Inactivation of the major hemolysin gene influences expression of the nonribosomal peptide synthetase gene swrA in the insect pathogen Serratia sp. strain SCBI

Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens. The role of the major hemolysin gene in the insect pathogen Serratia sp. SCBI was investigated using both forward and reverse genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in loss of hemolysis, but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility as well as increased antimicrobial activity. qRT-PCR analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly variable antibiotic activity, motility, virulence and hemolysis phenotypes that were dependent on the site of disruption within this 17.75 KB gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences expression of swrA, these results suggest swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI

Graft-versus-host disease after liver and small bowel transplantation in a child.

An 8-month-old child with an immunodeficiency disorder characterized by abnormal lymphocyte function and by low IgG and IgA levels had combined liver and small bowel transplantation under tacrolimus and steroid immunosuppression for the treatment of short gut syndrome and hepatic cirrhosis. The patient developed an early postoperative episode of Pneumocystis carinii pneumonia, and a subsequent surgical complication, prompting discontinuance of tacrolimus. A skin rash eventually shown to be graft-versus-host disease (GVHD) developed in the flank on the 12th post-transplant day and gradually became generalized. Peritonitis, sepsis, multisystem organ failure including the liver allograft led to death on the 23rd post-operative day. The mechanisms leading to post-transplant GVHD under the specific circumstances in this case are discussed

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity

The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon ?2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate surface domains with a level of hydrophobicity above a certain threshold

The rank reversal problem in multi-criteria decision making : a literature review

Despite the importance of multicriteria decision-making (MCDM) techniques for constructing effective decision models, there are many criticisms due to the occurrence of a problem called rank reversal. Nevertheless, there is a lack of a systematic literature review on this important subject which involves different methods. This study reviews the pertinent literature on rank reversal, based on 130 related articles published from 1980 to 2015 in international journals, which were gathered and analyzed according to the following perspectives: multicriteria technique, year and journal in which the papers were published, co-authorship network, rank reversal types, and research goal. Thus our survey provides recommendations for future research, besides useful information and knowledge regarding rank reversal in the MCDM field

Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+i/K+i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+i/K+i-sensitive genes. Among the ubiquitous Na+i/K+i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders

Subsampling effects in neuronal avalanche distributions recorded in vivo

Background Many systems in nature are characterized by complex behaviour where large cascades of events, or avalanches, unpredictably alternate with periods of little activity. Snow avalanches are an example. Often the size distribution f(s) of a system's avalanches follows a power law, and the branching parameter sigma, the average number of events triggered by a single preceding event, is unity. A power law for f(s), and sigma=1, are hallmark features of self-organized critical (SOC) systems, and both have been found for neuronal activity in vitro. Therefore, and since SOC systems and neuronal activity both show large variability, long-term stability and memory capabilities, SOC has been proposed to govern neuronal dynamics in vivo. Testing this hypothesis is difficult because neuronal activity is spatially or temporally subsampled, while theories of SOC systems assume full sampling. To close this gap, we investigated how subsampling affects f(s) and sigma by imposing subsampling on three different SOC models. We then compared f(s) and sigma of the subsampled models with those of multielectrode local field potential (LFP) activity recorded in three macaque monkeys performing a short term memory task. Results Neither the LFP nor the subsampled SOC models showed a power law for f(s). Both, f(s) and sigma, depended sensitively on the subsampling geometry and the dynamics of the model. Only one of the SOC models, the Abelian Sandpile Model, exhibited f(s) and sigma similar to those calculated from LFP activity. Conclusions Since subsampling can prevent the observation of the characteristic power law and sigma in SOC systems, misclassifications of critical systems as sub- or supercritical are possible. Nevertheless, the system specific scaling of f(s) and sigma under subsampling conditions may prove useful to select physiologically motivated models of brain function. Models that better reproduce f(s) and sigma calculated from the physiological recordings may be selected over alternatives

Layered control architectures in robots and vertebrates

We revieiv recent research in robotics, neuroscience, evolutionary neurobiology, and ethology with the aim of highlighting some points of agreement and convergence. Specifically, we com pare Brooks' (1986) subsumption architecture for robot control with research in neuroscience demonstrating layered control systems in vertebrate brains, and with research in ethology that emphasizes the decomposition of control into multiple, intertwined behavior systems. From this perspective we then describe interesting parallels between the subsumption architecture and the natural layered behavior system that determines defense reactions in the rat. We then consider the action selection problem for robots and vertebrates and argue that, in addition to subsumption- like conflict resolution mechanisms, the vertebrate nervous system employs specialized selection mechanisms located in a group of central brain structures termed the basal ganglia. We suggest that similar specialized switching mechanisms might be employed in layered robot control archi tectures to provide effective and flexible action selection

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21Waf1/Cip1 and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. Conclusion: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from Bothrops jararacussu

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells

Ouabain protects against adverse developmental programming of the kidney

The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards the formation of functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that ouabain, the Na,K-ATPase ligand, triggers a calcium–nuclear factor-κB signal, which protects kidney development from adverse effects of malnutrition. To mimic malnutrition, kidneys were serum deprived for 24 h. This resulted in severe retardation of nephron formation and a robust increase in apoptosis. In ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given a low-protein diet and treated with ouabain or vehicle throughout pregnancy. Thus, we have identified a survival signal and a feasible therapeutic tool to prevent adverse programming of kidney development