Diversidade e variação sazonal de moscas-das-frutas (Diptera: Tephritidae, Lonchaeidae) e seus parasitóides (Hymenoptera: Braconidae, Figitidae) em pomares de goiaba, nêspera e pêssego

This work was carried out in orchards of guava progenies, and loquat and peach cultivars, in Monte Alegre do Sul, SP, Brazil, in 2002 and 2003. Guavas and loquats were bagged and unbagged bi-weekly and weekly, respectively, for assessment of the infestation period. Peach was only bagged weekly. The assays started when the fruits were at the beginning of development, but still green. Ripe fruits were taken to the laboratory and placed individually into plastic cups. McPhail plastic traps containing torula yeast were hung from January 2002 to January 2004 to assess the fruit fly population in each orchard, but only the Ceratitis capitata population is here discussed. Five tephritid species were reared from the fruits: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi, and C. capitata, in addition to six lonchaeid species: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4, and Neosilba sp. 10 (both species are in the process of being described by P. C. Strikis), as well as some unidentified Neosilba species. Ten parasitoid species were obtained from fruit fly puparia, of which five were braconids: Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan, and Utetes anastrephae (Viereck), and five figitids: Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut), and Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata showed a seasonal behavior with population density peaking at the second semester of each year. Anastrepha and Neosilba species remained in the orchards throughout both years.Este trabalho foi realizado em três pomares em Monte Alegre do Sul, SP, em 2002 e 2003, representados por coleção de progênies de goiabeiras, de cultivares de nespereiras e de cultivares de pessegueiros. O período de infestação foi determinado por meio de ensacamento e desensacamento quinzenal e semanal de goiabas e nêsperas, respectivamente, e pelo ensacamento semanal de pêssegos. Os ensaios iniciaram-se com os frutos verdes (princípio de desenvolvimento). Os frutos maduros foram levados ao laboratório e acondicionados individualmente em copos plásticos. A flutuação populacional de Ceratitis capitata (Wiedemann) foi avaliada por meio de armadilhas plásticas modelo McPhail com torula em cada pomar, de janeiro/2002 a janeiro/2004. Dos frutos foram obtidas cinco espécies de tefritídeos: Anastrepha bistrigata Bezzi, A. fraterculus (Wiedemann), A. obliqua (Macquart), A. sororcula Zucchi e C. capitata e seis de lonqueídeos: Neosilba certa (Walker), N. glaberrima (Wiedemann), N. pendula (Bezzi), N. zadolicha McAlpine and Steyskal, Neosilba sp. 4 e Neosilba sp. 10, além de algumas espécies não-identificadas. Foram obtidas 10 espécies de parasitóides, cinco da família Braconidae - Asobara anastrephae (Muesebeck), Doryctobracon areolatus (Szépligeti), D. brasiliensis (Szépligeti), Opius bellus Gahan e Utetes anastrephae (Viereck) - e cinco da família Figitidae - Aganaspis pelleranoi (Brèthes), Dicerataspis grenadensis Ashmead, Lopheucoila anastrephae (Rhower), Leptopilina boulardi (Barbotin, Carlton and Kelner-Pillaut) e Trybliographa infuscata Diaz, Gallardo and Uchôa. Ceratitis capitata apresentou comportamento sazonal com picos populacionais durante o segundo semestre dos dois anos. As espécies de Anastrepha e de Neosilba permaneceram nos pomares durante os dois anos

Glucoamylase isoform (GAII) purified from a thermophilic fungus Scytalidium thermophilum 15.8 with biotechnological potential

Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose chromatographic column glucoamylases form II (GAII) was separated and purified from glucoamylases form I (GAI) that was previously purified and characterised (Cereia et al., 2000) when the filtrate of the culture medium was applied to a DEAE-Cellulose chromatographic column. GAII bound to the DEAECellulose and was eluted with a NaCl gradient, while GAI did not bind to the resin. GAII presentedelectrophoretic homogeneity in 6% denaturing and non-denaturing PAGE, separately, with a molecular mass of 83 kDa, after the second round DEAE-Cellulose purification step. The enzyme pI was 7.2.Optima pH and activity temperature were 5.5 and 55ºC respectively for starch and maltose as substrates, with a termostability of 2.5 min at 60ºC. Enzymatic activities were activated by 1 mM Na+, Mn2+ and Mg2+ or 10 mM NH4+, Ba2+ and Mg2+. The carbohydrate content was 10%. The kinetic parameters Km and Vmax with starch and maltose as substrate were 0.2 and 1.5 mg/ml, and 22.3 and 4.39 U/mg of protein, respectively. The amino acid sequence of GAII had 92% homology with theglucoamylase of Humicola grisea var. thermoidea after 13 cycles. Generally, GAII had different properties compared with GAI (Cereia et al., 2000)

Exposure of humans to the zoonotic nematode Dirofilaria immitis in Northern Portugal

Dirofilariosis caused by Dirofilaria immitis (heartworm) is a zoonosis, considered an endemic disease of dogs and cats in several countries of Western Europe, including Portugal. This study assesses the levels of D. immitis exposure in humans from Northern Portugal, to which end, 668 inhabitants of several districts belonging to two different climate areas (Csa: Bragança, Vila Real and Csb: Aveiro, Braga, Porto, Viseu) were tested for anti-D. immitis and anti-Wolbachia surface proteins (WSP) antibodies. The overall prevalence of seropositivity to both anti-D. immitis and WSP antibodies was 6.1%, which demonstrated the risk of infection with D. immitis in humans living in Northern Portugal. This study, carried out in a Western European country, contributes to the characterisation of the risk of infection with D. immitis among human population in this region of the continent. From a One Health point of view, the results of the current work also support the close relationship between dogs and people as a risk factor for human infectio

Liquid biopsy for disease monitoring in non-small cell lung cancer: The link between biology and the clinic

Introduction: Cell-free DNA (cfDNA) analysis offers a non-invasive method to identify sensitising and resistance mutations in advanced Non-Small Cell Lung Cancer (NSCLC) patients. Next-generation sequencing (NGS) of circulating free DNA (cfDNA) is a valuable tool for mutations detection and disease' s clonal monitoring. Material and methods: An amplicon-based targeted gene NGS panel was used to analyse 101 plasma samples of advanced non-small cell lung cancer (NSCLC) patients with known oncogenic mutations, mostly EGFR mutations, serially collected at different clinically relevant time points of the disease. Results: The variant allelic frequency (VAF) monitoring in consecutive plasma samples demonstrated different molecular response and progression patterns. The decrease in or the clearance of the mutant alleles was associated with response and the increase in or the emergence of novel alterations with progression. At the best response, the median VAF was 0% (0.0% to 3.62%), lower than that at baseline, with a median of 0.53% (0.0% to 9.9%) (p = 0.004). At progression, the VAF was significantly higher (median 4.67; range: 0.0–36.9%) than that observed at the best response (p = 0.001) and baseline (p = 0.006). These variations anticipated radiographic changes in most cases, with a median time of 0.86 months. Overall, the VAF evolution of different oncogenic mutations predicts clinical outcomes. Conclusion: The targeted NGS of circulating tumour DNA (ctDNA) has clinical utility to monitor treatment response in patients with advanced lung adenocarcinoma.This work was supported by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; and by FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), and “Transferencia horizontal de resistencia à terapia: mudança de paradigma na monitorização de pacientes com cancro” (PTDC/DTPPIC/2500/2014); and by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), in the framework of the project NORTE-01-0145-FEDER-000029

Targeted gene next-generation sequencing panel in patients with advanced lung adenocarcinoma: Paving the way for clinical implementation

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (¿ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.We acknowledge the work of the members of our department, especially doctors from the thoracic oncology unit, oncology and pulmonology nurses and, patients and their relatives. N. Martins would like to thank the Portuguese Foundation for Science and Technology (FCT-Portugal) for the Strategic project ref. UID/BIM/04293/2013 and "NORTE2020—Northern Regional Operational Program" (NORTE-01-0145-FEDER-000012)

Evaluation of urinary cysteinyl leukotrienes as biomarkers of severity and putative therapeutic targets in COVID-19 patients

Background Cysteinyl leukotrienes (CysLT) are potent inflammation-promoting mediators, but remain scarcely explored in COVID-19. We evaluated urinary CysLT (U-CysLT) relationship with disease severity and their usefulness for prognostication in hospitalized COVID-19 patients. The impact on U-CysLT of veno-venous extracorporeal membrane oxygenation (VV-ECMO) and of comorbidities such as hypertension and obesity was also assessed. Methods Blood and spot urine were collected in severe (n = 26), critically ill (n = 17) and critically ill on VV-ECMO (n = 17) patients with COVID-19 at days 1-2 (admission), 3-4, 5-8 and weekly thereafter, and in controls (n = 23) at a single time point. U-CysLT were measured by ELISA. Routine markers, prognostic scores and outcomes were also evaluated. Results U-CysLT did not differ between groups at admission, but significantly increased along hospitalization only in critical groups, being markedly higher in VV-ECMO patients, especially in hypertensives. U-CysLT values during the first week were positively associated with ICU and total hospital length of stay in critical groups and showed acceptable area under curve (AUC) for prediction of 30-day mortality (AUC: 0.734, p = 0.001) among all patients. Conclusions U-CysLT increase during hospitalization in critical COVID-19 patients, especially in hypertensives on VV-ECMO. U-CysLT association with severe outcomes suggests their usefulness for prognostication and as therapeutic targets.This work was supported by a RESEARCH 4 COVID-19 grant (project 519, reference number: 613690173) from FCT-Fundacao para a Ciencia e a Tecnologia (special support for rapid implementation projects for innovative response solutions to COVID-9 pandemic). CS-P is a recipient of a Ph.D. fellowship from FCT and MedInUP (UI/BD/150816/2020). P-PT was supported by a research contract within the scope of the RIFF-HEART project funded by FEDER via COMPETE, Portugal 2020-Operational Programme for Competitiveness and Internationalization (POCI) (POCI-01-0145-FEDER-032188) and by FCT (PTDC/MEC-CAR/32188/2017). Open access funding provided by FCT|FCCN (b-on)

Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis