Intraoral Radiographic Interpretation Agreement Between Veterinary Students, Veterinary Dental Residents and Veterinary Dental Specialists.

This study evaluated observer variations in the interpretation of radiographic evidence for periodontal disease, tooth resorption, and endodontic disease in dogs. Forty dental radiographs were evaluated for 12 different parameters (periapical destruction of bone, wider than expected root canal, narrower than expected root canal, apical root resorption, loss of alveolar bone, external surface resorption, external replacement resorption, external inflammatory resorption, external cervical root resorption, internal surface resorption, internal replacement resorption, internal inflammatory resorption). Interpretations by 20 veterinary dentists, 10 veterinary dental residents, and 10 veterinary students were analyzed for consistency within groups and between groups by intraclass correlation coefficients (ICCs [95% CI]). Additionally, the agreement rate between groups compared to histopathological diagnosis, when available, and to a consensus group were evaluated. The results showed fair to good interobserver agreement for all participants when looking at all questions simultaneously (0.578 [0.515-0.635]) and with the consensus group (0.483 [0.451-0.517]). However, questions pertaining to various types of tooth resorption scored the lowest ICCs ranging from 0.005 (-0.311 to 0.321) to 0.189 (-0.105 to 0.402) across individual groups. Students had the lowest agreement compared to the consensus group for all questions (0.383 [0.347-0.421]) with fair to good agreement involving groups of residents (0.501 [0.465-0.538]), recently boarded diplomates (0.541 [0.506-0.578]), and more experienced diplomates (0.545 [0.510-0.582]). While dental radiographs are essential for clinical decision making, this study shows that interpretation of radiographs is highly subjective.

Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer.

INTRODUCTION: Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together. METHODS: Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients. RESULTS: Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression. CONCLUSIONS: A standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.

Acute skin exposure to ultraviolet light triggers neutrophil-mediated kidney inflammation.

Photosensitivity to ultraviolet (UV) light affects up to ∼80% of lupus patients. Sunlight exposure can exacerbate local as well as systemic manifestations of lupus, including nephritis, by mechanisms that are poorly understood. Here, we report that acute skin exposure to UV light triggers a neutrophil-dependent injury response in the kidney characterized by upregulated expression of endothelial adhesion molecules as well as inflammatory and injury markers associated with transient proteinuria. We showed that UV light stimulates neutrophil migration not only to the skin but also to the kidney in an IL-17A-dependent manner. Using a photoactivatable lineage tracing approach, we observed that a subset of neutrophils found in the kidney had transited through UV light-exposed skin, suggesting reverse transmigration. Besides being required for the renal induction of genes encoding mediators of inflammation (vcam-1, s100A9, and Il-1b) and injury (lipocalin-2 and kim-1), neutrophils significantly contributed to the kidney type I interferon signature triggered by UV light. Together, these findings demonstrate that neutrophils mediate subclinical renal inflammation and injury following skin exposure to UV light. Of interest, patients with lupus have subpopulations of blood neutrophils and low-density granulocytes with similar phenotypes to reverse transmigrating neutrophils observed in the mice post-UV exposure, suggesting that these cells could have transmigrated from inflamed tissue, such as the skin.

Experimental and bioinformatics considerations in cancer application of single cell genomics.

Single cell genomics offers an unprecedented resolution to interrogate genetic heterogeneity in a patient's tumour at the intercellular level. However, the DNA yield per cell is insufficient for today's sequencing library preparation protocols. This necessitates DNA amplification which is a key source of experimental noise. We provide an evaluation of two protocols using micro-fluidics based amplification for whole exome sequencing, which is an experimental scenario commonly used in single cell genomics. The results highlight their respective biases and relative strengths in identification of single nucleotide variations. Towards this end, we introduce a workflow SoVaTSiC, which allows for quality evaluation and somatic variant identification of single cell data. As proof of concept, the framework was applied to study a lung adenocarcinoma tumour. The analysis provides insights into tumour phylogeny by identifying key mutational events in lung adenocarcinoma evolution. The consequence of this inference is supported by the histology of the tumour and demonstrates usefulness of the approach.

Predictors of hyponatremia following elective primary unilateral knee arthroplasty at a tertiary centre: A retrospective observational cohort and predictive model.

INTRODUCTION: Hyponatremia is a common electrolyte disorder. This can be associated with nausea, disorientation and in more serious cases a decreased level of consciousness or neurological deficits. These symptoms may lead to increases in the cost of hospital care and significant morbidity. The purpose of this retrospective, observational cohort study is to investigate the impact of hyponatremia on patient and systems specific outcome measures in those undergoing elective, unilateral total knee arthroplasty (TKA) at two hospitals in Auckland, New Zealand over a twelve-month period. MATERIALS AND METHODS: Patients were stratified into two groups based on the presence or absence of post-operative hyponatremia (defined as a blood sodium of <135 mmol/L with a concurrent decrease of ≥5 mmol/L between the pre- and post-operative recordings). Outcomes collected included Quality of Recovery - 15 (QOR) scores, time to assisted mobilisation, discharge ICD-10 complication codes and hospital length of stay. RESULTS: During the study period 236 patients underwent surgery. Eighty-six (36.4%) patients met criteria for post-operative hyponatremia. This finding was associated with prolongation of the hospital length of stay (4.17 (3.26-5.18) versus 4.28 (3.31-5.45) days, p = 0.031) and a reduction in the QOR score on the second post-operative day (113.0 (99.5-126.5) versus 105.0 (94.0-118.0), p = 0.039). There was no difference in the time to assisted mobilisation. CONCLUSIONS: Hyponatremia is a common finding following TKA. This abnormality is associated with small changes in patient specific outcome measures. These implications of these findings may become more significant in settings where same day or rapid discharge from hospital is targeted.

The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent.

Most systemic lupus erythematosus (SLE) patients are photosensitive and ultraviolet B light (UVB) exposure worsens cutaneous disease and precipitates systemic flares of disease. The pathogenic link between skin disease and systemic exacerbations in SLE remains elusive. In an acute model of UVB-triggered inflammation, we observed that a single UV exposure triggered a striking IFN-I signature not only in the skin, but also in the blood and kidneys. The early IFN-I signature was significantly higher in female compared to male mice. The early IFN-I response in the skin was almost entirely, and in the blood partly, dependent on the presence of cGAS, as was skin inflammatory cell infiltration. Inhibition of cGAMP hydrolysis augmented the UVB-triggered IFN-I response. UVB skin exposure leads to cGAS-activation and both local and systemic IFN-I signature and could contribute to acute flares of disease in susceptible subjects such as patients with SLE.

Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury.

Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.

Concurrent Single-Cell RNA and Targeted DNA Sequencing on an Automated Platform for Comeasurement of Genomic and Transcriptomic Signatures.

BACKGROUND: The comeasurement of both genomic and transcriptomic signatures in single cells is of fundamental importance to accurately assess how the genetic information correlates with the transcriptomic phenotype. However, existing technologies have low throughput and laborious work flows. METHODS: We developed a new method for concurrent sequencing of the transcriptome and targeted genomic regions (CORTAD-seq) within the same single cell on an automated microfluidic platform. The method was compatible with the downstream library preparation, allowing easy integration into existing next-generation sequencing work flows. We incorporated a single-cell bioinformatics pipeline for transcriptome and mutation analysis. RESULTS: As proof of principle, we applied CORTAD-seq to lung cancer cell lines to dissect the cellular consequences of mutations that result in resistance to targeted therapy. We obtained a mean detection of 6000 expressed genes and an exonic rate of 50%. The targeted DNA-sequencing data achieved a 97.8% detection rate for mutations and allowed for the identification of copy number variations and haplotype construction. We detected expression signatures of tyrosine kinase inhibitor (TKI) resistance, epidermal growth factor receptor (EGFR) amplification, and expansion of the T790M mutation among resistant cells. We also identified characteristics for TKI resistance that were independent of EGFR T790M, indicating that other alterations are required for resistance in this context. CONCLUSIONS: CORTAD-seq allows assessment of the interconnection between genetic and transcriptomic changes in single cells. It is operated on an automated, commercially available single-cell isolation platform, making its implementation straightforward.

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care.

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Wearable multi-channel microelectrode membranes for elucidating electrophysiological phenotypes of injured myocardium.

Understanding the regenerative capacity of small vertebrate models has provided new insights into the plasticity of injured myocardium. Here, we demonstrate the application of flexible microelectrode arrays (MEAs) in elucidating electrophysiological phenotypes of zebrafish and neonatal mouse models of heart regeneration. The 4-electrode MEA membranes were designed to detect electrical signals in the aquatic environment. They were micro-fabricated to adhere to the non-planar body surface of zebrafish and neonatal mice. The acquired signals were processed to display an electrocardiogram (ECG) with high signal-to-noise-ratios, and were validated via the use of conventional micro-needle electrodes. The 4-channel MEA provided signal stability and spatial resolution, revealing the site-specific electrical injury currents such as ST-depression in response to ventricular cryo-injury. Thus, our polymer-based and wearable MEA membranes provided electrophysiological insights into long-term conduction phenotypes for small vertebral models of heart injury and regeneration with a translational implication for monitoring cardiac patients.