DYSCALCULIA, a Venus flytrap mutant without the ability to count action potentials.

The Venus flytrap Dionaea muscipula estimates prey nutrient content by counting trigger hair contacts initiating action potentials (APs) and calcium waves traveling all over the trap.1,2,3 A first AP is associated with a subcritical rise in cytosolic calcium concentration, but when the second AP arrives in time, calcium levels pass the threshold required for fast trap closure. Consequently, memory function and decision-making are timed via a calcium clock.3,4 For higher numbers of APs elicited by the struggling prey, the Ca2+ clock connects to the networks governed by the touch hormone jasmonic acid (JA), which initiates slow, hermetic trap sealing and mining of the animal food stock.5 Two distinct phases of trap closure can be distinguished within Dionaea's hunting cycle: (1) very fast trap snapping requiring two APs and crossing of a critical cytosolic Ca2+ level and (2) JA-dependent slow trap sealing and prey processing induced by more than five APs. The Dionaea mutant DYSC is still able to fire touch-induced APs but does not snap close its traps and fails to enter the hunting cycle after prolonged mechanostimulation. Transcriptomic analyses revealed that upon trigger hair touch/AP stimulation, activation of calcium signaling is largely suppressed in DYSC traps. The observation that external JA application restored hunting cycle progression together with the DYSC phenotype and its transcriptional landscape indicates that DYSC cannot properly read, count, and decode touch/AP-induced calcium signals that are key in prey capture and processing.

An AHP based approach to forecast groundwater level at potential recharge zones of Uckermark District, Brandenburg, Germany.

Uckermark, a district of the state Brandenburg, Germany is situated in one of the driest regions of Germany. The district is known for its agricultural activities and natural resources. But in recent times the district is being prone to groundwater deficit due to the dryness of its climate. In this research initiative, a GIS and Remote Sensing based approach has been made to detect the potential groundwater recharge zones of Uckermark district and observe the groundwater level condition over a period of 21 years (2000-2020). Analytic Hierarchy Process has been used to locate the potential groundwater recharge zones and later a Long Short-Term Memory (LSTM) based model has been developed to forecast the seasonal groundwater level for the upcoming five years in the potential groundwater recharge zones based on observation data from groundwater measurement points. This enabled us to see the groundwater condition of Uckermark in near future and point out the necessary steps to be taken.

Gland cell responses to feeding in Drosera capensis, a carnivorous plant.

Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

Sulforaphane promotes C. elegans longevity and healthspan via DAF-16/DAF-2 insulin/IGF-1 signaling.

The broccoli-derived isothiocyanate sulforaphane inhibits inflammation, oxidative stress and cancer, but its effect on healthspan and longevity are unclear. We used the C. elegans nematode model and fed the wildtype and 9 mutant strains ±sulforaphane. The lifespan, phenotype, pharyngeal pumping, mobility, lipofuscin accumulation, and RNA and protein expression of the nematodes were assessed by using Kaplan-Meier survival analysis, in vivo live imaging, fluorescence microscopy, and qRT-PCR. Sulforaphane increased the lifespan and promoted a health-related phenotype by increasing mobility, appetite and food intake and reducing lipofuscin accumulation. Mechanistically, sulforaphane inhibited DAF-2-mediated insulin/insulin-like growth factor signaling and its downstream targets AGE-1, AKT-1/AKT-2. This was associated with increased nuclear translocation of the FOXO transcription factor homolog DAF-16. In turn, the target genes sod-3, mtl-1 and gst-4, known to enhance stress resistance and lifespan, were upregulated. These results indicate that sulforaphane prolongs the lifespan and healthspan of C. elegans through insulin/IGF-1 signaling. Our results provide the basis for a nutritional sulforaphane-enriched strategy for the promotion of healthy aging and disease prevention.

Combining industrial and urban water-reuse concepts for increasing the water resources in water-scarce regions.

Water scarcity is a huge challenge for industrial and urban developments. As such developments are based on a secure water supply, strategies to ensure the required water quantities must be put into effect. In this context, sustainability is becoming an increasingly important factor due to the worsening of pollution and climate change. The integrated industrial-urban water-reuse concept (IU-WA-RE) links gray and green infrastructures by providing reuse water for different infrastructural purposes. Municipal and industrial wastewater is treated separately in different water resource recovery facilities. As a baseline the SEMIZENTRAL approach with the Resource and Recovery Center (RRC) and the Industrial Wastewater Management Concept with a focus on Reuse (IW2 MC→R) for the industrial wastewater treatment are taken into account. These approaches are new concepts for wastewater treatment "fit for purpose." IU-WA-RE combines the water-reuse concepts by linking reuse water flows between the urban area and the adjacent industrial park, but focuses not on a production internal water reuse. The concept is designed to offer a holistic strategy to increase the water-reuse potential and thus the water resources. It offers a solution to cover the lack of water requirements in urban areas. It is therefore possible to drive sustainable urban developments. PRACTITIONER POINTS: The water-reuse potential increases enormously by combining industrial and municipal wastewater flows. Industrial wastewater should be treated "fit for purpose" and applied in the urban area since the municipal wastewater is not sufficient to cover its own water requirements for infrastructural purposes. Water-reuse for infrastructural purposes increases water resources. The application of reuse water drives sustainable urban developments.

Air quality in the operating room: Surgical site infections, HVAC systems and discipline - position paper of the German Society of Hospital Hygiene (DGKH).

In recent years, there has been an ongoing discussion about the value of laminar airflow (LAF=low turbulence displacement ventilation) in the operating room for prevention of surgical site infections (SSI). Some publications, e.g., from the WHO, issued the demand to no longer build LAF ceilings in operating rooms. The present statement deals critically with this position and justifies the use of LAF ceilings in different ways: Many of the papers cited by the WHO and others for the case against LAF do not provide reliable data.The remaining studies which might be used for answering the question give quite different results, also in favor of LAF.The size of the LAF ceiling in many studies is not given or mostly too small in comparison to actual technical requirements.LAF in different countries can mean quite different techniques (e.g., the US in comparison to Germany) so that the results of studies that do not take this into account may not be comparable.LAF has positive effects in terms of reducing particulate and bacterial load, associated with increased airflow in the surgical working area. A reduction of carcinogenic substances in the air may also be assumed, which would increase workers' safety. Thus, this paper recommends building LAF ceilings in the future as well, depending on the operations intended. Further, this paper gives an overview of possible reasons for surgical site infections and highlights the importance of discipline in the operating theatre.

Establishment and Characterization of a Novel Cell Line, ASAN-PaCa, Derived From Human Adenocarcinoma Arising in Intraductal Papillary Mucinous Neoplasm of the Pancreas.

OBJECTIVES: Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion. METHODS: The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling. RESULTS: The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes. CONCLUSIONS: ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.

Quantitative T2'-mapping in acute ischemic stroke.

BACKGROUND AND PURPOSE: Quantitative T2'-mapping detects regional changes in the relation of oxygenated and deoxygenated haemoglobine and might reflect areas with increased oxygen extraction. T2'-mapping in conjunction with an elaborate algorithm for motion correction was performed in patients with acute large-vessel stroke, and quantitative T2'-values were determined within the diffusion-weighted imaging lesion and perfusion-restricted tissue. METHODS: Eleven patients (median age, 71 years) with acute middle cerebral or internal carotid artery occlusion underwent MRI before scheduled endovascular treatment. MR-examination included diffusion- and perfusion-weighted imaging and quantitative, motion-corrected mapping of T2'. Time-to-peak maps were thresholded for different degrees of perfusion delays (eg, ≥0 s, ≥ 2s) when compared with a reference time-to-peak value from healthy contralateral tissue. Mean T2'-values in areas with reduced apparent diffusion coefficient and in areas with impaired perfusion were compared with T2'-values in corresponding contralateral areas. RESULTS: Median time between symptom onset and MRI was 238 minutes. T2'-values were significantly reduced within the apparent diffusion coefficient -lesion when compared with contralateral healthy tissue (83 ms [67, 97] versus 97 ms [91, 111]; P<0.003). In perfusion-restricted tissue, T2'-values were also significantly lower when compared with contralateral healthy tissue (ie, for time to peak, ≥0 s 93 ms [86, 102] versus 104 [90, 110]; P=0.008) but were significantly higher than within the apparent diffusion coefficient lesion. The severity of the perfusion impairment had no influence on median T2'-values. CONCLUSIONS: Motion-corrected T2'-mapping reveals significant and gradually declining values from healthy to perfusion-disturbed to apparent diffusion coefficient-restricted tissue. Current T2'-mapping can differentiate between the ischemic core and the perfusion-impaired areas but not on its own between penumbral and oligemic tissue.

Endocytotic uptake of nutrients in carnivorous plants.

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.

Actinin-4 expression in primary and metastasized pancreatic ductal adenocarcinoma.

OBJECTIVES: Actinin-4 is an actin-bundling protein that probably has a tumor-promoting potential in several solid tumors. The present study analyzed the expression of actinin-4 in the pancreas, in localized and metastasized pancreatic ductal adenocarcinoma (PDAC), and the correlation with clinical outcome. METHODS: Pancreatic ductal adenocarcinoma tissue from 38 patients, 15 lymph node and 10 liver metastases, normal pancreas, and 4 PDAC cell lines, were examined by immunohistochemistry, and actinin-4 expression was quantified by immunofluorescence analysis. RESULTS: In the normal pancreas, actinin-4 was most prominently expressed in ductal cells. In PDAC, tumor cells exhibited strong but differential cytoplasmic immunoreactivity for actinin-4. A multivariate analysis revealed actinin-4 immunoreactivity, advanced age, and undifferentiated grade as significant prognostic factors associated with worse survival after PDAC resection. Cells metastasized to lymph nodes or to the liver exhibited no significant increase of actinin-4 compared with the primary tumors. A nuclear staining was observed neither in any of the PDAC samples nor in the 4 cell lines. In PDAC cells, actinin-4 localized to dynamic actin structures and to invadopodia. CONCLUSIONS: Actinin-4 expression levels significantly correlate with worse survival after PDAC resection. Although actinin-4 has been reported to promote lymph node metastases, there was no enhanced expression in PDAC metastases.

Comparative analysis of tumorbiology and CD133 positivity in primary and recurrent pancreatic ductal adenocarcinoma.

In over 70% of the cases, patients with curative surgery and adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC) develop recurrent tumors. The cancer stem cell (CSC) hypothesis suggests that CSCs are chemoresistant and enriched in recurrent tumors. This study analyzes tumorbiology, expression of the metastasis-promoting CXCR4 and actinin-4, and of the CSC marker CD133 in primary and recurrent PDAC. Twenty-six patients underwent resection for primary and recurrent PDAC and most developed tumor recurrence within 2 years. In 81% the histologic tumor grade was unchanged. Immunohistochemistry could be performed with 15 pairs of primary and recurrent PDAC. The mean Ki-67 proliferation index increased (P = 0.06). About 30% of tumor cells were positive for CXCR4 and almost all tumor cells expressed actinin-4, but there were neither significant changes in the expression levels in recurrent PDAC, nor specifically enhanced levels in metastases. The prominent CD133 pattern was an apical membrane staining of inflammatorily altered, non-neoplastic ductal structures equally observed in primary and recurrent PDAC. The membrane CD133 positivity was consistently absent in neoplastic PDAC cells. Cytoplasmic CD133 positivity was extremely rare (0.85 and 0.34 cells/cm(2) in primary and recurrent PDAC, respectively; P = 0.07). Tumor grade is mainly unchanged and the expression of CXCR4, actinin-4 and CD133 are not enhanced in recurrent PDAC. The apical membrane CD133 positivity of normal and inflammatorily altered ductal structures and its lack in tumor cells bring the role of CD133 as a specific CSC marker in PDAC into question.

Fludarabine induces apoptosis, activation, and allogenicity in human endothelial and epithelial cells: protective effect of defibrotide.

Fludarabine is a nonmyeloablative immunosuppressant increasingly used as a component of alternative conditioning regimens before allogeneic bone marrow transplantation. It is expected to reduce conditioning-related toxicity and proinflammatory activation of the host tissues. However, in our in vitro study, we provide evidence that 2-fluoroadenine 9-beta-D-arabinofuranoside (F-Ara) as the active metabolized form of fludarabine damages human microvascular endothelial cells (HMECs) and dermal and alveolar epithelial cell lines after 48 hours of culture when it is used in pharmacologically relevant concentrations (range, 10 microg/mL-1 microg/mL). In addition, flow cytometric analyses revealed a significant up-regulation of intercellular adhesion molecule 1 and major histocompatibility complex (MHC) class I molecules by F-Ara, suggesting a proinflammatory activation of HMECs. Cytotoxicity assays demonstrated that target HMECs pretreated with F-Ara (10 microg/mL) showed increased lysis by allogeneic MHC class I-restricted cytotoxic T lymphocytes from healthy human donors. We conclude that, beside its immunosuppressive activities, F-Ara can be harmful for target tissues of transplantation-related complications and can even stimulate allogeneic immune responses. We identified the pharmaceutical compound defibrotide as protective against F-Ara- induced apoptosis and alloactivation, importantly, without affecting the antileukemic effect of F-Ara. This observation argues for a potential clinical usage of defibrotide in pretransplantation conditioning.

The genetic structure of Escherichia coli populations in primary and secondary habitats.

Escherichia coli were recovered from the members of two two-person households and their associated septic tanks. The E. coli were isolated using selective and non-selective isolation techniques, characterized using the method of multi-locus enzyme electrophoresis and screened for the presence of virulence factors associated with extra-intestinal disease by using PCR. The growth rate-temperature relationships of strains from the two habitats were also determined. Temporal variation explained 25% of the observed electrophoretic type (ET) diversity in the humans. Among-host variation accounted for 29% of the observed allelic diversity. In one household, ET diversity of the E. coli population in the septic tank was significantly lower than ET diversity in the humans providing the inputs to the septic tank. Molecular analysis of variance revealed that, on average, strains recovered from the septic tank of this household were genetically distinct from strains recovered from the humans providing the faecal inputs to the septic tank. Further, the growth rate-temperature response of strains differed between strains isolated from the septic tank and strains isolated from the humans. Septic tank isolates grew better at low temperatures than strains isolated from humans, but more slowly at high temperatures compared to the human isolates. By contrast, no real differences in ET diversity, allelic diversity, or the growth characteristics of strains could be detected between strains from the humans and strains from the septic tank of the other household. The results of this study suggest there are strains of E. coli that are better "adapted" to conditions found in the external environment compared to strains isolated from the gastrointestinal habitat. Further, the finding that the numerically dominant clones and clonal diversity in secondary habitats can differ substantially from those found in the source populations will confound efforts to identify the sources of faecal pollution in the environment.