Massively parallel phenotyping of coding variants in cancer with Perturb-seq.

Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings.

Amycolatopsis spp. infection with correlative histopathological findings from the paw of a cat.

This report describes the clinical presentation and diagnosis of a deep cutaneous Amycolatopsis spp. infection in a cat. Diagnosis was based on a combination of methods including culture, 16s rRNA sequencing and histopathological evaluation. Histopathological findings demonstrated unique melanin production. This report highlights the potential for infection by Actinomycetales beyond Nocardia and Actinomyces.

Ce rapport décrit la présentation clinique et le diagnostic d'une infection cutanée profonde à Amycolatopsis spp. chez un chat. Le diagnostic était basé sur une combinaison de méthodes comprenant la culture, le séquençage de l'ARNr 16s et l'évaluation histopathologique. Les résultats histopathologiques ont démontré une production unique de mélanine. Ce rapport met en évidence le potentiel d'infection par Actinomycetales au-delà de Nocardia et Actinomyces.

Este artículo describe la presentación clínica y el diagnóstico de una infección cutánea profunda con Amycolatopsis spp. en un gato. El diagnóstico se basó en una combinación de métodos que incluían cultivo, secuenciación del RNAr 16s y evaluación histopatológica. Los hallazgos histopatológicos demostraron una producción llamativa de melanina. Este informe destaca el potencial de infección por otros Actinomycetales distintos de Nocardia y Actinomyces.

Este relato descreve a apresentação clínica e o diagnóstico de uma infecção cutânea profunda por Amycolatopsis spp. em um gato. O diagnóstico foi baseado em uma combinação de métodos incluindo cultura, sequenciamento de 16S rRNA e avaliação histopatológica. Os achados histopatológicos demonstraram distinta produção de melanina. Este relato destaca o potencial de infecção por Actynomycetales além de Nocardia e Actinomyces.

Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

Validation of a flash glucose monitoring system in outpatient diabetic cats.

BACKGROUND: Interstitial glucose (IG) concentration measurement using a flash glucose monitoring system (FGMS) is a noninvasive, affordable, and informative method to regulate patients with diabetes mellitus (DM) but has not been fully validated in outpatient cats with DM. OBJECTIVES: To further validate the FreeStyle Libre FGMS in outpatient diabetic cats. ANIMALS: Eight client-owned cats with DM. METHODS: Prospective observational validation study. Tissue glue was used to attach the sensor to the cat. Lin's concordance correlation coefficient (ρc ) was used to compare IG concentrations measured by the FGMS to blood glucose concentrations measured using an automated biochemistry analyzer (ABA) and point-of-care glucometer (POCG). RESULTS: Data from 15 sensor placements in 8 cats were analyzed. Paired IG and ABA glucose concentrations (139 samples) had excellent correlation (ρc  = 0.96) as did IG and POCG glucose concentrations (142 samples, ρc  = 0.92). Sensor failure or displacement were recorded for 12/15 (80%) sensor placements. Median time of sensor activity was 7 days (range, 2-13 days). CONCLUSIONS AND CLINICAL IMPORTANCE: In outpatient cats with DM, the FGMS-measured IG concentration correlated well with ABA-measured blood glucose concentration, but a high rate of sensor failures was observed.

Assessment of postprandial hyperglycemia and circadian fluctuation of glucose concentrations in diabetic dogs using a flash glucose monitoring system.

BACKGROUND: Postprandial hyperglycemia (PPH) and circadian glucose concentration fluctuations recorded in the home environment of dogs with naturally occurring diabetes mellitus (DM) have not been reported. OBJECTIVES: To determine if a flash glucose monitoring system (FGMS; FreeStyle Libre) can detect PPH and circadian fluctuations in glucose concentrations in dogs with variably controlled DM. ANIMALS: Fourteen client-owned dogs with DM. METHODS: Prospective observational study. Interstitial glucose (IG) concentrations measured by the FGMS during a 13-day study period were analyzed. RESULTS: A total of 17, 446 FGMS IG concentrations were analyzed. For all dogs analyzed together, median IG concentration measured within 30 (288 mg/dL), 60 (286 mg/dL), 90 (285 mg/dL), and 120 (285 mg/dL) minutes of meals was each significantly higher than the median IG concentration at all other times (260 mg/dL, 259 mg/dL, 258 mg/dL, and 257 mg/dL, respectively; range, 40-500 mg/dL; P < .001 for each). Median night-time IG concentration measured from all dogs on 3,547 samples recorded between 1:00 am and 6:00 am (268 mg/dL; range, 40-500 mg/dL) was significantly higher than median IG measured on 13, 899 samples at all other time points (259 mg/dL; range, 40-500 mg/dL; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The FGMS can be used for future studies of PPH and circadian fluctuations of glucose concentrations in dogs with DM in their home environment.

Identification and evaluation of volatile organic compounds evolved during solarization with almond hull and shell amendments.

Biosolarization is a fumigation alternative that combines solarization with organic amendments to suppress pests and pathogens in agricultural soils. The generation of volatile biopesticides in the soil, stemming from biodegradation of carbon-rich amendments, contributes to pest inactivation. The purpose of this study was to (1) profile volatiles that may contribute to pest control under field conditions and (2) measure volatile compounds that may present nuisance or exposure risks for humans near biosolarized fields where larger-scale anaerobic degradation of residues occurs. Biosolarization was performed using prominent agricultural waste products, hulls and shells from several almond varieties as soil amendments. After 8 days of biosolarization, soil samples were analyzed using solid phase microextraction-gas chromatography coupled to mass spectrometry. Volatile fatty acids and ketones made up 85% of biosolarized soil headspace, but terpenes, alcohols, aldehydes, esters, and sulfides were detected as well. Different almond variety residues produced distinct volatile profiles, and nonpareil-amended soils had a much richer and more diverse profile, as well as a fivefold greater VOC abundance, than pollinator-amended soil. Identified volatiles with low US recommended exposure limits were quantified via internal and external standards, including acetic acid, 2-butanone, butanal, hexanal, and phenylethyl alcohol. Across biosolarization treatments, headspace concentrations of selected compounds did not exceed 1 mg/m3. This study demonstrates that almond processing residues recycled into the soil as biosolarization substrates produce a high diversity of bioactive degradation compounds on a field scale, with low potential of non-target risks to humans.Implications: This manuscript has implications for two policy goals in the state of California: to reduce landfill disposal of organic waste, and to reduce emissions from soil fumigants. Almond hulls and shells are an increasing source of organic waste, and novel recycling strategies must be developed. Here, recycling almond residues as soil amendments promoted the rapid formation of VOCs which may act as alternatives to chemical fumigants. Headspace concentrations of potentially deleterious VOCs produced from treated soil were low, on the order of parts per billion. These results will help achieve policy goals by expanding waste usage and fumigation alternatives.

Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages.

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

Almond processing residues as a source of organic acid biopesticides during biosolarization.

Biosolarization utilizes organic amendments to produce biopesticide compounds in soil that can work in tandem with other stresses to inactivate agricultural pests. The prospect of using by-products from industrial almond processing as amendments for biosolarization was assessed. Soil mesocosms were used to simulate biosolarization using various almond by-products, application rates, and incubation times. Several potentially biopesticidal organic acids were identified and quantified in the soil, and the toxicity of soil extracts was evaluated for the root lesion nematode (Pratylenchus vulnus). It was determined that both almond hulls and a mixture of hulls and shells harbored several acids, the concentration of which was enhanced 1-7 fold via fermentation by native soil microbes. Organic acid concentration in the soil showed a significant linear relationship with the quantity of waste biomass amended. Extracts from soils containing at least 2.5% incorporated biomass by dry weight showed a 84-100% mortality of nematodes, which corresponded to acid concentrations 0.75 mg/g (2.0 g/L) or greater. This study showed that almond processing by-products - hulls and a hull and shell mixture - were suitable amendments for control of P. vulnus and potentially other soil agricultural pests in the context of biosolarization.

Vesicovaginal fistula in a dog with urinary incontinence.

CASE DESCRIPTION: A 5-year-old spayed female Maltese mixed-breed dog was referred for evaluation because of severe urinary incontinence refractory to medical management. CLINICAL FINDINGS: Physical examination revealed constant dribbling of urine and urine scalding. Culture of a urine sample yielded methicillin-resistant Staphylococcus pseudintermedius and Proteus mirabilis. Abdominal ultrasonographic examination revealed absence of the left kidney, a small, nondistended urinary bladder, and diffuse hepatopathy. Urinary incontinence persisted despite appropriate antimicrobial treatment. Cystourethroscopy and vaginoscopy were subsequently performed and revealed a hypoplastic bladder and a vesicovaginal fistula with urinary leakage through the vaginal diverticulum; no left ureterovesicular junction was identified, consistent with suspected left renal aplasia. TREATMENT AND OUTCOME: Exploratory laparotomy was performed, and the cranial aspect of the vagina was circumferentially ligated immediately caudal to the fistula. The urinary incontinence resolved immediately after surgery, and lower urinary tract signs improved over the next 2 weeks. Moderate urinary incontinence recurred approximately 6 months later, and a urinary tract infection with Escherichia coli was subsequently identified and treated; clinical signs resolved ≤ 48 hours after treatment was initiated. CLINICAL RELEVANCE: To the author's knowledge, vesicovaginal fistulas in dogs have not been previously described and should be considered a differential diagnosis for persistent urinary incontinence and recurrent urinary tract infections in female dogs. Vaginoscopy in addition to cystourethroscopy was required to identify the abnormality in this patient. Because multiple concurrent anomalies can be present, both procedures should be performed in female dogs with these clinical signs, even if an abnormality is identified cystoscopically.

Heart Transplant in Children: What a Primary Care Provider Needs to Know.

Heart transplantation is offered to children with heart failure that is not amenable to medical or surgical therapy. Indications for heart transplant include unrepairable congenital heart disease, failed palliation of congenital heart disease, and cardiomyopathies. There has been tremendous progress in this field since the first heart transplant was performed in 1967. Each year, approximately 500 pediatric heart transplants take place worldwide. Pediatric heart transplant survivors are living longer with their initial transplant. Many pediatric practitioners are faced with caring for these patients before as well as after the heart transplant and, therefore, they should be knowledgeable about basic principles related to heart transplant. There are certain complications that are unique to this population, and medication side-effects, evaluation and management of a febrile illness, opportunistic infections, vaccination, pregnancy, and exercise recommendations are areas that require special consideration. [Pediatr Ann. 2018;47(4):e172-e178.].

NetSig: network-based discovery from cancer genomes.

Methods that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes; but such approaches are challenging to validate at scale, and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks with data from 4,742 tumor exomes. NetSig can accurately classify known driver genes in 60% of tested tumor types and predicts 62 new driver candidates. Using a quantitative experimental framework to determine in vivo tumorigenic potential in mice, we found that NetSig candidates induce tumors at rates that are comparable to those of known oncogenes and are ten-fold higher than those of random genes. By reanalyzing nine tumor-inducing NetSig candidates in 242 patients with oncogene-negative lung adenocarcinomas, we find that two (AKT2 and TFDP2) are significantly amplified. Our study presents a scalable integrated computational and experimental workflow to expand discovery from cancer genomes.

Survival analysis of hypotensive cats admitted to an intensive care unit with or without hyperlactatemia: 39 cases (2005-2011).

OBJECTIVE To examine the association between blood lactate concentration and survival to hospital discharge in critically ill hypotensive cats. DESIGN Retrospective case series. ANIMALS 39 cats admitted to an intensive care unit of a university veterinary hospital between January 2005 and December 2011 for which blood lactate concentration was recorded ≤ 1 hour before or after a Doppler-derived arterial blood pressure measurement ≤ 90 mm Hg (ie, hypotension) was obtained. PROCEDURES Medical records of each cat were reviewed to assess survival to hospital discharge, illness severity, duration of hospitalization, age, body weight, and PCV. Results were compared between hypotensive cats with and without hyperlactatemia (blood lactate concentration ≥ 2.5 mmol/L). RESULTS 6 of 39 (15%) hypotensive cats survived to hospital discharge. Twelve (31%) cats were normolactatemic (blood lactate concentration < 2.5 mmol/L), and 27 (69%) were hyperlactatemic. Hypotensive cats with normolactatemia had a higher blood pressure and higher survival rate than hypotensive cats with hyperlactatemia. Five-day Kaplan-Meier survival rates were 57% for normolactatemic cats and 17% for hyperlactatemic cats. Age, body weight, duration of hospitalization, PCV, and illness severity did not differ significantly between hypotensive cats with and without hyperlactatemia. CONCLUSIONS AND CLINICAL RELEVANCE Hypotensive, normolactatemic cats in an intensive care unit had a significantly greater chance of survival to hospital discharge than their hyperlactatemic counterparts. Blood lactate concentration may be a useful prognostic indicator for this patient population when used in conjunction with other clinical and laboratory findings.

Chemical Analysis of Extracts from Newfoundland Berries and Potential Neuroprotective Effects.

Various species of berries have been reported to contain several polyphenolic compounds, such as anthocyanins and flavonols, which are known to possess high antioxidant activity and may be beneficial for human health. To our knowledge, a thorough chemical analysis of polyphenolics in species of these plants native to Newfoundland, Canada has not been conducted. The primary objective of this study was to determine the polyphenolic compounds present in commercial extracts from Newfoundland berries, which included blueberries (V. angustifolium), lingonberries (V. vitis-idaea) and black currant (Ribes lacustre). Anthocyanin and flavonol glycosides in powdered extracts from Ribes lacustre and the Vaccinium species were identified using the high performance liquid chromatographic (HPLC) separation method with mass spectrometric (MS) detection. The identified compounds were extracted from dried berries by various solvents via ultrasonication followed by centrifugation. A reverse-phase analytical column was employed to identify the retention time of each chemical component before submission for LC-MS analysis. A total of 21 phenolic compounds were tentatively identified in the three species. Further, we tested the effects of the lingonberry extract for its ability to protect neurons and glia from trauma utilizing an in vitro model of cell injury. Surprisingly, these extracts provided complete protection from cell death in this model. These findings indicate the presence of a wide variety of anthocyanins and flavonols in berries that grow natively in Newfoundland. These powdered extracts maintain these compounds intact despite being processed from berry fruit, indicating their potential use as dietary supplements. In addition, these recent findings and previous data from our lab demonstrate the ability of compounds in berries to protect the nervous system from traumatic insults.