Bacterial growth properties at low optical densities

A method for accurate quantification of growth rate and yield of bacterial populations at low densities was developed with a modified version of a stepwise linear model for fitting growth curves based on optical density measurements, and adapted to measurements at low optical densities in 96-well microtiter plates. The method can be used for rapid and precise estimates of growth rate and yield, based on optical density measurements of large numbers of cultures of Escherichia coli. E. coli B lines were serially propagated at low glucose concentration during a long-term evolution experiment. Growth rate and yield of populations sampled from each of 12 lines that evolved for 20,000 generations under these conditions and two ancestral clones was measured. Populations were grown at three different glucose concentrations. Consistent with earlier findings, statistical analysis showed that both exponential growth rate and yield per unit of glucose differed significantly between the three glucose concentrations tested. Significant adaptation of the evolved populations to the nutrient conditions in which they evolved for 20,000 generations was observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Differential Cellular Effects of Electroporation and Electrochemotherapy in Monolayers of Human Microvascular Endothelial Cells

In vivo electroporation of tumours shows disruption of blood flow and creates a vascular effect with an initial rapid and transient vasoconstriction phase and a much longer lasting phase with changed microvascular endothelium. These changes are not well understood but are presumed to involve the cytoskeleton. The paper presents for the first time differential in vitro effects describing cytoskeleton changes and monolayer integrity changes by both electroporation and electrochemotherapy of monolayers of human microvascular endothelial cells (HMEC-1). After the application of electric field pulses, the morphology of cells, and both the F-actin and Beta-tubulin cytoskeleton proteins were affected. During both electroporation and electrochemotherapy, the initial phase of cellular damage was noticed at 10 min as swollen cells and honeycomb-like actin bundles. The electroporation-induced cellular effects, observed from electric pulses >150 V, were voltage-dependent and within 24 hrs partly recoverable. The electrochemotherapy-induced cellular effects developed at 2 hrs in spindle-like cells, and more densely packed F-actin and Beta-tubulin were observed, which were dependent on the amount of bleomycin and the voltages applied (>50 V). In addition, for electrochemotherapy with electric pulses >150 V cellular changes were not recoverable within 24 hrs. The effects on monolayer integrity were reflected in the enhanced monolayer permeability, with the electrochemotherapy showing an earlier onset and synergy. We conclude that electrochemotherapy as compared to electroporation leads within 24 hrs to a quicker and more pronounced monolayer integrity damage and endothelial cell death, which together provide further insight into the cellular changes of the vascular disruption of electrochemotherapy.     

Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.     

A single-centre experience with octreotide in the treatment of different hypersecretory syndromes in patients with functional gastroenteropancreatic neuroendocrine tumors

The aim of this research was to assess the clinical and biochemical efficacy of the octreotide in the treatment of patients with various functional gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The study included 14 patients treated with octreotide for 6 months. They were diagnosed with VIPoma, glucagonoma, gastrinoma, medullary thyroid carcinoma (solitary and as a part of MEN-II syndrome), pancreatic carcinoids (solitary and as a part of multiple endocrine neoplasia type-1 syndrome-MEN-1 syndrome) and midgut carcinoids. The patients presented with Verner-Morrison, glucagonoma, Zollinger Ellison and carcinoid syndrome respectively. All had a metastatic disease at the time of diagnosis and a positive octreoscan finding. Initially elevated chromogranin A (CgA) levels were detected in 11 (78.6%) and elevated 5-hydroxyindolacetic acid (5-HIAA) levels in 8 (57.1%) patients. Symptomatic efficacy assessments were made by diarrhea reductions during treatment course, and laboratory efficacy was assessed through changes in 5-HIAA and CgA levels. Assessments were made initially and following 6 months of therapy. Median urinary 5-HIAA and the number of stools decreased significantly (p = 0.016 and p = 0.009 respectively, p < 0.05) while CgA levels had the decreasing tendency but not statistically significant (p = 0.14). There was a positive correlation between the 5-HIAA reduction and the decrease in stool number at baseline and during treatment course (p < 0.05). No correlation was observed between 5-HIAA and CgA levels and also there was no correlation between CgA reduction and symptomatic improvement. The results prove octreotide to be effective in reducing symptoms and biochemical markers associated with hypersecretory syndromes of GEP-NETs.     

Antibody profile of pregnant women with antiphospholipid syndrome and pregnancy outcome after treatment with low dose aspirin and low-weight-molecular heparin

The aim of the research was to show our diagnostic and therapeutic experience with antiphospholipid syndrome (APS) in pregnant women. 36 pregnant women suspect on APS were included in the study: 32 with primary antiphospholipd syndrome (PAPS) and 4 with secondary antiphospholipid syndrome (SAPS). All pregnant women received low-molecular-weight-heparin (LMWH) and low dose aspirin (LDA) therapy. Control group represented 26 women with SAPS and previous bad reproductive anamnesis. Average pregnancy lasted 37.06 +/- 0.707 weeks. LMWH and LDA therapy was successful in 97.22%. Lupus anticoagulant (LA) was found to be more frequent in PAPS group (71.87%). Anticardiolipin antibodies (aCL) were found to be more frequent in SAPS (26.66%). For three patients (3.37%), PAPS was diagnosed due to a fact that they had positive antibeta2-glycoproteinl (antibeta-GP1). To make APS diagnosis, it is of great importance to search for all antiphospholipid antibodies. LMWH and low dose of acetylsalicylic acid should be the first choice therapy.     

Pheochromocytoma presenting as a pancreatic mass: Avoiding a dangerous pitfall in endoscopic ultrasound-guided fine needle aspiration biopsy using GATA3 immunostain

Pheochromocytomas and sympathetic paragangliomas are rare tumours arising from chromaffin cells, producing catecholamines in various amounts. Fatal hypertensive episodes may occur perioperatively, which are preventable by alpha adrenergic receptor blockers. The perioperative mortality rate of diagnosed versus undiagnosed catecholamine-producing tumours is significant, considering that only a minority of tumours develop metastasis. Herein we describe a case of a primary adrenal pheochromocytoma referred to as a pancreatic tumour, successfully diagnosed by endoscopic ultrasound-guided fine needle aspiration biopsy, with a distinct morphology (prominent nuclear anisonucleosis, intranuclear pseudoinclusions, and multinucleation) and immunohistochemical signature.     

Multi‐causality and spatial non‐stationarity in the determinants of groundwater crustacean diversity in Europe

The recognition of multi‐causality and spatial non‐stationarity in the determinants of large‐scale biodiversity patterns requires to consider the role of multiple mechanisms, their interactions, and how these mechanisms vary in strength relative to each other across geographical space. Here, we challenge the view that historical climate stability primarily drives European patterns of groundwater crustacean diversity by testing also the role of spatial heterogeneity and productive energy. First, we predicted that the three mechanisms would be equally important at continental scale when analyzed separately, but that the importance of historical climate variability would weaken in joint analyses due to co‐variation with the two other mechanisms. Second, we predicted that the three mechanisms would exhibit predictable latitudinal changes in their relative strength. To test these predictions, we selected predictors representing each mechanism and analyzed separately and jointly their effects and interactions using global regression models. We further mapped the independent and overlapping effects of mechanisms across Europe using partial geographically weighted regressions. When analyzed separately, the three mechanisms explained the same amount of variation in species richness, but in the joint analysis, the influence of historical climate stability became hidden in the variation shared with the other mechanisms. Topographic heterogeneity interacted synergistically with actual evapotranspiration and habitat heterogeneity on species richness. Spatial non‐stationarity in the independent and overlapping effects of the three mechanisms was the most plausible explanation for the hump‐shaped latitudinal pattern of crustacean species richness. Productive energy and spatial heterogeneity were important predictors at mid and southern latitudes, whereas historical climate stability overlapped with the two other mechanisms in northern Europe and productive energy in southern Europe. Multi‐causality and spatial non‐stationarity provide a broader perspective of groundwater biodiversity determinants that revives the importance of spatial heterogeneity and the strong dependence of subterranean communities on food supply from the surface.