Publications by authors named "William Wallace"

212 Publications

Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library.

J Proteome Res 2021 Mar 8;20(3):1612-1629. Epub 2021 Feb 8.

Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8362, Gaithersburg, Maryland 20899, United States.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of "hard-to-find" disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.
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http://dx.doi.org/10.1021/acs.jproteome.0c00823DOI Listing
March 2021

Assessment of Waldeyer's ring in pediatric and adolescent Hodgkin lymphoma patients-Importance of multimodality imaging: Results from the EuroNet-PHL-C1 trial.

Pediatr Blood Cancer 2021 Apr 3;68(4):e28903. Epub 2021 Feb 3.

Department of Radiology, Medical Faculty of the Martin-Luther-University, Halle (Saale), Germany.

Background: In the EuroNet Pediatric Hodgkin Lymphoma (EuroNet-PHL) trials, decision on Waldeyer's ring (WR) involvement is usually based on clinical assessment, that is, physical examination and/or nasopharyngoscopy. However, clinical assessment only evaluates mucosal surface and is prone to interobserver variability. Modern cross-sectional imaging technology may provide valuable information beyond mucosal surface, which may lead to a more accurate WR staging.

Patients, Materials, And Methods: The EuroNet-PHL-C1 trial recruited 2102 patients, of which 1752 underwent central review including reference reading of their cross-sectional imaging data. In 14 of 1752 patients, WR was considered involved according to clinical assessment. In these 14 patients, the WR was re-assessed by applying an imaging-based algorithm considering information from F-fluorodeoxyglucose positron emission tomography, contrast-enhanced computed tomography, and/or magnetic resonance imaging. For verification purposes, the imaging-based algorithm was applied to 100 consecutive patients whose WR was inconspicuous on clinical assessment.

Results: The imaging-based algorithm confirmed WR involvement only in four of the 14 patients. Of the remaining 10 patients, four had retropharyngeal lymph node involvement and six an inconspicuous WR. Applying the imaging-based algorithm to 100 consecutive patients with physiological appearance of their WR on clinical assessment, absence of WR involvement could be confirmed in 99. However, suspicion of WR involvement was raised in one patient.

Conclusions: The imaging-based algorithm was feasible and easily applicable at initial staging of young patients with Hodgkin lymphoma. It increased the accuracy of WR staging, which may contribute to a more individualized treatment in the future.
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http://dx.doi.org/10.1002/pbc.28903DOI Listing
April 2021

Pitfalls and Artifacts of DaTscan Imaging in Parkinsonian Syndromes- A quality improvement teaching tool.

J Nucl Med Technol 2020 Dec 24. Epub 2020 Dec 24.

University of Mississippi Medical Center, United States.

The aim of the current article is image quality improvement and a teaching tool on I Ioflupane SPECT (DaTscan). The imaging uses the radiopharmaceutical I Ioflupane (I-FP-CIT) to visualize the nigrostriatal pathway. Parkinson's disease and Parkinsonian syndromes are movement disorders that exhibit nigrostriatal degeneration, with a decreased Dopamine transporter level in the pathway and thus a decreased I Ioflupane distribution. Other non-Parkinson's movement disorders, such as essential tremor, will have intact dopaminergic neurons and exhibit a normal distribution of the radiopharmaceutical throughout the striata. Parkinson's disorders are usually diagnosed clinically. However, DaTscan (GE Healthcare) can be a valuable tool when the clinical features are not sufficiently clear. DaTscan image interpretation is not always straightforward. Many pitfalls, including biological factors, technical factors, medications, and various other factors, including age, race, ethnicity, body habitus, can make the interpretation challenging. The technologist and nuclear radiologist must identify the expected imaging findings to avoid the most common mistakes related to artifacts. Our main goal is to improve image quality by reviewing the most common pitfalls and artifacts of DaTscan that can compromise an accurate diagnosis and lead to misinterpretation.
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http://dx.doi.org/10.2967/jnmt.120.258491DOI Listing
December 2020

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer.

BMC Med 2020 12 4;18(1):374. Epub 2020 Dec 4.

MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK.

Background: Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues.

Methods: We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24-240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified.

Results: Cisplatin exposure resulted in a significant reduction in the total number of germ cells (- 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (- 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (- 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure.

Conclusions: This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.
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http://dx.doi.org/10.1186/s12916-020-01844-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716476PMC
December 2020

Tissue-Specific Immunopathology in Fatal COVID-19.

Am J Respir Crit Care Med 2021 01;203(2):192-201

Centre for Inflammation Research, Queen's Medical Research Institute, and.

In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown. To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury. Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence. Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.
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http://dx.doi.org/10.1164/rccm.202008-3265OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874430PMC
January 2021

Reproductive Complications in Childhood Cancer Survivors.

Pediatr Clin North Am 2020 12;67(6):1187-1202

Department of Pediatric Haematology and Oncology, Royal Hospital for Sick Children, Edinburgh, Scotland.

Gonadal dysfunction and infertility after cancer treatment are major concerns for childhood cancer survivors and their parents. Uncertainty about fertility or being diagnosed with infertility has a negative impact on quality of survival. In this article, determinants of gonadal damage are reviewed and consequences for fertility and pregnancies are discussed. Recommendations for screening and treatment of gonadal function are provided. These should enable timely treatment of gonadal insufficiency aiming to improve linear growth, pubertal development, and sexual functioning. Options for fertility preservation are discussed.
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http://dx.doi.org/10.1016/j.pcl.2020.08.003DOI Listing
December 2020

Single-cell RNA-sequencing reveals dysregulation of molecular programs associated with SARS-CoV-2 severity and outcomes in patients with chronic lung disease.

bioRxiv 2020 Oct 20. Epub 2020 Oct 20.

Rationale: Patients with chronic lung disease have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes.

Objectives: To identify molecular characteristics of diseased lung epithelial and immune cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases.

Methods: We analyzed the transcriptomes of 605,904 single cells isolated from healthy (79 samples) and diseased human lungs (31 chronic obstructive pulmonary disease (COPD), 82 idiopathic pulmonary fibrosis (IPF) and 18 non-IPF interstitial lung disease samples).

Measurements And Main Results: Cellular distribution and relative expression of SARS-CoV-2 entry factors ( , ) was similar in disease and control lungs. Epithelial cells isolated from diseased lungs expressed higher levels of genes linked directly to efficiency of viral replication and the innate immune response. Unique correlated gene sets were identified for each diagnosis group in the type II alveolar cells. Diseased lungs have a significant increase in the proportion of CD4, CD8 and NK cells compared to control lungs. Components of the interferon pathway, the IL6 cytokine pathway and the major histocompatibility complex (MHC) class II genes are upregulated in several diseased immune cell types. These differences in inflammatory gene expression programs highlight how chronic lung disease alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung.

Conclusions: Chronic lung disease is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence innate and adaptive immune responses to SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.10.20.347187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587778PMC
October 2020

Development of a histopathology scoring system for the pulmonary complications of organophosphorus insecticide poisoning in a pig model.

PLoS One 2020 14;15(10):e0240563. Epub 2020 Oct 14.

Pharmacology, Toxicology & Therapeutics, Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom.

Organophosphorus (OP) insecticide self-poisoning causes over 100,000 global deaths annually. Around a third of patients are intubated and up to half of these can die. Post-mortem analysis of OP poisoned patients' lungs reveals consolidation, edema and hemorrhage, suggesting that direct or indirect lung damage may contribute to mortality. The lung injury caused by these formulated agricultural preparations is poorly characterised in humans, and a valid histopathology scoring system is needed in a relevant animal model to further investigate the disease and potential treatments. We conducted two pilot studies in anesthetized minipigs, which are commonly used for toxicological studies. In the first, pigs were given 2.5 mL/kg of either OP (n = 4) or saline (n = 2) by gavage and compared with positive controls (iv oleic acid n = 2). The second study simulated ingestion followed by gastric content aspiration: mixtures of OP (n = 3) or saline (n = 2) (0.63-0.71mL/kg) were placed in the stomach, and then small volumes of the gastric content were placed in the lung. At post-mortem examination, lungs were removed and inflation-fixed with 10% neutral buffered formalin. Samples (n = 62) were taken from cranial and caudal regions of both lungs. Two experienced lung histopathologists separately scored these samples using 8 proposed features of damage and their scores related (Kendall rank order). Two elements had small and inconsistent scores. When these were removed, the correlation increased from 0.74 to 0.78. Eight months later, a subset of samples (n = 35) was re-scored using the modified system by one of the previous histopathologists, with a correlation of 0.88. We have developed a reproducible pulmonary histopathology scoring system for OP poisoning in pigs which will assist future toxicological research and improve understanding and treatment of human OP poisoning.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240563PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556475PMC
December 2020

Impacts of platinum-based chemotherapy on subsequent testicular function and fertility in boys with cancer.

Hum Reprod Update 2020 11;26(6):874-885

MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.

Background: Children with cancer often face infertility as a long-term complication of their treatment. For boys, compromised testicular function is common after chemotherapy and currently there are no well-established options to prevent this damage. Platinum-based agents are used to treat a wide variety of childhood cancers. However, platinum agents are not currently included in the cyclophosphamide equivalent dose (CED), which is used clinically to assess the risks to fertility posed by combination chemotherapy in children with cancer.

Objective And Rationale: This was a systematic search of the literature designed to determine the evidence for effects of platinum-based cancer treatment on the prepubertal human testis in relation to subsequent testicular function and fertility.

Search Methods: PubMed and EMBASE were searched for articles published in English between 01 January 1966 and 05 April 2020 using search terms including 'cancer treatment', 'chemotherapy', 'human', 'prepubertal', 'testis', 'germ cells', 'testosterone' and related terms. Abstracts were screened and full-text articles were obtained for those that met the three major inclusion criteria (age ≤12 years at treatment, exposure to platinum-based chemotherapeutic and measure of reproductive function). Screening of bibliographies for full-text articles was used to identify additional studies.

Outcomes: Our initial search identified 1449 articles of which 20 (1.3%) studies (n = 13 759 males) met all inclusion criteria. A control group (healthy individuals or siblings) was included for 5/20 (25%) studies. A total of 10/20 (50%) studies provided sub-analysis of the relative gonadotoxicity of platinum-based agents.The primary outcome measures were: pregnancies and fatherhood; semen analysis; and hormonal function. For pregnancies and fatherhood, three studies (n = 10 453 males) reported negative associations with platinum-agents, including the largest (n = 5640) controlled study (hazard ratio = 0.56, P = 0.0023), whilst two other studies (n = 1781) with platinum sub-analysis reported no association. For semen analysis (based on World Health Organization criteria), platinum-based chemotherapy was associated with azoospermia in one study (n = 129), whilst another (n = 44) found no association and the remainder did not perform platinum-based sub-analysis. For hormone analysis, conflicting results were obtained regarding potential associations between platinum-based agents and elevated FSH (a proxy for impaired spermatogenesis); however, the majority of these studies were based on low numbers of patients receiving platinum-based chemotherapy.

Wider Implications: Overall, these results indicate that platinum-based chemotherapy should be included in clinical calculators, for example CED, used to determine gonadotoxicity for childhood cancer treatment. These findings have important implications for clinicians regarding counselling patients and their carer(s) on fertility risk, guiding requirements for fertility preservation strategies (e.g. testicular tissue cryopreservation) and modification of treatments to reduce or eliminate the risk of infertility in childhood cancer survivors.
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http://dx.doi.org/10.1093/humupd/dmaa041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600277PMC
November 2020

Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells.

Cell Stem Cell 2020 10 4;27(4):663-678.e8. Epub 2020 Sep 4.

Stem Cell Program and Divisions of Hematology/Oncology and Pulmonary Medicine, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.
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http://dx.doi.org/10.1016/j.stem.2020.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541765PMC
October 2020

Triple Traumatic Hernia.

Am Surg 2020 Aug 31:3134820945252. Epub 2020 Aug 31.

Department of Surgery, Mercer University School of Medicine, The Medical Center Navicent Health, Macon, GA, USA.

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http://dx.doi.org/10.1177/0003134820945252DOI Listing
August 2020

Spatial and temporal trends in physiological biomarkers of adult eastern oysters, Crassostrea virginica, within an urban estuary.

Mar Environ Res 2020 Oct 20;161:105122. Epub 2020 Aug 20.

Biology Program, The Graduate Center, City University of New York, 365 Fifth Ave., New York, NY, 07305, USA; Department of Natural Science, Baruch College, 17 Lexington Ave, New York, NY, 07305, USA.

Heavy metal contamination and water quality may alter reproductive capacity of oysters in highly urbanized, eutrophic ecosystems. This study assessed physiological biomarkers and heavy metal body burdens in adult oysters, Crassostrea virginica, placed at a highly urban and reference site. Condition index and Vitellogenin-like proteins were significantly different between sites, but protein concentration and activity of the electron transport system were not. Accumulation of Cd and Hg occurred at both sites, and Cd body burden was greater at the urban site. There was a negative relationship between condition index and Cd body burden at the urban site, while no relationship was found between physiological biomarkers and metal burden at the reference site. The results suggest that oyster condition and reproductive potential may be negatively influenced by the biotic and abiotic factors typically found within urban, eutrophic ecosystems.
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http://dx.doi.org/10.1016/j.marenvres.2020.105122DOI Listing
October 2020

A tricentric medical student perspective on open book examinations.

Med Teach 2021 02 11;43(2):241. Epub 2020 Aug 11.

Imperial College London, London, UK.

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http://dx.doi.org/10.1080/0142159X.2020.1800614DOI Listing
February 2021

Early Operative Ligation in the Management of Bilateral Chylothoraces After Blunt Thoracic Injury.

Am Surg 2020 Aug 10:3134820942148. Epub 2020 Aug 10.

Department of Surgery, Mercer University School of Medicine, The Medical Center Navicent Health, Macon, GA, USA.

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http://dx.doi.org/10.1177/0003134820942148DOI Listing
August 2020

Reframing Histological Risk Assessment of Oral Squamous Cell Carcinoma in the Era of UICC 8th Edition TNM Staging.

Head Neck Pathol 2021 Mar 13;15(1):202-211. Epub 2020 Jul 13.

Royal Infirmary of Edinburgh, 51 Little France Crescent , Old Dalkeith Road, Edinburgh, EH16 4SA, Scotland.

Objectives: To assess whether application of the risk model originally proposed by Brandwein-Gensler, influences survival and disease progression in patients treated for oral squamous cell carcinoma (OSCCs) MATERIALS AND METHODS: Tumours from 134 T1 and T2 OSCC resections (7th edition) were scored independently by 3 histopathologists according to worst pattern of invasion (WPOI), lymphocytic host response (LHR) and perineural invasion (PNI) and categorised according to risk score. Local recurrence, locoregional recurrence, disease progression and overall survival were study endpoints. Interobserver variability of pathologist scoring was also assessed.

Results: Seventy-two patients (54%) were classified with low or intermediate risk and 62 (46%) patients were 'high risk'. The inter-observer agreement was in moderate to strong agreement with the consensus scores (k range = 0.45-0.82). There was statistical significance between distant metastasis and 'high risk' tumours. Thirty tumours were upstaged to T3 in the 8th edition TNM staging, of which 83% had high risk scores. Overall risk score and TNM8 T stage has significant correlation with overall survival in comparison to the TNM 7 T stage.

Conclusion: 'High risk' tumours were significantly associated with distant metastasis possibly due to the greater likelihood of aggressive features such as WPOI and PNI. Primary tumours are more likely to express high risk features with increasing T stage. None of the patients classified as 'low risk' died perhaps suggesting these tumours represent a rare variant of OSCC with excellent prognosis.
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http://dx.doi.org/10.1007/s12105-020-01201-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010015PMC
March 2021

Mass Spectral Library of Acylcarnitines Derived from Human Urine.

Anal Chem 2020 05 23;92(9):6521-6528. Epub 2020 Apr 23.

Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology (NIST), Gaithersburg, Maryland 20899, United States.

We describe the creation of a mass spectral library of acylcarnitines and conjugated acylcarnitines from the LC-MS/MS analysis of six NIST urine reference materials. To recognize acylcarnitines, we conducted in-depth analyses of fragmentation patterns of acylcarnitines and developed a set of rules, derived from spectra in the NIST17 Tandem MS Library and those identified in urine, using the newly developed hybrid search method. Acylcarnitine tandem spectra were annotated with fragments from carnitine and acyl moieties as well as neutral loss peaks from precursors. Consensus spectra were derived from spectra having similar retention time, fragmentation pattern, and the same precursor / and collision energy. The library contains 157 different precursor masses, 586 unique acylcarnitines, and 4 332 acylcarnitine consensus spectra. Furthermore, from spectra that partially satisfied the fragmentation rules of acylcarnitines, we identified 125 conjugated acylcarnitines represented by 987 consensus spectra, which appear to originate from Phase II biotransformation reactions. To our knowledge, this is the first report of conjugated acylcarnitines. The mass spectra provided by this work may be useful for clinical screening of acylcarnitines as well as for studying relationships among fragmentation patterns, collision energies, structures, and retention times of acylcarnitines. Further, these methods are extensible to other classes of metabolites.
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http://dx.doi.org/10.1021/acs.analchem.0c00129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657438PMC
May 2020

FRA1 contributes to MEK-ERK pathway-dependent PD-L1 upregulation by KRAS mutation in premalignant human bronchial epithelial cells.

Am J Transl Res 2020 15;12(2):409-427. Epub 2020 Feb 15.

Lung Cancer Research Program, Jonsson Comprehensive Cancer Center Los Angeles, CA, USA.

Oncogenic KRAS mutations are frequently found in non-small cell lung carcinoma (NSCLC) and cause constitutive activation of the MEK-ERK pathway. Many cancer types have been shown to overexpress PD-L1 to escape immune surveillance. FRA1 is a MEK/ERK-dependent oncogenic transcription factor and a member of the AP-1 transcriptional factor superfamily. This study assesses the hypothesis that KRAS mutation directly regulates PD-L1 expression through the MEK-ERK pathway mediated by FRA1. Premalignant human bronchial epithelial cell (HBEC) lines harboring the KRAS mutation, EGFR mutation, p53 knock-down, or both KRAS mutation and p53 knock-down were tested for levels of PD-L1, FRA1, and ERK activation (pERK). Our results showed that KRAS mutation alone, but not other genetic alterations, induced significantly higher expression of PD-L1 compared to its vector counterparts. The increased PD-L1 expression in the KRAS mutated cells was dramatically reduced by inhibition of ERK activation. Furthermore, the MEK-ERK pathway-dependent PD-L1 expression was markedly reduced by FRA1 silencing. Interestingly, FRA1 silencing led to inhibition of ERK activation, indicating that FRA1 plays a role in PD-L1 regulation via positive feedback of ERK activation. Correlation of PD-L1 and FRA1 mRNA expression was validated using human lung cancer specimens from The Cancer Genome Atlas (TCGA) and established NSCLC cell lines from Cancer Cell Line Encyclopedia (CCLE). FRA1 expression was significantly associated with PD-L1 expression, and high FRA1 expression was correlated with poor overall survival. Our findings suggest that oncogenic KRAS-driven PD-L1 expression is dependent on MEK-ERK and FRA1 in high risk, premalignant HBEC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061839PMC
February 2020

Lower DLco% identifies exercise pulmonary hypertension in patients with parenchymal lung disease referred for dyspnea.

Pulm Circ 2020 Jan-Mar;10(1):2045894019891912. Epub 2020 Feb 19.

Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Exercise pulmonary hypertension is an underappreciated form of physical limitation related to early pulmonary vascular disease. A low diffusing capacity of lungs for carbon monoxide (DLco) can be seen in patients with resting pulmonary hypertension as well as parenchymal lung disease. It remains unclear whether low DLco% identifies early pulmonary vascular disease. We hypothesize that a reduced DLco% differentiates the presence of exercise pulmonary hypertension in patients with parenchymal lung disease. Fifty-six patients referred for unexplained exertional dyspnea with pulmonary function tests within six months of hemodynamic testing underwent exercise right heart catheterization. Exclusion criteria included resting pulmonary arterial or venous hypertension. Receiver operator characteristic curve determined the optimal DLco% cutoffs based on the presence or absence of parenchymal lung disease. Twenty-one (37%) patients had parenchymal lung disease, most common manifesting as chronic obstructive lung disease or interstitial lung disease. In patients with parenchymal lung disease, a DLco of 46% demonstrated 100% sensitivity and 73% specificity for detecting exercise pulmonary hypertension. In patients without parenchymal lung disease, a DLco of 73% demonstrated 58% sensitivity and 94% specificity for detecting exercise pulmonary hypertension. In both cohorts, DLco% below the optimum cutoffs were associated with higher peak mean pulmonary arterial pressure and peak total pulmonary resistance consistent with the hemodynamic definition of exercise pulmonary hypertension. Patients with a DLco < 46% were more often treated with pulmonary vasodilators and had a trend to higher mortality and lung transplant. DLco% is a simple non-invasive screening test for the presence of exercise pulmonary hypertension in our mixed referral population with progressive exertional dyspnea. DLco < 46% with parenchymal lung disease and DLco < 73% without parenchymal lung disease may play a role in differentiating the presence of pulmonary vascular disease prior to invasive hemodynamic testing.
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http://dx.doi.org/10.1177/2045894019891912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031800PMC
February 2020

Neurorobotics Workshop for High School Students Promotes Competence and Confidence in Computational Neuroscience.

Front Neurorobot 2020 13;14. Epub 2020 Feb 13.

Backyard Brains, Inc., Ann Arbor, MI, United States.

Understanding the brain is a fascinating challenge, captivating the scientific community and the public alike. The lack of effective treatment for most brain disorders makes the training of the next generation of neuroscientists, engineers and physicians a key concern. Over the past decade there has been a growing effort to introduce neuroscience in primary and secondary schools, however, hands-on laboratories have been limited to anatomical or electrophysiological activities. Modern neuroscience research labs are increasingly using computational tools to model circuits of the brain to understand information processing. Here we introduce the use of neurorobots - robots controlled by computer models of biological brains - as an introduction to computational neuroscience in the classroom. Neurorobotics has enormous potential as an education technology because it combines multiple activities with clear educational benefits including neuroscience, active learning, and robotics. We describe a 1-week introductory neurorobot workshop that teaches high school students how to use neurorobots to investigate key concepts in neuroscience, including spiking neural networks, synaptic plasticity, and adaptive action selection. Our do-it-yourself (DIY) neurorobot uses wheels, a camera, a speaker, and a distance sensor to interact with its environment, and can be built from generic parts costing about $170 in under 4 h. Our Neurorobot App visualizes the neurorobot's visual input and brain activity in real-time, and enables students to design new brains and deliver dopamine-like reward signals to reinforce chosen behaviors. We ran the neurorobot workshop at two high schools ( = 295 students total) and found significant improvement in students' understanding of key neuroscience concepts and in students' confidence in neuroscience, as assessed by a pre/post workshop survey. Here we provide DIY hardware assembly instructions, discuss our open-source Neurorobot App and demonstrate how to teach the Neurorobot Workshop. By doing this we hope to accelerate research in educational neurorobotics and promote the use of neurorobots to teach computational neuroscience in high school.
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http://dx.doi.org/10.3389/fnbot.2020.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033397PMC
February 2020

Risk and Response Adapted Treatment Guidelines for Managing First Relapsed and Refractory Classical Hodgkin Lymphoma in Children and Young People. Recommendations from the EuroNet Pediatric Hodgkin Lymphoma Group.

Hemasphere 2020 02 10;4(1):e329. Epub 2020 Jan 10.

University of Edinburgh and Department of Pediatrics, Royal Hospital for Sick Children, Edinburgh, Scotland, UK.

The objective of this guideline is to aid clinicians in making individual salvage treatment plans for pediatric and adolescent patients with first relapse or refractory (R/R) classical Hodgkin lymphoma (cHL). While salvage with standard dose chemotherapy followed by high dose chemotherapy and autologous stem cell transplant is often considered the standard of care in adult practice, pediatric practice adopts a more individualized risk stratified and response adapted approach to salvage treatment with greater use of non-transplant salvage. Here, we present on behalf of the EuroNet Pediatric Hodgkin Lymphoma group, evidence and consensus-based guidelines for standardized diagnostic, prognostic and response procedures to allocate children and adolescents with R/R cHL to stratified salvage treatments.
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http://dx.doi.org/10.1097/HS9.0000000000000329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000476PMC
February 2020

Digital Whole Slide Imaging Compared With Light Microscopy for Primary Diagnosis in Surgical Pathology.

Arch Pathol Lab Med 2020 10;144(10):1245-1253

The Department of Pathology and Laboratory Medicine, Hospital for Special Surgery, Weill Cornell Medical College, New York, New York (TW Bauer).

Context.—: The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility.

Objective.—: To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology.

Design.—: The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review.

Results.—: The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities.

Conclusions.—: WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.
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http://dx.doi.org/10.5858/arpa.2019-0569-OADOI Listing
October 2020

Capillary Proliferation in Systemic-Sclerosis-Related Pulmonary Fibrosis: Association with Pulmonary Hypertension.

ACR Open Rheumatol 2019 Mar 15;1(1):26-36. Epub 2019 Mar 15.

University of California Los Angeles.

Objective: We sought to determine if any histopathologic component of the pulmonary microcirculation can distinguish systemic sclerosis (SSc)-related pulmonary fibrosis (PF) with and without pulmonary hypertension (PH).

Methods: Two pulmonary pathologists blindly evaluated 360 histologic slides from lungs of 31 SSc-PF explants or autopsies with (n = 22) and without (n = 9) PH. The presence of abnormal small arteries, veins, and capillaries (pulmonary microcirculation) was semiquantitatively assessed in areas of preserved lung architecture. Capillary proliferation (CP) within the alveolar walls was measured by its distribution, extent (CP % involvement), and maximum number of layers (maximum CP). These measures were then evaluated to determine the strength of their association with right heart catheterization-proven PH.

Results: Using consensus measures, all measures of CP were significantly associated with PH. Maximum CP had the strongest association with PH ( = 0.013; C statistic 0.869). Maximum CP 2 or more layers and CP % involvement 10% or greater were the optimal thresholds that predicted PH, both with a sensitivity of 56% and specificity of 91%. The CP was typically multifocal rather than focal or diffuse and was associated with a background pattern of usual interstitial pneumonia. There was a significant but weaker relationship between the presence of abnormal small arteries and veins and PH.

Conclusion: In the setting of advanced SSc-PF, the histopathologic feature of the pulmonary microcirculation best associated with PH was capillary proliferation in architecturally preserved lung areas.
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http://dx.doi.org/10.1002/acr2.1003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858021PMC
March 2019

Nanoharvesting of bioactive materials from living plant cultures using engineered silica nanoparticles.

Mater Sci Eng C Mater Biol Appl 2020 Jan 11;106:110190. Epub 2019 Sep 11.

Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506, USA. Electronic address:

Plant secondary metabolites are valuable therapeutics not readily synthesized by traditional chemistry techniques. Although their enrichment in plant cell cultures is possible following advances in biotechnology, conventional methods of recovery are destructive to the tissues. Nanoharvesting, in which nanoparticles are designed to bind and carry biomolecules out of living cells, offers continuous production of metabolites from plant cultures. Here, nanoharvesting of polyphenolic flavonoids, model plant-derived therapeutics, enriched in Solidago nemoralis hairy root cultures, is performed using engineered mesoporous silica nanoparticles (MSNPs, 165 nm diameter and 950 m/g surface area) functionalized with both titanium dioxide (TiO, 425 mg/g particles) for coordination binding sites, and amines (NH, 145 mg/g particles) to promote cellular internalization. Intracellular uptake and localization of the nanoparticles (in Murashige and Skoog media) in hairy roots were confirmed by tagging the particles with rhodamine B isothiocyanate, incubating the particles with hairy roots, and quenching bulk fluorescence using trypan blue. Nanoharvesting of biologically active flavonoids was demonstrated by observing increased antiradical activity (using 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay) by nanoparticles after exposure to hairy roots (indicating general antioxidant activity), and by the displacement of the radio-ligand [H]-methyllycaconitine from rat hippocampal nicotinic receptors by solutes recovered from nanoharvested particles (indicating pharmacological activity specific to S. nemoralis flavonoids). Post-nanoharvesting growth suggests that the roots are viable after nanoharvesting, and capable of continued flavonoid synthesis. These observations demonstrate the potential for using engineered nanostructured particles to facilitate continuous isolation of a broad range of biomolecules from living and functioning plant cultures.
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http://dx.doi.org/10.1016/j.msec.2019.110190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935263PMC
January 2020

Ferumoxytol-enhanced MRI in patients with prior cardiac transplantation.

Open Heart 2019;6(2):e001115. Epub 2019 Oct 3.

Clinical Research Imaging Centre, University of Edinburgh, Edinburgh, UK.

Objectives: Ultra-small superparamagnetic particles of iron oxide (USPIO)-enhanced MRI can detect cellular inflammation within tissues and may help non-invasively identify cardiac transplant rejection. Here, we aimed to determine the normal reference values for USPIO-enhanced MRI in patients with a prior cardiac transplant and examine whether USPIO-enhanced MRI could detect myocardial inflammation in patients with transplant rejection.

Methods: Ten volunteers and 11 patients with cardiac transplant underwent T2, T2* and late gadolinium enhancement 1.5T MRI, with further T2* imaging at 24 hours after USPIO (ferumoxytol, 4 mg/kg) infusion, at baseline and 3 months.

Results: Ten patients with clinically stable cardiac transplantation were retained for analysis. Myocardial T2 values were higher in patients with cardiac transplant versus healthy volunteers (53.8±5.2 vs 48.6±1.9 ms, respectively; p=0.003). There were no differences in the magnitude of USPIO-induced change in R2* in patients with transplantation (change in R2*, 26.6±7.3 vs 22.0±10.4 s in healthy volunteers; p=0.28). After 3 months, patients with transplantation (n=5) had unaltered T2 values (52.7±2.8 vs 52.12±3.4 ms; p=0.80) and changes in R2* following USPIO (29.42±8.14 vs 25.8±7.8 s; p=0.43).

Conclusion: Stable patients with cardiac transplantation have increased myocardial T2 values, consistent with resting myocardial oedema or fibrosis. In contrast, USPIO-enhanced MRI is normal and stable over time suggesting the absence of chronic macrophage-driven cellular inflammation. It remains to be determined whether USPIO-enhanced MRI may be able to identify acute cardiac transplant rejection.

Trial Registration Number: NCT02319278349 (https://clinicaltrials.gov/ct2/show/NCT02319278) Registered 03.12.2014 EUDraCT 2013-002336-24.
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http://dx.doi.org/10.1136/openhrt-2019-001115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802993PMC
February 2021

Sensitive Method for the Confident Identification of Genetically Variant Peptides in Human Hair Keratin.

J Forensic Sci 2020 Mar 31;65(2):406-420. Epub 2019 Oct 31.

Biomolecular Measurement Division, Mass Spectrometry Data Center, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899.

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair-derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel-based method employed clearly shows the high degree of cross-linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non-cross-linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.
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http://dx.doi.org/10.1111/1556-4029.14229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064992PMC
March 2020

Everolimus worsening chronic proteinuria in patient with diabetic nephropathy post liver transplantation.

Saudi J Kidney Dis Transpl 2019 Jul-Aug;30(4):989-994

Department of Medicine, Division of Digestive Disease, Hepatology, University of California Los Angeles David Geffen School of Medicine; Department of Surgery, University of California Los Angeles David Geffen School of Medicine, Los Angeles, CA, USA.

Mammalian target of rapamycin (mTOR) inhibitors are used in renal sparing protocols and transplant immunosuppression in patients with solid organ and stem cell transplants. They cause various side effects, including proteinuria, which is mediated by blockade of the vascular endothelial growth factor receptor pathway. There have been various reports of mTOR inhibitors causing proteinuria or worsening proteinuria form preexisting renal glomerulo-pathies. We report a 73-year old male with diabetic glomerulosclerosis, acute liver failure due to Budd-Chiari syndrome, chronic low platelets, and worsening proteinuria from 0.46 g protein/g creatinine to 2.2 g protein/g creatinine. Workup revealed no thrombotic microangiopathy through skin biopsy, and a renal biopsy confirmed only clinically suspected diabetic and hypertensive glomerulosclerosis and possible calcineurin inhibitors. On discontinuation of everolimus urine protein decreased back to 0.6 g/g creatinine. We review the mechanism of mTOR-induced proteinuria and how this may affect diabetic nephropathy secondarily. We also consider the clinical implications of this in transplant patients receiving these agents.
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http://dx.doi.org/10.4103/1319-2442.265481DOI Listing
February 2020

Secondary membranous nephropathy in a patient with myasthenia gravis without thymic disease, and partial remission induced by adrenocorticotropic hormone therapy.

SAGE Open Med Case Rep 2019 8;7:2050313X19869764. Epub 2019 Aug 8.

Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.

Membranous glomerulonephritis is the most common glomerular disease in adults. Its primary form has been characterized with formation of phospholipase A2 receptor antibodies. Malignancy, infections, and autoimmune disorders are the most common causes of secondary membranous glomerulonephritis. We present a case of a 55-year-old African American female who presented with nephrotic range proteinuria and diagnosed with secondary membranous glomerulonephritis based on distinct pathological features on kidney biopsy and absence of serum phospholipase A2 receptor antibodies. She initially underwent extensive workup for malignancies, infections, and common autoimmune disorders which were all negative. Her proteinuria remained resistant to steroid treatment and she was treated with subcutaneous adrenocorticotropic hormone injections. Meanwhile, she was also diagnosed with the anti-muscle specific kinase antibody variant of myasthenia gravis. In literature, there are few case reports of myasthenia gravis as a cause of secondary membranous glomerulonephritis. In our case, the lack of other inciting factors also suggested this association.
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http://dx.doi.org/10.1177/2050313X19869764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689923PMC
August 2019

Examining the relationship between metal exposure (Cd and Hg), subcellular accumulation, and physiology of juvenile Crassostrea virginica.

Environ Sci Pollut Res Int 2019 Sep 4;26(25):25958-25968. Epub 2019 Jul 4.

The Graduate Center, City University of New York, 365 Fifth Ave., New York, NY, 10016, USA.

To assess the toxicity and accumulation (total and subcellular partitioning) of cadmium (Cd) and mercury (Hg), juvenile eastern oysters, Crassostrea virginica, were exposed for 4 weeks to a range of concentrations (Control, Low (1×), and High (4×)). Despite the 4-fold increase in metal concentrations, oysters from the High-Cd treatment (2.4 μM Cd) attained a body burden that was only 2.4-fold greater than that of the Low-Cd treatment (0.6 μM Cd), while oysters from the High-Hg treatment (0.056 μM Hg) accumulated 8.9-fold more Hg than those from the Low-Hg treatment (0.014 μM Hg). This fold difference in total Cd burdens was, in general, mirrored at the subcellular level, though binding to heat-denatured proteins in the High-Cd treatment was depressed (only 1.6-fold higher than the Low-Cd treatment). Mercury did not appear to appreciably partition to the subcellular fractions examined in this study, with the fold difference in accumulation between the Low- and High-Hg treatments ranging from 1.5-fold (heat-stable proteins) to 4.6-fold (organelles). Differences in toxicological impairments (reductions in condition index, protein content, and ETS activity) exhibited by oysters from the High-Cd treatment may be partially due to the nature of how different metals partition to subcellular components in the oysters, though exact mechanisms will require further examination.
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http://dx.doi.org/10.1007/s11356-019-05860-1DOI Listing
September 2019

Black lungs in the general population: a new look at an old dispute.

J R Coll Physicians Edinb 2019 Jun;49(2):165-170

University of Aberdeen, Aberdeen, UK.

Almost from the time that autopsies were first routinely carried out, darkening of lungs with increasing age was described. Different explanations for the origin of the accumulating black pigment arose and by the early nineteenth century three hypotheses had emerged: 1) soot inhaled into the lungs from the air; 2) carbon accumulating in the lungs from abnormal pulmonary carbon dioxide metabolism; and, 3) pigment derived from the blood. In 1813 the English physician and chemist George Pearson published a paper in which he described the recovery of the black pigment from lungs and its chemical analysis. Pearson declared the black pigment to be airborne carbon/soot from the burning of coal and wood. He described these particles depositing in 'black spots' in the terminal airways and accumulating in the peribronchial lymph nodes, forming 'black glands'. Despite Pearson's prescient account, debate continued and the true explanation, given in that paper, was not fully accepted until the late nineteenth century.
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http://dx.doi.org/10.4997/JRCPE.2019.219DOI Listing
June 2019