Methionine Restriction Reduces Lung Cancer Progression and Increases Chemotherapy Response.

Targeting tumor metabolism through dietary interventions is an area of growing interest, and may help to improve the significant mortality of aggressive cancers, including non-small cell lung cancer (NSCLC). Here we show that the restriction of methionine in the aggressive KRAS /Lkb1- mutant NSCLC autochthonous mouse model drives decreased tumor progression and increased carboplatin treatment efficacy. Importantly, methionine restriction during early stages of tumorigenesis prevents the lineage switching known to occur in the model, and alters the tumor immune microenvironment (TIME) to have fewer tumor-infiltrating neutrophils. Mechanistically, mutations in LKB1 are linked to anti-oxidant production through changes to cystathionine-ß-synthase (CBS) expression. Human cell lines with rescued LKB1 show increased CBS levels and resistance to carboplatin, which can be partially rescued by methionine restriction. Furthermore, LKB1 rescued cells, but not mutant cells, show less G2- M arrest and apoptosis in high methionine conditions. Knock-down of CBS sensitized both LKB1 mutant and non-mutated lines to carboplatin, again rescuing the carboplatin resistance of the LKB1 rescued lines. Given that immunotherapy is commonly combined with chemotherapy for NSCLC, we next wanted to understand if T cells are impaired by MR. Therefore, we examined the ability of T cells from MR and control tumor bearing mice to proliferate in culture and found that T cells from MR treated mice had no defects in proliferation, even though we continued the MR conditions ex vivo . We also identified that CBS is most highly correlated with smoking, adenocarcinomas with alveolar and bronchiolar features, and adenosquamous cell carcinomas, implicating its roles in oxidative stress response and lineage fate in human tumors. Taken together, we have shown the importance of MR as a dietary intervention to slow tumor growth and improve treatment outcomes for NSCLC.

A dose titration protocol for once-daily insulin glargine 300 U/mL for the treatment of diabetes mellitus in dogs.

BACKGROUND: In purpose-bred dogs, insulin glargine 300 U/mL (IGla300) has long duration of action, peakless time-action profile, and low potency, making it suitable for use as a basal insulin. HYPOTHESIS: To evaluate IGla300 in client-owned diabetic dogs monitored using a flash glucose monitoring system (FGMS). ANIMALS: Ninety-five client-owned diabetic dogs, newly diagnosed or previously treated with other insulin formulations, with or without concurrent diseases. METHODS: Prospective multi-institutional study. Clinical signs and standardized assessment of FGMS data, using treatment and monitoring guidelines established a priori, guided dose adjustments and categorization into levels of glycemic control. RESULTS: The initial IGla300 dose was 0.5 U/Kg q24h for newly diagnosed dogs and (median dose [range]) 0.8 U/Kg (0.2-2.5) q24h for all dogs. Glycemic control was classified as good or excellent in 87/95 (92%) dogs. The IGla300 was administered q24h (1.9 U/kg [0.2-5.2]) and q12h (1.9 U/kg/day [0.6-5.0]) in 56/95 (59%) and 39/95 (41%) dogs, respectively. Meal-time bolus injections were added in 5 dogs (0.5 U/kg/injection [0.3-1.0]). Clinical hypoglycemia occurred in 6/95 (6%) dogs. Dogs without concurrent diseases were more likely to receive IGla300 q24h than dogs with concurrent diseases (72% vs 50%, respectively; P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: Insulin glargine 300 U/mL can be considered a suitable therapeutic option for once-daily administration in diabetic dogs. Clinicians should be aware of the low potency and wide dose range of IGla300. In some dogs, twice-daily administration with or without meal-time bolus injections may be necessary to achieve glycemic control. Monitoring with FGMS is essential for dose titration of IGla300.

Polycomb deficiency drives a FOXP2-high aggressive state targetable by epigenetic inhibitors.

Inhibitors of the Polycomb Repressive Complex 2 (PRC2) histone methyltransferase EZH2 are approved for certain cancers, but realizing their wider utility relies upon understanding PRC2 biology in each cancer system. Using a genetic model to delete Ezh2 in KRAS-driven lung adenocarcinomas, we observed that Ezh2 haplo-insufficient tumors were less lethal and lower grade than Ezh2 fully-insufficient tumors, which were poorly differentiated and metastatic. Using three-dimensional cultures and in vivo experiments, we determined that EZH2-deficient tumors were vulnerable to H3K27 demethylase or BET inhibitors. PRC2 loss/inhibition led to de-repression of FOXP2, a transcription factor that promotes migration and stemness, and FOXP2 could be suppressed by BET inhibition. Poorly differentiated human lung cancers were enriched for an H3K27me3-low state, representing a subtype that may benefit from BET inhibition as a single therapy or combined with additional EZH2 inhibition. These data highlight diverse roles of PRC2 in KRAS-driven lung adenocarcinomas, and demonstrate the utility of three-dimensional cultures for exploring epigenetic drug sensitivities for cancer.

Successful multimodal treatment of extreme hypertriglyceridemia in a juvenile diabetic dog.

OBJECTIVE: To describe the therapeutic protocol used to normalize severe hypertriglyceridemia in a dog. CASE SUMMARY: A 7-month-old, 1.2-kg female Pomeranian presented with acute polyuria, polydipsia, and ocular discoloration. Diagnoses included diabetic ketosis, severe hypertriglyceridemia (>225 mmol/L [>20,000 mg/dl]), lipemia retinalis, and bilateral uveitis. The triglyceride concentration was near normal within 2 days of initiating treatment with fenofibrate, regular insulin constant rate infusion (CRI), manual therapeutic plasma exchange (TPE), and a low-fat diet. All clinical signs resolved. The dog has had no relapse of hypertriglyceridemia at the time of writing the manuscript, 6 months later, with continued treatment of diabetes mellitus. NEW OR UNIQUE INFORMATION PROVIDED: This is the first case report documenting the combination of fenofibrate, insulin CRI, and manual TPE for treatment of severe hyperlipidemia in a dog. Detailed protocols for manual TPE and a novel insulin CRI are provided. A discussion of multiple spurious biochemical and hematologic errors associated with the severe hypertriglyceridemia is also provided.

Dysregulated Polycomb Repressive Complex 2 contributes to chronic obstructive pulmonary disease by rewiring stem cell fate.

Aberrant lung cell differentiation is a hallmark of many lung diseases including chronic obstructive pulmonary disease (COPD). The EZH2-containing Polycomb Repressive Complex 2 (PRC2) regulates embryonic lung stem cell fate, but its role in adult lung is obscure. Histological analysis of patient tissues revealed that loss of PRC2 activity was correlated with aberrant bronchiolar cell differentiation in COPD lung. Histological and single-cell RNA-sequencing analyses showed that loss of EZH2 in mouse lung organoids led to lowered self-renewal capability, increased squamous morphological development, and marked shifts in progenitor cell populations. Evaluation of in vivo models revealed that heterozygosity of Ezh2 in mice with ovalbumin-induced lung inflammation led to epithelial cell differentiation patterns similar to those in COPD lung. We also identified cystathionine-ß-synthase as a possible upstream factor for PRC2 destabilization. Our findings suggest that PRC2 is integral to facilitating proper lung stem cell differentiation in humans and mice.

Comparison of biochemical and hematologic values obtained via jugular venipuncture and peripheral intravenous catheters in dogs.

BACKGROUND: Sampling from a peripheral intravenous catheter (PIVC) might be a more efficient and less traumatic collection of blood for serum biochemistry (SB) or CBC than direct venipuncture (DV). Agreement between results of samples obtained by these methods has not been evaluated in dogs. OBJECTIVES: The primary objectives were to determine whether sampling from PIVC could be used in place of DV for dogs. We hypothesized DV and PIVC samples would have clinically equivalent SB and CBC results. ANIMALS: Sixty-one client-owned dogs were included in each study arm. METHODS: This was a partially randomized method-comparison study. Paired DV and PIVC samples obtained within 1 to 2 minutes after, or approximately 24 hours after, placement of a PIVC in a cephalic vein were evaluated for agreement and bias using percentage difference plots (with a priori application of consensus total allowable error), Bland-Altman analysis, Passing-Bablok regression analysis, Wilcoxon signed rank test, and McNemar's test. RESULTS: There was statistically and clinically acceptable agreement and no bias between sampling methods for the majority of results. Analytes with the most frequent disagreement were aspartate aminotransferase, total bilirubin, potassium, bicarbonate, and leukocyte differential counts, as well as red blood cell count, hemoglobin, hematocrit, and packed cell volume in the hospitalized PIVC sampling group. Few observed differences would change clinical decision making. CONCLUSIONS AND CLINICAL IMPORTANCE: PIVC sampling can provide generally acceptable SB and CBC results for most dogs, but clinicians should be aware of a few values for which disparate results might occasionally be obtained.

EZH2 inhibition confers PIK3CA-driven lung tumors enhanced sensitivity to PI3K inhibition.

Members of the PI3K signaling pathway, especially PIK3CA, the gene encoding the catalytic subunit of the PI3K complex, are highly mutated and amplified in various cancer types, including non-small cell lung cancer. Although PI3K inhibitors have been used in clinics for follicular lymphoma and chronic lymphocytic leukemia, no agents targeting PI3K aberrations in lung cancer have been approved by the FDA so far. In this study, we observed that PIK3CA-E545K, the most common mutation in lung cancer, harbored a modest induction of stem-like properties in lung epithelial cells, and drove development of adenocarcinoma autochthonously when paired with p53 loss in a murine mouse model. We also found that PIK3CA-mutant of amplified lung cancer cells were sensitive to EZH2 inhibition. EZH2 inhibition synergized with PI3K inhibition in human cancer cells in vitro and worked together efficiently in vivo. Mechanistically, EZH2 inhibition cooperated with PI3K inhibition to produce a more potent suppression of phospho-AKT downstream of PI3K. This study suggests a promising combination therapy to combat lung cancers with PIK3CA mutation or amplification. Both copanlisib, the PI3K inhibitor, and tazemetostat, the EZH2 inhibitor, are FDA-approved, which should enhance the clinical translation of this work.

Pulmonary Sarcomatoid Carcinoma Associated with Arterial Thromboembolism in a Cat.

A 14-year-old, neutered male domestic shorthair cat presented for acute monoparesis with physical exam findings and biochemical data supportive of a distal arterial thromboembolism. Thoracic radiographs revealed an alveolar pattern in the right middle lung lobe and multifocal nodules in other lung lobes. A pulmonary mass was found on necropsy, which was composed of both carcinomatous and sarcomatous components, confirmed with cytokeratin and vimentin immunohistochemistry. Using the World Health Organization classification scheme for mixed pulmonary tumors, this tumor would be characterized as a pleomorphic squamous cell carcinoma under the umbrella term of pulmonary sarcomatoid carcinoma. The World Health Organization classification of mixed pulmonary tumors and its application to previously reported mixed pulmonary tumors in companion animals is discussed. This is the first reported case of this tumor type in a cat, as well as the first report of this tumor type associated with an arterial thromboembolism in any veterinary species.

Reproductive strategy and gamete development of an invasive fanworm, Sabella spallanzanii (Polychaeta: Sabellidae), a field study in Gulf St Vincent, South Australia.

Several reproductive strategies have been identified as key factors that contribute to the establishment and dispersal of invasive species in new environments. These strategies include early maturity, high reproductive capacity and flexibility in timing of reproduction. It is therefore critical to investigate the reproductive biology of target exotic species to understand their potential for population increase and invasive spread, and to inform management control strategies. The European fanworm, Sabella spallanzanii (Gmelin 1791), has established invasive populations along the southern coast of Australia. Gamete development and reproductive periodicity of this worm were investigated in two populations in Gulf St Vincent in South Australia over a 1 year period (July 2012 to June 2013). Samples of worms were collected monthly and dissected for histological analysis. Most individuals reached reproductive maturity at 70 mm body length (thorax and abdomen). Individuals from both populations contained mature and developing gametes year-round and a distinct spawning season was not observed. This may indicate sustained spawning by the population over the year, which provides a constant supply of new recruits to the area. Body length and egg size of worms from these populations were smaller than conspecifics in its native range and other invasive locations. Reproduction and development of S. spallanzanii differs not only between native and invasive locations, but also within invasive locations. This study has shown that S. spallanzanii exhibits a higher phenotypic plasticity and reproductive flexibility than previously known.

Running for exercise mitigates age-related deterioration of walking economy.

INTRODUCTION: Impaired walking performance is a key predictor of morbidity among older adults. A distinctive characteristic of impaired walking performance among older adults is a greater metabolic cost (worse economy) compared to young adults. However, older adults who consistently run have been shown to retain a similar running economy as young runners. Unfortunately, those running studies did not measure the metabolic cost of walking. Thus, it is unclear if running exercise can prevent the deterioration of walking economy. PURPOSE: To determine if and how regular walking vs. running exercise affects the economy of locomotion in older adults. METHODS: 15 older adults (69 ± 3 years) who walk ≥ 30 min, 3x/week for exercise, "walkers" and 15 older adults (69 ± 5 years) who run ≥ 30 min, 3x/week, "runners" walked on a force-instrumented treadmill at three speeds (0.75, 1.25, and 1.75 m/s). We determined walking economy using expired gas analysis and walking mechanics via ground reaction forces during the last 2 minutes of each 5 minute trial. We compared walking economy between the two groups and to non-aerobically trained young and older adults from a prior study. RESULTS: Older runners had a 7-10% better walking economy than older walkers over the range of speeds tested (p = .016) and had walking economy similar to young sedentary adults over a similar range of speeds (p =  .237). We found no substantial biomechanical differences between older walkers and runners. In contrast to older runners, older walkers had similar walking economy as older sedentary adults (p =  .461) and ∼ 26% worse walking economy than young adults (p<.0001). CONCLUSION: Running mitigates the age-related deterioration of walking economy whereas walking for exercise appears to have minimal effect on the age-related deterioration in walking economy.

Concordancia de antígenos de dengue en el ELISA de captura de IgM (MAC ELISA) en el IICS-UNA / Concordance of dengue antigens M antibody-capture ELISA (MAC ELISA) in the IICS-UNA

El dengue es una enfermedad aguda grave considerada actualmente como infección reemergente, cuyo vector principal es el Aedes aegypti. En Paraguay en el 2007 fueron reportados 28.181 casos, 55 se clasificaron como fiebre hemorrágica del dengue de los cuales 7 fallecieron. El 90% de los casos fueron de Asunción y del Departamento Central,10% del resto del país. En los últimos años se han desarrollado diferentes sistemas inmunoenzimáticos para el diagnóstico del dengue, entre ellos el ELISA de captura de IgM (MAC ELISA). El objetivo de este estudio observacional analítico de corte transverso fu ecomparar la prueba del MAC ELISA desarrollada en el Instituto de Investigaciones en Ciencias de la Salud (IICS) utilizando antígenos suministrados por el Instituto Pedro Kouri(IPK) de Cuba y el Evandro Chagas de Brasil, con el kit comercial ELISA IgM por capturapara virus del dengue (Focus Diagnostics Inc. Cypress, CA, USA). Fueron seleccionados alazar 92 sueros de pacientes codificados que concurrieron al IICS con sospecha de dengue, respetándose la confidencialidad de los mismos. La concordancia obtenida fue del94.6% (Índice Kappa: 0.891) utilizando el antígeno del IPK y 96.7% (Índice Kappa:0.9350) con el antígeno del Evandro Chagas, mostrándose alta significancia estadística(p<0.00001) en ambos casos. La excelente concordancia obtenida con los dos antígenos indica que los mismos pueden ser utilizados indistintamente en la prueba del MAC ELISA desarrollada en el IICS, a fin de apoyar el diagnóstico del dengue a menor costo y quesería de producción local.

Dengue is an acute disease currently considered a re-emerging infection, whose main vector is Aedes aegypti. In 2007, 28,181 cases were reported in Paraguay, 55 were classified as dengue hemorrhagic fever and seven of them died. Ninety percent of the cases were from Asunción and the Central Department and the remaining 10% from the rest of the country. In recent years various immunoenzymatic systems have been developed immunoassay for the diagnosis of dengue, including the M antibody captureELISA (MAC ELISA). The aim of this cross-sectional observational study was to compare the MAC ELISA test developed at the Instituto de Investigaciones en Cienciad de la Salud(IICS) using antigens supplied by the Instituto Pedro Kouri (IPK) of Cuba and Evandro Chagas of Brazil with a commercial kit of M antibody capture ELISA for dengue virus (Focus Diagnostics Inc. Cypress, CA, USA). Ninety two coded serum samples wererandomly selected from patients who attended the IICS with suspected dengue, respecting their confidentiality. The concordance obtained was 94.6% (Kappa Index: 0.891) using the IPK antigen and 96.7% (Kappa index: 0.9350) with the antigen from Evandro Chagas showing high statistical significance (p<0.00001) in both cases. The excellent concordance obtained with the two antigens indicates that they can be used indistinctly in the MAC ELISA test developed in the IICS to support the diagnosis of dengue at a lower cost and would be locally produced.

Concordancia entre las pruebas de ELISA avidez IgG desarrollados en el Instituto de Investigaciones en Ciencias de la Salud y un ELISA avidez comercial / Concordance between the IgG avidity ELISA tests developed at the Instituto de Investigaciones en Ciencias de la Salud and a commercial avidity ELISA

La infección aguda de toxoplasmosis en la mujer durante el embarazo en su mayor parte es asintomática y detectable solo por anticuerpos lo que podría afectar severamenteal feto si no es diagnosticada y tratada precozmente. En este estudio observacionalanalítico de corte transverso se comparó la prueba de ELISA Avidez IgG paratoxoplasmosis desarrollado en el Instituto de Investigaciones en Ciencias de la Salud(IICS) con un test comercial de avidez InmunoLISA Organics USA. Para la concordanciase utilizó 41 sueros mantenidos a -20 ºC procedentes de la seroteca del Departamento de Producción-Bioquímica seleccionados al azar. La concordancia obtenida fue de 92.7% y un índice de Kappa de 0.820 con IC95% (0.6-1) y p<0.0001. El índice bajo de avidez sugiere una infección aguda pero para el diagnóstico debería estar acompañado de otras pruebas serológicas y la clínica del paciente. En cambio un índice alto es diagnóstico de una infección crónica.

The acute infection caused by Toxoplasma gondii in pregnant women is mostly asymptomatic and detectable only by antibodies that could severely affect the fetus if the infection is not diagnosed and treated precociously. In this cross-sectional observational,the analytical the IgG avidity ELISA test for toxoplasmosis, developed at the Instituto de Investigaciones en Ciencias de la Salud (IICS), was compared to a commercial avidity kit InmunoLISA (Organics, USA). For the concordance, 41 serum samples kept at -20ºC atthe Department of Production-Biochemistry of IICS were tested. The concordance obtained was 92.7% and a Kappa index of 0.820 with IC95% (0.6-1) and p <0.0001. The low avidity index suggests an acute infection but for diagnosis this result should beaccompanied by other serologic tests and clinical symptoms. Instead, a high avidity index suggests a chronic infection.

Mantenimiento y titulación del virus de dengue para obtención de antígeno viral / Maintenance and titration of dengue virus for the obtainment of viral antigens

El dengue es un grave problema de salud pública que no posee vacuna ni tratamiento específico. El método de diagnóstico más utilizado es el serológico, específicamente, la detección de anticuerpos IgM anti-dengue. Los antígenos virales utilizados en este método pueden ser preparados en cultivo de células de Aedes albopictus (C6/36). El objetivo de este trabajo fue el mantenimiento de los cuatro serotipos virales (D1 (RIO), D2 (RIO), D3 (H-87), D4 (BV)) en células C6/36 para la futura preparación de antígenos virales. Las células C6/36 fueron cultivadas en medio L-15 con 10% de SFB a 28ºC, e infectadas con 50 ml de cada uno de los serotipos virales por 5 a 7 días. Una vez confirmada la infección por inmunoflurescencia indirecta, los virus fueron titulados por la técnica de placa de lisis. Los títulos de los serotipos fueron D1 (RIO) (2,9 x 106 PFU/ml), D2 (RIO) (4,4 x107 PFU/ml), D3 (H87) (6,4 x 107 PFU/ml) y D4 (BV) (5,1 x106 PFU/ml). La producción de antígenos virales es de gran importancia dado que los mismos pueden ser utilizados en diversos métodos diagnósticos.

Dengue is a serious public health problem that has neither vaccine nor specific treatment. Serology is the most frequently used diagnosis method, specifically the anti-dengue IgM detection. The viral antigens employed in this method could be prepared from Aedes albopictus cell cultures (C6/36). The objective of this study was to maintain the four viral serotypes (D1 (RIO), D2 (RIO), D3 (H-87), D4 (BV)) on C6/36 cells for the preparation of viral antigen in the future. The C6/36 cells were cultured in L-15 medium supplemented with 10% FCS, infected with 50 µl of each viral serotype and then incubated for 5-7 days at 28°C. After confirmation of the infection by indirect immunofluorescence (IIF), viral titration was performed by lysis plaque assay. The serotypes titres obtained were as follows: [2.9 x 106 PFU/ml] for D1 (RIO), (4.4 x107 PFU/ml) for D2 (RIO), (6.4 x 10 7 PFU/ml) for D3 (H87) and (5.1 x106 PFU/ml) for D4 (BV). The production of viral antigens is very important because they could be used in several diagnosis methods.

Estudio de Fiebre Amarilla en primates en áreas de brote de los departamentos de San Pedro y Central del Paraguay / Study of Yellow Fever in primates in outbreaks areas of the departments of San Pedro and Central in Paraguay

La Fiebre Amarilla (FA) es una de las más importantes zoonosis que afecta a poblaciones humanas. La FA silvestre es imposible de ser erradicada, manteniéndose activa en zonas tropicales en África y Sudamérica. Todas las especies de primates son susceptibles y se consideran reservorios en el medio silvestre. La mortalidad es baja, se desconoce su valor con precisión, sin embargo existen epizootias con alta mortalidad, en humanos varía entre 20-50%. El objetivo de este trabajo fue buscar evidencias de FA en primates capturados en áreas de brote de FA de los departamentos de San Pedro y Central del Paraguay mediante la técnica de Neutralización por reducción de placas para FA cepa vacunal 17 D. Los resultados en los 35 primates estudiados fueron negativos, quizás por lo tardío del momento en la toma de muestras y bajo número de primates capturados.

Yellow Fever (YF) is one of the most important zoonotic diseases affecting human population. It is impossible to eradicate wild YF remaining active in tropical zones of Africa and South America. All species of primates are susceptible and are considered reservoirs in wild regions. Mortality is low and its precise value is unknown though there are epizootics with high mortality rates and in humans varies between 20-50%. The objective of this study was to search for evidence of YF in primates caught in YF outbreaks areas of the departments of San Pedro and Central in Paraguay through the neutralization technique by plates reduction for YF vaccine strain 17 D. The results in the 35 primates studied were negative, perhaps because of the lateness of the time sampling and the low number of captured primates.

Relaxation: mapping an uncharted world.

Nine hundred and forty practitioners of massage, abbreviated progressive muscle relaxation (PMR), yoga stretching, breathing, imagery meditation, and various combination treatments described their technique experiences on an 82-item wordlist. Factor analysis yielded 10 interpretable relaxation categories: Joyful Affects and Appraisals (Joyful), Distant, Calm, Aware, Prayerful, Accepted, Untroubled, Limp, Silent, and Mystery The relaxation response and cognitive/somatic specificity models predict Calm and Limp, which account for only 5.5% of the variance of relaxation experience. Unlike much of previous relaxation research, we found important technique differences. PMR and massage are associated with Distant and Limp; yoga stretching, breathing, and meditation with Aware; meditation with Prayerful and all techniques except PMR with Joyful. Results are consistent with cognitive-behavioral relaxation theory and have implications for relaxation theory, treatment, training, assessment, and research. We close with a revised model of relaxation that posits three global dimensions; tension-relief, passive disengagement, and passive engagement.