Schmidt technique that enables repetitive simultaneous determination of CBF, CMR(O2), CMRglc and CMRlac on awake, non-stressed animals. After constant intravenous infusion with 133Xenon, tracer infusion is terminated, and systemic arterial blood and cerebral venous blood are continuously withdrawn for 9 min. In this paper, we evaluate if the assumptions applied with the Kety-Schmidt technique are fulfilled with our application of the method. When measured twice in the same animal, the intra-individual variation for CBF, CMR(O2), and CMRglc were 10% (SD: 25%), 8% (SD: 25%), and 9% (SD: 28%), respectively. In the awake rat the values obtained for CBF, CMR(O2) and CMRglc were 106 mL [100 g](-1) min(-1), 374 micromole [100 g](-1) min(-1) and 66 micromole [100 g](-1) min(-1), respectively. The glucose taken up by the brain during wakefulness was fully accounted for by oxidation and cerebral lactate efflux. Anaesthesia with pentobarbital induced a uniform reduction of cerebral blood flow and metabolism by approximately 40%. During halothane anaesthesia CBF and CMRglc increased by approximately 50%, while CMR(O2) was unchanged.     

Comparison of Danish isolates of Salmonella enterica serovar enteritidis PT9a and PT11 from hedgehogs (Erinaceus europaeus) and humans by plasmid profiling and pulsed-field gel electrophoresis

During the years 1994 to 1998, 10 strains of Salmonella enterica serovar Enteritidis phage type 11 (PT11) and 6 PT9a strains were isolated from Danish hedgehogs, together with 7 strains that did not yield phage susceptibility patterns conforming with any known phage type (routine dilution no conformity [RDNC]). From 1995 to 1998, five Danish patients were reported infected with serovar Enteritidis PT11 and two with PT9a. All serovar Enteritidis PT11, PT9a, and RDNC isolates from hedgehogs and humans were analyzed by pulsed-field gel electrophoresis (PFGE), plasmid profiling, and restriction fragment length polymorphism (RFLP) of plasmids. By use of S1 nuclease and HindIII, the PT11 and PT9a isolates had identical plasmid profiles and RFLP patterns, which differed from the RDNC profiles. The PFGE profiles were identical for all serovar Enteritidis PT11 and PT9a strains from hedgehogs, four of five human strains of serovar Enteritidis PT11, and two human strains of serovar Enteritidis PT9a, irrespective of restriction enzyme, whereas the last human strain deviated slightly when NotI was used but not when XbaI or SpeI was used. The results indicate that serovar Enteritidis PT9a and PT11 are closely related and that PT11 and PT9a from Danish hedgehogs and humans belong to the same clonal lineage.     

New peptide receptor radionuclide therapy of invasive cancer cells: in vivo studies using (177)Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts

The proposition of uPAR as a potential target in cancer therapy is advanced by its predominant expression at the invasive front of colorectal cancer (CRC) and its value as prognostic biomarker for poor survival in this disease. In this study, we provide the first in vivo proof-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model.MethodsA DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with ⁶⁴Cu and ¹⁷⁷Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29 cells were inoculated in Nude mice and treated with ¹⁷⁷Lu-DOTA-AE105 once a visible tumor had formed. To evaluate the true effect of the targeted radiotherapy, two controls groups were included in this study, one receiving a ¹⁷⁷Lu-labeled non-binding control peptide and one receiving vehicle. All animals were treated day 0 and 7. A parallel ¹⁸F-FLT PET/CT study was performed on day 0, 1, 3 and 6. Dosimetry calculations were based on a biodistribution study, where organs and tissue of interest were collected 0.5, 1.0, 2.0, 4.0 and 24 h post injection of ¹⁷⁷Lu-DOTA-AE105. Toxicity was assessed by recording mouse weight and by H&;E staining of kidneys in each treatment group.ResultsuPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using ⁶⁴Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using ¹⁷⁷Lu-DOTA-AE105, where a significant effect on tumor size and the number of uPAR-positive cells in the tumor was found (p < 0.05). Evaluations of biodistribution and dosimetry revealed highest accumulation of radioactivity in kidneys and tumor tissue. ¹⁸F-FLT PET/CT imaging study revealed a significant correlation between ¹⁸F-FLT tumor uptake and efficacy of the radionuclide therapy. A histological examination of the kidneys from one animal in each treatment group did not reveal any gross abnormalities and the general performance of all treated animals also showed no indications of radioactivity-induced toxicity.ConclusionThese findings document for the first time the in vivo efficacy of an uPAR-targeted radionuclide therapeutic intervention on both tumor size and its content of uPAR expressing cells thus setting the stage for future translation into clinical use.     

Peak antifactor xa activity produced by dalteparin treatment in patients with renal impairment compared with controls

STUDY OBJECTIVE: To evaluate the relationship between impaired renal function and antifactor Xa activity in patients receiving dalteparin. DESIGN: Open-label prospective study. SETTING: Inpatient and outpatient units of a large teaching hospital. SUBJECTS: Eleven patients with renal impairment and 11 control subjects with normal renal function. INTERVENTION: Subjects were administered dalteparin at a dosage of approximately 100 U/kg subcutaneously every 12 hours. MEASUREMENTS AND MAIN RESULTS: Peak steady-state antifactor Xa levels were compared between the groups. Mean +/- SD levels were similar for patients with and without renal impairment: 0.47 +/- 0.25 and 0.55 +/- 0.20 U/ml, respectively. A test of equivalency showed statistical significance (p=0.0001). CONCLUSION: No meaningful difference in peak antifactor Xa activity was found between patients with renal impairment and control subjects. To the extent that peak antifactor Xa levels can be used as markers for adjusting doses of dalteparin, these data suggest that such adjustments are not necessary for patients with renal impairment who are not receiving dialysis.     

Potent 4-aryl- or 4-arylalkyl-substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling

We have previously described a series of competitive GABA(A) antagonists derived from the low-efficacy partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 4). The 2-naphthylmethyl analogue, 4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol (5), provided affinity for the GABA(A) receptor site higher than that of the standard GABA(A) receptor antagonist, SR 95531 (3). Molecular modeling studies of these compounds exposed a cavity at the receptor recognition site capable of accommodating aromatic groups of substantial size in the 4-position in the 3-isoxazolol ring. Here we present a series of analogues of 5, with various substituents in different positions in the naphthyl ring system (6a-k), and compounds with aromatic substituents directly attached to the 4-position of the 3-isoxazolol ring (7l-n). The compounds have been pharmacologically characterized using receptor-binding assays and electrophysiological whole-cell patch-clamp techniques. All of the tested compounds show affinity for the GABA(A) receptor site. While the 5-, 7-, and 8-bromo analogues, 6b-d, showed receptor affinities (K(i) = 45, 109, and 80 nM, respectively) comparable with that of 5 (K(i) = 49 nM), the 1-bromo analogue, 6a, provided the highest receptor affinity of the series (K(i) = 10 nM). Introduction of a series of different substituents in the 1-position in the 2-naphthyl ring system led to compounds, 6e-k, with retained high affinity for the GABA(A) receptor (K(i) = 16-250 nM). Introduction of a phenyl ring directly into the 4-position on the 3-isoxazolol ring gave a 41-fold increase in affinity relative to that of 4-PIOL. In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine, 6a showing antagonist potency (IC(50) = 42 nM) markedly higher than that of 3 (IC(50) = 240 nM). Molecular modeling studies, based on the compounds described, emphasized the importance of the distal ring in 5 for receptor affinity and the considerable dimensions of the proposed receptor cavity. Furthermore, the phenyl rings in 7l and in 6k were shown to represent highly favorable positions for an aromatic ring in previously unexplored receptor regions in terms of a pharmacophore model.     

Comparison of agricultural injuries reported in the media and census of fatal occupational injuries

The Bureau of Labor Statistics (BLS) publishes annual statistics on occupational injuries and fatalities in the United States. The BLS fatality data include all agricultural workers while the non-fatal injury data only cover hired employees on large farms. In 2012, the Central States Center for Agricultural Safety and Health (CS-CASH) began collecting regional media monitoring data of agricultural injury incidents to augment national statistics. The aims of this report were: a) to compare CS-CASH injury and fatality data collected via print and online sources to data reported in previous studies, and b) to compare fatality data from media monitoring to BLS Census of Fatal Occupational Injuries (CFOI) data. CS-CASH media monitoring data were collected from a news clipping service and an internet detection and notification system. These data covered years 2012–2017 in seven Midwestern states (Iowa, Kansas, Minnesota, Missouri, Nebraska, North Dakota, and South Dakota). CS-CASH occupational fatality data were compared with aggregate CFOI data for the region during 2012–2015. Media monitoring captured 1048 injury cases; 586 (56%) were non-fatal and 462 (44%) were fatal. The numbers of occupational fatality cases from media monitoring and CFOI were nearly identical (280 vs. 282, respectively), and the distributions by type of injury were similar. Findings suggest that media monitoring can capture equal numbers of fatalities compared to CFOI. Non-fatal injuries, not captured by national surveillance systems, can be collected and tracked using print and electronic media. Risk factors, identified in media sources, such as gender, age, time, and source of the incident are consistent with previously reported data. Media monitoring can provide timely access to detailed information on individual cases, which is important for detecting unique and emerging hazards, designing interventions and for setting policy and guiding national strategies.     

Synthesis and evaluation of silanediols as highly selective uncompetitive inhibitors of human neutrophil elastase

Chronic obstructive pulmonary disease (COPD) is an increasing health problem and is estimated to be the fifth leading cause of death in 2020 according to the World Health Organization. Current treatments are only palliative, and therefore the development of new medicine for the treatment of COPD is urgent. Human Neutrophil Elastase (HNE) is a serine protease that is heavily involved in the progression of COPD through inflammatory breakdown of lung tissue. Consequently, inhibitors of HNE are of great interest as therapeutics. In this article, the development of silanediol peptide isosters as inhibitors of HNE is presented. Kinetic studies revealed that incorporation of a silanediol isoster in the inhibitor structure resulted in an uncompetitive mechanism of inhibition, which further resulted in excellent selectivity. The peculiar mechanism of inhibition and the resulting selectivity makes the presented inhibitors promising leads for the development of new HNE-inhibitor-based therapeutics for the treatment of COPD.     

Antidepressant or Antipsychotic Overdose in the Intensive Care Unit – Identification of Patients at Risk

It is often advised that patients who have ingested an overdose of antidepressants (AD) or antipsychotics (AP) are monitored with continuous ECG for minimum of 12–24 hr. These patients are often observed in an ICU. Our aim was to identify the number of patients with AD and/or AP overdose without adverse signs at hospital admission that turned out to need intensive care treatment. The effect of the antidepressants overdose risk assessment (ADORA) system was evaluated in patients with antidepressant as well as antipsychotic overdose. Our hypothesis was that patients with low ADORA do not need intensive care treatment. This retrospective study was conducted in adult patients admitted to the ICU at Odense University Hospital after an overdose with AP and/or AD between 1 January 2009 and 1 September 2014. Patients with predefined adverse signs in the emergency department were excluded due to obvious need of intensive care. Of the 157 patients included, 12 patients (8%) developed events during the ICU stay. Only 3 patients received intubation, vasoactive drugs and/or dialysis. None developed ventricular dysrhythmias. There were no fatalities. All the patients with low‐risk assessment by ADORA within the first 6 hr did not develop events within the first 24 hr after hospital admission. The vast majority of patients with AD and/or AP overdose and no adverse signs at admission did not require intensive care treatment. Low‐risk ADORA identified patients with antidepressant as well as antipsychotic overdose who would not require initial intensive care treatment. This is the first time the ADORA system has been evaluated in patients with antidepressant as well as antipsychotic overdose.