Molecular identity of human outer radial glia during cortical development.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

Zika virus alters centrosome organization to suppress the innate immune response.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD.

OBJECTIVE: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD. METHODS: Immunoglobulin G (IgG) from pediatric ROHHAD patients (n = 9), non-inflammatory individuals (n = 100) and relevant pediatric controls (n = 25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively. RESULTS: Autoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed. INTERPRETATION: Our results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

Persistence of Ambigrammatic Narnaviruses Requires Translation of the Reverse Open Reading Frame.

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as "ambigrammatic" harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb "Robin" RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Can Radiologists Predict the Presence of Ductal Carcinoma In Situ and Invasive Breast Cancer?

OBJECTIVE: We hypothesize that radiologists' estimated percentage likelihood assessments for the presence of ductal carcinoma in situ (DCIS) and invasive cancer may predict histologic outcomes. MATERIALS AND METHODS: Two hundred fifty cases categorized as BI-RADS category 4 or 5 at four University of California Medical Centers were retrospectively reviewed by 10 academic radiologists with a range of 1-39 years in practice. Readers assigned BI-RADS category (1, 2, 3, 4a, 4b, 4c, or 5), estimated percentage likelihood of DCIS or invasive cancer (0-100%), and confidence rating (1 = low, 5 = high) after reviewing screening and diagnostic mammograms and ultrasound images. ROC curves were generated. RESULTS: Sixty-two percent (156/250) of lesions were benign and 38% (94/250) were malignant. There were 26 (10%) DCIS, 20 (8%) invasive cancers, and 48 (19%) cases of DCIS and invasive cancer. AUC values were 0.830-0.907 for invasive cancer and 0.731-0.837 for DCIS alone. Sensitivity of 82% (56/68), specificity of 84% (153/182), positive predictive value (PPV) of 66% (56/85), negative predictive value (NPV) of 93% (153/165), and accuracy of 84% ([56 + 153]/250) were calculated using an estimated percentage likelihood of 20% or higher as the prediction threshold for invasive cancer for the radiologist with the highest AUC (0.907; 95% CI, 0.864-0.951). Every 20% increase in the estimated percentage likelihood of invasive cancer increased the odds of invasive cancer by approximately two times (odds ratio, 2.4). For DCIS, using a threshold of 40% or higher, sensitivity of 81% (21/26), specificity of 79% (178/224), PPV of 31% (21/67), NPV of 97% (178/183), and accuracy of 80% ([21 + 178]/250) were calculated. Similarly, these values were calculated at thresholds of 2% or higher (BI-RADS category 4) and 95% or higher (BI-RADS category 5) to predict the presence of malignancy. CONCLUSION: Using likelihood estimates, radiologists may predict the presence of invasive cancer with fairly high accuracy. Radiologist-assigned estimated percentage likelihood can predict the presence of DCIS, albeit with lower accuracy than that for invasive cancer.

Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids.

Rubella virus is an important human pathogen that can cause neurological deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.

Quantitative reverse transcription PCR assay to detect a genetic marker of pyrethroid resistance in Culex mosquitoes.

Pyrethroid insecticides are widely used to control mosquitoes that transmit pathogens such as West Nile virus (WNV) to people. Single nucleotide polymorphisms (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene in Culex mosquitoes are associated with knockdown resistance to pyrethroids. RNAseq was used to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. The cDNA sequences were used to develop a quantitative reverse transcriptase PCR assay that detects the L1014F kdr mutation in the Vgsc. Because this locus is conserved, the assay was used successfully in six Culex spp. The resulting Culex RTkdr assay was validated using quantitative PCR and sequencing of PCR products. The accuracy of the Culex RTkdr assay was 99%. The L1014F kdr mutation associated with pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and was more prevalent in mosquitoes collected near farmland. The Culex RTkdr assay takes advantage of the RNA that vector control agencies routinely isolate to assess arbovirus prevalence in mosquitoes. We anticipate that public health and vector control agencies may employ the Culex RTkdr assay to define the geographic distribution of the L1014F kdr mutation in Culex species and improve the monitoring of insecticide resistance that will ultimately contribute to effective control of Culex mosquitoes.

Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway.

Single mosquito metatranscriptomics identifies vectors, emerging pathogens and reservoirs in one assay.

Mosquitoes are major infectious disease-carrying vectors. Assessment of current and future risks associated with the mosquito population requires knowledge of the full repertoire of pathogens they carry, including novel viruses, as well as their blood meal sources. Unbiased metatranscriptomic sequencing of individual mosquitoes offers a straightforward, rapid, and quantitative means to acquire this information. Here, we profile 148 diverse wild-caught mosquitoes collected in California and detect sequences from eukaryotes, prokaryotes, 24 known and 46 novel viral species. Importantly, sequencing individuals greatly enhanced the value of the biological information obtained. It allowed us to (a) speciate host mosquito, (b) compute the prevalence of each microbe and recognize a high frequency of viral co-infections, (c) associate animal pathogens with specific blood meal sources, and (d) apply simple co-occurrence methods to recover previously undetected components of highly prevalent segmented viruses. In the context of emerging diseases, where knowledge about vectors, pathogens, and reservoirs is lacking, the approaches described here can provide actionable information for public health surveillance and intervention decisions.

Upper airway gene expression reveals a more robust innate and adaptive immune response to SARS-CoV-2 in children compared with older adults.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults and only rarely in children. To investigate whether differences in the upper airway immune response could contribute to this disparity, we compared nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes (ISGs) was robustly activated in both children and adults with SARS-CoV-2 compared to the respective non-viral groups, with only relatively subtle distinctions. Children, however, demonstrated markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including TNF, IFNγ, IL-2 and IL-4 production. Cell type deconvolution confirmed greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibited a decrease in proportions of ciliated cells, the primary target of SARS-CoV-2, upon infection with the virus. These findings demonstrate that children elicit a more robust innate and adaptive immune response to SARS-CoV-2 infection in the upper airway that likely contributes to their protection from severe disease in the lower airway.

Genome Sequence of a Divergent Avian Metapneumovirus from a Monk Parakeet (Myiopsitta monachus).

Here, we report the coding-complete genome sequence of an avian metapneumovirus from a monk parakeet (Myiopsitta monachus), identified by metagenomic next-generation sequencing during an investigation into a disease outbreak in a captive parrot breeding facility. Based on divergence from known strains, this sequence represents a new subgroup of avian metapneumovirus.

An exploration of ambigrammatic sequences in narnaviruses.

Narnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of ~3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are 'ambigrammatic' and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The >3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development.

METAGENOMIC NEXT-GENERATION SEQUENCING REVEALS MIAMIENSIS AVIDUS (CILIOPHORA: SCUTICOCILIATIDA) IN THE 2017 EPIZOOTIC OF LEOPARD SHARKS ( TRIAKIS SEMIFASCIATA) IN SAN FRANCISCO BAY, CALIFORNIA, USA.

During March to August of 2017, hundreds of leopard sharks ( Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, US. Similar mass stranding events occurred in 1967 and 2011, but analysis of those epizootics was incomplete, and no etiology was confirmed. Our investigation of the 2017 epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. We pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening. We showed that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard ( n=12) and other shark species ( n=2) that stranded in San Francisco Bay but was absent in leopard sharks caught elsewhere. This ciliated protozoan has been implicated in devastating outbreaks in teleost marine fish but not in wild elasmobranchs. Our results highlight the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease.

Exploratory proteomic analysis implicates the alternative complement cascade in primary CNS vasculitis.

OBJECTIVE: To identify molecular correlates of primary angiitis of the CNS (PACNS) through proteomic analysis of CSF from a biopsy-proven patient cohort. METHODS: Using mass spectrometry, we quantitatively compared the CSF proteome of patients with biopsy-proven PACNS (n = 8) to CSF from individuals with noninflammatory conditions (n = 11). Significantly enriched molecular pathways were identified with a gene ontology workflow, and high confidence hits within enriched pathways (fold change >1.5 and concordant Benjamini-Hochberg-adjusted p < 0.05 on DeSeq and t test) were identified as differentially regulated proteins. RESULTS: Compared to noninflammatory controls, 283 proteins were differentially expressed in the CSF of patients with PACNS, with significant enrichment of the complement cascade pathway (C4-binding protein, CD55, CD59, properdin, complement C5, complement C8, and complement C9) and neural cell adhesion molecules. A subset of clinically relevant findings were validated by Western blot and commercial ELISA. CONCLUSIONS: In this exploratory study, we found evidence of deregulation of the alternative complement cascade in CSF from biopsy-proven PACNS compared to noninflammatory controls. More specifically, several regulators of the C3 and C5 convertases and components of the terminal cascade were significantly altered. These preliminary findings shed light on a previously unappreciated similarity between PACNS and systemic vasculitides, especially anti-neutrophil cytoplasmic antibody-associated vasculitis. The therapeutic implications of this common biology and the diagnostic and therapeutic utility of individual proteomic findings warrant validation in larger cohorts.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Indigenous Values and Health Systems Stewardship in Circumpolar Countries.

Circumpolar regions, and the nations within which they reside, have recently gained international attention because of shared and pressing public policy issues such as climate change, resource development, endangered wildlife and sovereignty disputes. In a call for national and circumpolar action on shared areas of concern, the Arctic states health ministers recently met and signed a declaration that identified shared priorities for international cooperation. Among the areas for collaboration raised, the declaration highlighted the importance of enhancing intercultural understanding, promoting culturally appropriate health care delivery and strengthening circumpolar collaboration in culturally appropriate health care delivery. This paper responds to the opportunity for further study to fully understand indigenous values and contexts, and presents these as they may apply to a framework that will support international comparisons and systems improvements within circumpolar regions. We explored the value base of indigenous peoples and provide considerations on how these values might interface with national values, health systems values and value bases between indigenous nations particularly in the context of health system policy-making that is inevitably shared between indigenous communities and jurisdictional or federal governments. Through a mixed methods nominal consensus process, nine values were identified and described: humanity, cultural responsiveness, teaching, nourishment, community voice, kinship, respect, holism and empowerment.

Inter-reader Variability in the Use of BI-RADS Descriptors for Suspicious Findings on Diagnostic Mammography: A Multi-institution Study of 10 Academic Radiologists.

RATIONALE AND OBJECTIVES: The study aimed to determine the inter-observer agreement among academic breast radiologists when using the Breast Imaging Reporting and Data System (BI-RADS) lesion descriptors for suspicious findings on diagnostic mammography. MATERIALS AND METHODS: Ten experienced academic breast radiologists across five medical centers independently reviewed 250 de-identified diagnostic mammographic cases that were previously assessed as BI-RADS 4 or 5 with subsequent pathologic diagnosis by percutaneous or surgical biopsy. Each radiologist assessed the presence of the following suspicious mammographic findings: mass, asymmetry (one view), focal asymmetry (two views), architectural distortion, and calcifications. For any identified calcifications, the radiologist also described the morphology and distribution. Inter-observer agreement was determined with Fleiss kappa statistic. Agreement was also calculated by years of experience. RESULTS: Of the 250 lesions, 156 (62%) were benign and 94 (38%) were malignant. Agreement among the 10 readers was strongest for recognizing the presence of calcifications (k = 0.82). There was substantial agreement among the readers for the identification of a mass (k = 0.67), whereas agreement was fair for the presence of a focal asymmetry (k = 0.21) or architectural distortion (k = 0.28). Agreement for asymmetries (one view) was slight (k = 0.09). Among the categories of calcification morphology and distribution, reader agreement was moderate (k = 0.51 and k = 0.60, respectively). Readers with more experience (10 or more years in clinical practice) did not demonstrate higher levels of agreement compared to those with less experience. CONCLUSIONS: Strength of agreement varies widely for different types of mammographic findings, even among dedicated academic breast radiologists. More subtle findings such as asymmetries and architectural distortion demonstrated the weakest agreement. Studies that seek to evaluate the predictive value of certain mammographic features for malignancy should take into consideration the inherent interpretive variability for these findings.

Approaching Etuaptmumk--introducing a consensus-based mixed method for health services research.

With the recognized need for health systems' improvements in the circumpolar and indigenous context, there has been a call to expand the research agenda across all sectors influencing wellness and to recognize academic and indigenous knowledge through the research process. Despite being recognized as a distinct body of knowledge in international forums and across indigenous groups, examples of methods and theories based on indigenous knowledge are not well documented in academic texts or peer-reviewed literature on health systems. This paper describes the use of a consensus-based, mixed method with indigenous knowledge by an experienced group of researchers and indigenous knowledge holders who collaborated on a study that explored indigenous values underlying health systems stewardship. The method is built on the principles of Etuaptmumk or two-eyed seeing, which aim to respond to and resolve the inherent conflicts between indigenous ways of knowing and the scientific inquiry that informs the evidence base in health care. Mixed methods' frameworks appear to provide a framing suitable for research questions that require data from indigenous knowledge sources and western knowledge. The nominal consensus method, as a western paradigm, was found to be responsive to embedding of indigenous knowledge and allowed space to express multiple perspectives and reach consensus on the question at hand. Further utilization and critical evaluation of this mixed methodology with indigenous knowledge are required.

"Score the Core" Web-based pathologist training tool improves the accuracy of breast cancer IHC4 scoring.

Hormone receptor status is an integral component of decision-making in breast cancer management. IHC4 score is an algorithm that combines hormone receptor, HER2, and Ki-67 status to provide a semiquantitative prognostic score for breast cancer. High accuracy and low interobserver variance are important to ensure the score is accurately calculated; however, few previous efforts have been made to measure or decrease interobserver variance. We developed a Web-based training tool, called "Score the Core" (STC) using tissue microarrays to train pathologists to visually score estrogen receptor (using the 300-point H score), progesterone receptor (percent positive), and Ki-67 (percent positive). STC used a reference score calculated from a reproducible manual counting method. Pathologists in the Athena Breast Health Network and pathology residents at associated institutions completed the exercise. By using STC, pathologists improved their estrogen receptor H score and progesterone receptor and Ki-67 proportion assessment and demonstrated a good correlation between pathologist and reference scores. In addition, we collected information about pathologist performance that allowed us to compare individual pathologists and measures of agreement. Pathologists' assessment of the proportion of positive cells was closer to the reference than their assessment of the relative intensity of positive cells. Careful training and assessment should be used to ensure the accuracy of breast biomarkers. This is particularly important as breast cancer diagnostics become increasingly quantitative and reproducible. Our training tool is a novel approach for pathologist training that can serve as an important component of ongoing quality assessment and can improve the accuracy of breast cancer prognostic biomarkers.