Genetic context and mobilization of class 1 integrons in Pseudomonas aeruginosa : are plasmids redundant?

University of Technology, Sydney. Faculty of Science.Antibiotic resistance is a global problem with some predicting a return to the pre antibiotic era where a bacterial infection was commonly fatal. Pseudomonas aeruginosa is one example of this problem. This bacterium is a major cause of infection especially in cystic fibrosis sufferers and in burns victims. The rising rates of adverse outcomes are partly a consequence of strains commonly displaying multi-drug resistance (MDR) profiles. MDR is driven by a number of intrinsic mechanisms in P. aeruginosa clinical isolates as well as by the capture of diverse resistance-mediating genes by Lateral Gene Transfer (LGT). LGT and intrinsic factors often act cooperatively to generate complex MDR phenotypes. While these complex interactions have been examined in a small number of isolates there has not been a comprehensive survey of strains on a global scale. Thus it is not clear what mechanisms and genes may be important in influencing the evolution of MDR at regional or global levels. Also, in some isolates, resistance profiles cannot always be explained by identifying the common resistance determining pathways, suggesting that additional mechanisms of resistance may be emerging in P. aeruginosa. The focus of this project was to comprehensively study the major mechanisms responsible for antibiotic resistance in P. aeruginosa strains from diverse geographical areas. Pathogenic P. aeruginosa isolates from four countries (Australia and three South American countries) were characterized by PCR to identify mobile elements and their genetic context. Also, quantitative expression analysis for activity of several pathways that influence antibiotic resistance was assessed and culture experiments were conducted to test how random movement of mobile elements during growth may influence resistance to some antibiotics. Data presented in this thesis indicated that, in most strains, antibiotic resistance was being driven by changes in multiple pathways (including overexpression of AmpC and two efflux pumps) and by the presence or absence of genes acquired by Lateral Gene Transfer (LGT). Class 1 integrons, elements important in the spread of antibiotic resistance genes in Gram-negative bacteria, were most frequently recovered in South American countries. Many class 1 integrons were mapped to a specific location within the genome. Regardless of country of origin all these mapped integrons were found to be in the chromosome, often in Genomic islands, and not on a plasmid despite data in the literature implying the opposite. The association of class 1 integrons with genomic islands may be an important mechanism driving LGT in P. aeruginosa. Also, a newly emerging mechanism involving the insertion sequence IS26 was identified that is capable of mobilizing resistance and other genes. This IS26-mediated mechanism may allow phenotype switching in clonal lines in a way that is likely to further exacerbate the treatment of infections mediated by P. aeruginosa. Data presented here suggested that P. aeruginosa strains are evolving to become multidrug resistant in increasingly complex ways. This is occurring by single strains acquiring changes in numerous known pathways as well as by newly emerging resistance mechanisms in this species

Artificial light pollution influences behavioral and physiological traits in a keystone predator species, Concholepas concholepas

Artificial Light At Night (ALAN) is an increasing global problem that, despite being widely recognized in terrestrial systems, has been studied much less in marine habitats. In this study we investigated the effect of ALAN on behavioral and physiological traits of Concholepas concholepas, an important keystone species of the south-eastern Pacific coast. We used juveniles collected in intertidal habitats that had not previously been exposed to ALAN. In the laboratory we exposed them to two treatments: darkness and white LED (Lighting Emitting Diodes) to test for the impacts of ALAN on prey-searching behavior, self-righting time and metabolism. In the field, the distribution of juveniles was observed during daylight-hours to determine whether C. concholepas preferred shaded or illuminated microhabitats. Moreover, we compared the abundance of juveniles collected during day- and night-time hours. The laboratory experiments demonstrated that juveniles of C. concholepas seek out and choose their prey more efficiently in darkened areas. White LED illuminated conditions increased righting times and metabolism. Field surveys indicated that, during daylight hours, juveniles were more abundant in shaded micro-habitats than in illuminated ones. However, during darkness hours, individuals were not seen to aggregate in any particular microhabitats. We conclude that the exposure to ALAN might disrupt important behavioral and physiological traits of small juveniles in this species which, as a mechanism to avoid visual predators, are mainly active at night. It follows that ALAN in coastal areas might modify the entire community structure of intertidal habitats by altering the behavior of this keystone species

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Abstract:  Excess dietary n6 polyunsaturated fatty acids (PUFA) affects various reproductive parameters. Objective: to evaluate the effect of diets exceeded in linoleic acid (LA)-n6 on placental vascularization, vitality and fetal growth. Albino swiss mice were fed from gestation day 0.5 (GD 0.5) with diets: control (C, commercial diet, LA=1.6%, n=25) or C with 10% of soybean or sunflower oils (SOD, LA=6.68%, n=24 and SFOD, LA=7.68%, n=26). At GD 16.5, mothers were sacrificed, and the following parameters were assessed: placental and fetal weights, fetal vitality. Placental areas and placental necrosis areas were measured in histological sections with H/E. Immunohistochemistry for nitric oxide synthase III (eNOS) in placental labyrinth was performed. For vascular endothelial delineation and natural killers (NK) cell labelling, histochemical analysis was performed with lectins: BSA-I and DBA, respectively. The labelled area and staining intensity were quantified through reciprocal intensity and number of NK cells, using FIJI software. Statistics: ANOVA, Kruskall-Wallis and Chi-square, p≤0.05. SFOD placentas were significantly lighter than SOD and C (SFOD=0.13±0.02 vs SOD=0.14±0.02 and C=0.15±0.02, p≤0.005). Significantly lower fetal weights in treated females (SOD=0.59±0.01, n=84 and SFOD=0.68±0.01, n=115 vs C=0.72±0.02, n=52; p≤0.005). Lower foetal vitality in treated females: SFOD= 69.59% and SOD=73.95% vs C=92.86%; p≤0.05. No significant differences in placental areas. Greater area of necrosis in decidua of SFOD placentas (SFOD=122.05µm2±32.38 vs SOD=52.39µm2±28.59 and C=35.78µm2±11.91; p≤0.05). No significant differences in eNOS-labelled area. However, staining intensity was lower in SFOD (SFOD=78.44±3.80 vs SOD=110.61±9.07 and C=100.21±4.76; p≤0.05). The labelled area with BSA-I was significantly greater in the treated females (SFOD=87.37µm2±0.88 and SOD=88.47µm2±2.07 vs C=80.01µm2±2.47; p≤0.05); however, the staining intensity was higher in the control group (C=98.33±2.4 vs SFOD=67.43±3.6 and SOD=73.83±7.69; p≤0.05). No significant differences in NK quantification. The lower staining intensity of eNOS and BSA-I staining demonstrates alterations in vascularization in group SFOD, which contains more n6. These changes would reduce nutrients supply to the foetus, affecting its growth and vitality. Further determinations will be made in order to study NK cells involved in placental angiogenesis and trophoblast development.Resumen:  El exceso de n6 dietario afecta diversos parámetros reproductivos. Nos propusimos evaluar el efecto de dietas excedidas en ácido linoleico (AL)-n6 sobre la vascularización placentaria, la vitalidad y el crecimiento fetal. Ratones Albino swiss se alimentaron desde el día de gestación (DG 0,5) 0,5 con dietas: control (C, dieta comercial, AL=1,6%, n=25) o C con 10% de aceites de soja o girasol (S, AL=6,68%, n=24 y G, AL=7,68%, n=26). Al DG 16,5 se evaluó: pesos placentario y fetal, vitalidad fetal. Se midieron áreas placentarias y áreas de necrosis en cortes histológicos de placentas con H/E. Se realizó inmunohistoquímica para sintasa de óxido nítrico III (eNOS) en laberinto placentario. Para la delimitación del endotelio vascular y la marcación de células natural killers (NK) se realizó análisis histoquímico con lectinas:BSA-I y DBA, respectivamente. Se cuantificaron área marcada e intensidad de tinción a través de intensidad recíproca y número de NK, con programa FIJI. Estadística: ANOVA, Kruskall-Wallis, Chi-cuadrado, p≤0,05. Las placentas G fueron significativamente más livianas que S y C (G=0,13±0,02 vs S=0,14±0,02 y C=0,15±0,02; p≤0,005). Pesos fetales significativamente menores en hembras tratadas (S=0,59±0,01; n=84; G=0,68±0,01;n=115 y C=0,72±0,02;n=52; p≤0,005). Vitalidad fetal menor en hembras tratadas: G= 69,59% y S=73,95% vs C=92,86%; p≤0,05. Sin diferencias en las áreas placentarias. Mayor área de necrosis en deciduas de hembras G (G=122,05µm2±32,38 vs S=52,39µm2±28,59 y C=35,78µm2±11,91; p≤0,05). Sin diferencias significativas en el área marcada con eNOS. Sin embargo, la intensidad de tinción fue menor en G (G=78,44±3,80 vs S=110,61±9,07 y C=100,21±4,76; p≤0,05). El área marcada con BSA-I fue significativamente mayor en las hembras tratadas (G=87,37µm2±0,88 y S=88,47µm2±2,07 vs C=80,01µm2±2,47; p≤0,05); sin embargo, la intensidad de tinción fue mayor en el grupo control (C=98,33±2,4 vs G=67,43±3,6 y S=73,83±7,69; p≤0,05). Sin diferencias significativas en la cuantificación de NK. La menor intensidad de tinción con eNOS y BSA-I demuestran alteraciones en la vascularización en el grupo G, que contiene mayor cantidad de n6. Estos cambios reducirían el aporte de nutrientes al feto, afectando su crecimiento y vitalidad.  Se realizarán más determinaciones a fin de estudiar con profundidad las NK, células implicadas en la angiogénesis placentaria y el desarrollo trofoblástico.

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Abstract:  The finding of melanoma metastasis in sentinel lymph node is an ominous prognostic sign and a strong predictor of overall survival. Current data have shown that patients with nevus cells in sentinel lymph nodes do not require additional therapy as their prognosis has been shown to be similar to that of patients with negative lymph nodes. Distinguishing between benign capsular nevi and metastatic melanoma is problematic from a diagnostic point of view. The use of an immunohistochemical panel for melanocytic lesions is usually of limited value in differentiating with metastatic melanoma and the diagnosis is usually based mainly on histomorphological findings. 53-year-old man, diagnosed with nodular variant of melanoma with BRAF V600E mutation in exon 15, located on the back and excised in November 2020; with surgical margin extension in February 2021 due to compromised surgical limit and with sentinel lymph node removal. Right and left axillary nodes were removed, the first measured 1.5 x 1 x 0.6 cm and the left ones were two: the largest measured 2 x 1.5 x 1 cm and the smallest 1 x 0.5 x 0.5 cm. Microscopy: reactive lymphoid hyperplasia and abundant black tattoo ink at sinus location were observed in the left nodes, while in the right one a proliferation of nevus-like cells arranged in nests was observed, at intracapsular location, and immunohistochemistry was Melan-A and HMB-45 positive, with a cell proliferation index (% Ki-67) of 1 - 5%. Consultation with local dermatopathologists was made and the diagnosis was intracapsular melanocytic nevus. The lack of standardized criteria to distinguish between nevus cells and melanoma poses a diagnostic problem. To avoid misdiagnosis and unnecessary therapy, obtaining a second opinion from additional histopathologists would be beneficial. The use of tissue morphology in conjunction with immunohistochemical staining may be the best way to make the most accurate diagnosis. The use of molecular tests to determine characteristic somatic mutations of melanoma will probably help in the near future to facilitate the distinction between capsular nevi and metastatic melanoma.Resumen:  El hallazgo de metástasis de melanoma en un ganglio linfático centinela es un signo de pronóstico ominoso y un fuerte predictor de supervivencia general.  los datos actuales han demostrado que los pacientes con células névicas en ganglio centinela no requieren terapia adicional, ya que se ha demostrado que su pronóstico es similar al de los pacientes con ganglios linfáticos negativos. Distinguir entre nevos capsulares benignos y melanoma metastásico, resulta problemático desde el punto de vista diagnóstico. La utilización de un panel de inmunohistoquímica para lesiones melanocíticas suele ser de valor limitado en la diferenciación con melanoma metastásico y el diagnóstico suele basarse principalmente en los hallazgos histomorfológicos.  Hombre de 53 años, con diagnóstico de melanoma variante nodular, BRAF mutado V600E en exón 15, localizado en espalda y extirpado en noviembre 2020; en febrero 2021 ampliación por límite quirúrgico comprometido y extirpación de ganglio centinela. Se realiza extirpación de ganglios axilares derecho e izquierdo, el primero de 1,5 x 1 x 0,6 cm y el izquierdo: 2 ganglios, el mayor de 2 x 1,5 x 1 cm y el menor de 1 x 0,5 x 0,5 cm. Microscopía: en ganglio izquierdo se observó hiperplasia linfoide reactiva y abundante tinta negra de tatuaje a nivel sinusal, mientras que en ganglio derecho se observó una proliferación de células de aspecto névico dispuestas en nidos, a nivel intracapsular, cuya inmunohistoquímica resultó Melan-A y HMB-4 positiva, con un índice de proliferación celular (%Ki-67)1- 5%. Se realizó interconsulta con dermatopatólogos locales y el  diagnóstico fue de nevo melanocítico intracapsular. La falta de criterios estandarizados para distinguir entre células névicas y melanoma plantea un problema diagnóstico. Para evitar un diagnóstico erróneo y realizar una terapia innecesaria, sería beneficioso obtener una segunda opinión de histopatólogos adicionales. El uso de la morfología de los tejidos junto con la tinción inmunohistoquímica puede ser la mejor manera de hacer el diagnóstico más preciso. El empleo de test moleculares para determinación de mutaciones somáticas propias del melanoma, probablemente ayudarán en un futuro cercano a facilitar la distinción entre nevo capsular y melanoma metastásico.

On an acute case of Chagas disease in a region under vector control in the state of São Paulo, Brazil

No vector transmitted cases of Chagas disease had been notified in the state of São Paulo since the 1970s. However, in March, 2006, the death of a six-year-old boy from the municipality of Itaporanga was notified to the Center for Epidemiological Survey of the São Paulo State Health Secretariat: an autochthonous case of acute Chagas disease. The postmortem histopathological examination performed in the Hospital das Clínicas of the Botucatu School of Medicine confirmed the diagnosis. Reference to hospital records, consultation with the health professionals involved in the case and interviews with members of the patient's family supplied the basis for this study. We investigated parasite route of transmission, probable local reservoirs and vectors. No further human cases of acute Chagas disease were diagnosed. No locally captured vectors or reservoirs were found infected with Trypanosoma cruzi. Alternative transmission hypotheses - such as the possible ingestion of foods contaminated with vector excreta - are discussed, as well as the need to keep previously endemic regions and infested houses under close surveillance. Clinicians should give due attention to such signs as uni- or bilateral palpebral edema, cardiac failure, myocarditis, pericarditis, anasarca and atypical signs of nephrotic syndrome or nephritis and consider the diagnostic hypothesis of Chagas disease.Desde a década de 1970 não se notificavam casos autóctones de doença de Chagas aguda em São Paulo. Em março de 2006 a Vigilância Epidemiológica registrou óbito por doença de Chagas aguda, em Itaporanga, de paciente de seis anos de idade. Exame histopatológico post mortem realizado no Hospital das Clínicas da Faculdade de Medicina de Botucatu confirmou o diagnóstico. Consultamos prontuários de hospitais e entrevistamos profissionais de saúde envolvidos além de familiares do paciente. Descrevemos medidas adotadas in loco para identificar a via de transmissão, reservatórios e vetores. Discutimos as possíveis fontes de infecção. Na região não foram identificados outros casos humanos, vetores ou reservatórios vertebrados infectados por Trypanosoma cruzi. Salientamos a importância de manter a vigilância, mesmo em áreas onde a transmissão de doença de Chagas está interrompida e naquelas ainda infestadas por triatomíneos. Deve-se admitir a hipótese diagnóstica de doença de Chagas quando observados: edema palpebral (uni ou bilateral), insuficiência cardíaca, miocardite, pericardite, anasarca, quadros similares aos de síndrome nefrótica ou glomerulonefrite sem causas outras aparentes, em pacientes com dados epidemiológicos positivos. Encontro, mesmo em raras ocasiões, de triatomíneos na região ou ainda contato com alimento contaminável com formas infectantes de T. cruzi

The Impact of Global Warming and Anoxia on Marine Benthic Community Dynamics: an Example from the Toarcian (Early Jurassic)

The Pliensbachian-Toarcian (Early Jurassic) fossil record is an archive of natural data of benthic community response to global warming and marine long-term hypoxia and anoxia. In the early Toarcian mean temperatures increased by the same order of magnitude as that predicted for the near future; laminated, organic-rich, black shales were deposited in many shallow water epicontinental basins; and a biotic crisis occurred in the marine realm, with the extinction of approximately 5% of families and 26% of genera. High-resolution quantitative abundance data of benthic invertebrates were collected from the Cleveland Basin (North Yorkshire, UK), and analysed with multivariate statistical methods to detect how the fauna responded to environmental changes during the early Toarcian. Twelve biofacies were identified. Their changes through time closely resemble the pattern of faunal degradation and recovery observed in modern habitats affected by anoxia. All four successional stages of community structure recorded in modern studies are recognised in the fossil data (i.e. Stage III: climax; II: transitional; I: pioneer; 0: highly disturbed). Two main faunal turnover events occurred: (i) at the onset of anoxia, with the extinction of most benthic species and the survival of a few adapted to thrive in low-oxygen conditions (Stages I to 0) and (ii) in the recovery, when newly evolved species colonized the re-oxygenated soft sediments and the path of recovery did not retrace of pattern of ecological degradation (Stages I to II). The ordination of samples coupled with sedimentological and palaeotemperature proxy data indicate that the onset of anoxia and the extinction horizon coincide with both a rise in temperature and sea level. Our study of how faunal associations co-vary with long and short term sea level and temperature changes has implications for predicting the long-term effects of “dead zones” in modern oceans

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

A missense TGFB2 variant p.(Arg320Cys) causes a paradoxical and striking increase in aortic TGFB1/2 expression.

Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder with a range of cardiovascular, skeletal, craniofacial and cutaneous manifestations. LDS type 4 is caused by mutations in TGFβ ligand 2 (TGFB2) and based on the family pedigrees described to date, appears to have a milder clinical phenotype, often presenting with isolated aortic disease. We sought to investigate its molecular basis in a new pedigree. We identified a missense variant p.(Arg320Cys) (NM_003238.3) in a highly evolutionary conserved region of TGFB2 in a new LDS type 4 pedigree with multiple cases of aortic aneurysms and dissections. There was striking upregulation of TGFB1 and TGFB2 expression on immunofluorescent staining, and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlights the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signaling in this emerging familial aortopathy.Raya Al Maskari has a PhD studentship funded by the Omani government.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ejhg.2016.14

The association of cold weather and all-cause and cause-specific mortality in the island of Ireland between 1984 and 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.This article has been made available through the Brunel Open Access Publishing Fund.Background This study aimed to assess the relationship between cold temperature and daily mortality in the Republic of Ireland (ROI) and Northern Ireland (NI), and to explore any differences in the population responses between the two jurisdictions. Methods A time-stratified case-crossover approach was used to examine this relationship in two adult national populations, between 1984 and 2007. Daily mortality risk was examined in association with exposure to daily maximum temperatures on the same day and up to 6 weeks preceding death, during the winter (December-February) and cold period (October-March), using distributed lag models. Model stratification by age and gender assessed for modification of the cold weather-mortality relationship. Results In the ROI, the impact of cold weather in winter persisted up to 35 days, with a cumulative mortality increase for all-causes of 6.4% (95%CI=4.8%-7.9%) in relation to every 1oC drop in daily maximum temperature, similar increases for cardiovascular disease (CVD) and stroke, and twice as much for respiratory causes. In NI, these associations were less pronounced for CVD causes, and overall extended up to 28 days. Effects of cold weather on mortality increased with age in both jurisdictions, and some suggestive gender differences were observed. Conclusions The study findings indicated strong cold weather-mortality associations in the island of Ireland; these effects were less persistent, and for CVD mortality, smaller in NI than in the ROI. Together with suggestive differences in associations by age and gender between the two Irish jurisdictions, the findings suggest potential contribution of underlying societal differences, and require further exploration. The evidence provided here will hope to contribute to the current efforts to modify fuel policy and reduce winter mortality in Ireland