Three-dimensional FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse beta cells

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is currently under debate. Here, we use Fib-Sem to image islet beta cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules and micro-tubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that micro-tubules form non-radial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus supportive role in insulin secretion

Evidence-based use of antibiotics in veal calves with diarrhea

Diarrhea is the leading cause of mortality in beef and dairy calves during the first week of life and results in substantial financial loss. Diarrhea is a multifactorial disease and can be infectious or non-infectious. However, in the majority of calves, infectious organisms, especially Cryptosporidium parvum, rotavirus, coronavirus, and E. coli, are the primary cause. The aim of this study was to generate a decision tree, based on prevalence, diagnostic testing and treatment and to estimate associated costs or risk. For each of the four main pathogens, two principal approaches are outlined and compared. The first approach relies on a detailed diagnostic workup and allows for specific etiological treatment. The second approach relies on the trial-and-error method, which involves the use of a first-choice antibiotic, followed by a second- and third-choice antibiotic if the previous ones failed to resolve the disease. In Switzerland, the prevalence of diarrheic calves infected with E. coli is approximately 1% suggesting that the use of antimicrobials for the treatment of scouring calves, in the absence of a diagnostic workup, is not always justified. However, for all four major pathogens, the trial-and-error method affords cheaper treatment compared with treatment based on an etiological diagnosis. This creates a quandary in view of the current worldwide efforts to reduce the use of antibiotics in animal agriculture

Congenital heart disease in school-aged children: Cognition, education, and participation in leisure activities

BACKGROUND Children with congenital heart disease (CHD) are at risk for neurodevelopmental deficits. This study aimed to investigate the impact of cognitive deficits on educational outcome and participation in leisure activities. METHODS A prospective cohort of 134 children with CHD who underwent cardiopulmonary bypass surgery (CPB) was examined at 10 years of age. IQ was assessed with the WISC-IV and executive functions with the BRIEF (parent- and teacher-report). Parents reported on type and level of education and educational support, and leisure activity participation. Ordinal regression analyses assessed the association between cognitive deficits and educational outcome and participation. RESULTS Total IQ (P = 0.023), working memory (P < 0.001), processing speed (P = 0.008), and teacher-reported metacognition (P = 0.022) were lower than norms. Regular school was attended by 82.4% of children with CHD compared to 97% of the general Swiss population (P < 0.001). Seventy-five percent of children participated in leisure activities. Lower total IQ and teacher-rated global executive functions were associated with more educational support and lower IQ was associated with less participation. CONCLUSION As school-aged children with CHD experience cognitive deficits, follow-up is required to provide optimal support with regard to educational outcome and participation in leisure activities. IMPACT Contemporary cohorts of children with congenital heart disease undergoing cardiopulmonary bypass surgery remain at increased risk for cognitive deficits. Cognitive deficits affect educational outcome and leisure activities. These findings underline the importance of early detection of cognitive deficits and recommend support with respect to cognitive functioning

Aortopulmonary collaterals in neonates with d-transposition of the great arteries - Clinical significance early after arterial switch operation

OBJECTIVES: Purpose of this study is to evaluate the clinical significance of major aortopulmonary collateral arteries (MAPCAs) during the early postoperative course after arterial switch operation (ASO) in d-transposition of the great arteries (dTGA). METHODS: Clinical data of 98 patients with simple dTGA between January 2007 and December 2016 at the University Children's Hospital Zurich, Switzerland were analyzed retrospectively. RESULTS: 37 from 98 patients (38%) required cardiac catheterization (CC) due to an early complicated postoperative course or difficult coronary transfer due to special coronary anatomy. In 15 (15%) patients, hemodynamically relevant MAPCAs were found during CC and coil embolization was performed. Patients with relevant MAPCAs had a significantly longer PICU stay (7 versus 6 days, p = 0.021), longer hospital stay (41 versus 27 days, p = 0.005), longer mechanical ventilation time (5 versus 3 days, p = 0.005), longer need for inotropic support (5 versus 4 days, p = 0.001) and delayed chest closure time (3 versus 2 days, p = 0.030) in those in whom it was left open in comparison to all other patients. In patients having CC, pre-surgery oxygen saturation was significantly lower in patients with relevant MAPCAs (58% vs 70%, p 0.019). Echocardiography had a sensitivity of 53% and a specificity of 100% in detecting relevant MAPCAs, accurately. CONCLUSIONS: MAPCAs are frequently found in dTGA patients and can be associated with lower baseline oxygen saturation and a prolonged postoperative course after ASO. Transthoracic echocardiography cannot replace CC as diagnostic tool. If significant MAPCAs are suspected, early CC should be performed for diagnostic and therapeutic reasons

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with ¹⁴C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of ¹⁴C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01)

3D FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse beta cells

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet beta cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion