Several studies have identified use of non-steroidal-anti-inflammatory drugs and statins for prevention of dementia, but their efficacy in slowing progression is not well understood. Cerebrovascular disturbances are common pathological feature of Alzheimer’s disease. We previously reported chronic ingestion of saturated fatty acids (SFA) compromises blood–brain barrier (BBB) integrity resulting in cerebral extravasation of plasma proteins and inflammation. However, the SFA-induced parenchymal accumulation of plasma proteins could be prevented by co-administration of some cholesterol lowering agents. Restoration of BBB dysfunction is clinically relevant, so the purpose of this study was to explore lipid-lowering agents could reverse BBB disturbances induced by chronic ingestion of SFA’s.Wild-type mice were fed an SFA diet for 12 weeks to induce BBB dysfunction, and then randomised to receive atorvastatin, pravastatin or ibuprofen in combination with the SFA-rich diet for 2 or 8 weeks. Abundance of plasma-derived immunoglobulin-G (IgG) and amyloid-β enriched apolipoprotein (apo)-B lipoproteins within brain parenchyme were quantified utilising immunofluorescence microscopy.Atorvastatin treatment for 2 and 8 weeks restored BBB integrity, indicated by a substantial reduction of IgG and apo B, particularly within the hippocampus. Pravastatin, a water-soluble statin was less effective than atorvastatin (lipid-soluble). Statin effects were independent of changes in plasma lipid homeostasis. Ibuprofen, a lipid-soluble cyclooxygenase inhibitor attenuated cerebral accumulation of IgG and apo B as effectively as atorvastatin. Our findings are consistent with the drug effects being independent of plasma lipid homeostasis.Our findings suggest that BBB dysfunction induced by chronic ingestion of SFA is reversible with timely introduction and sustained treatment with agents that suppress inflammation. Accumulating evidence is consistent with the concept that the onset and progression of Alzheimer’s disease (AD) is influenced by vascular-risk factors. A number of studies have demonstrated a positive association between AD and atherosclerosis, cardiovascular disease, dyslipidaemia, hypertension and insulin resistance,2. PopulAccumulating evidence suggests that cerebral capillary dysfunction precedes amyloidosis, a hallmark pathological protein marker for Alzheimer’s disease. Common Several lines of evidence are consistent with the hypothesis that suppressing cerebral capillary inflammation may confer benefit to AD onset, or disease progression. Reducing the plasma concentration of cytokines and pro-inflammatory proteins by the regular consumption of foods or vitamin supplements that suppress inflammation is associated with a delay for development of dementia-19. FurtPopulation studies also support a role for anti-inflammatory lipid lowering agents in the prevention of dementia. The 3-City Study represents a cohort of more than 9,000 subjects examining the association of plasma cholesterol and lipid-lowering agent intake with dementia prevalence. In thatPresently, there is insufficient evidence to recommend statins for the treatment of dementia once disease is established or advanced. Howeverin vivo. Despite this difficulty, proof-of-concept data using surrogate markers of cerebrovascular inflammation in relevant animal models would provide information on the putative efficacy of selected and targeted interventions. Our preliminary investigations demonstrated beneficial effects of atorvastatin in preventing saturated-fatty-acid (SFA) induced cerebrovascular dysfunction[In a clinical context restoration of cerebral capillary integrity would be therapeutically beneficial for slowing or delaying AD progression, although presently this is a difficult phenomenon to assess function. HoweverOur results confirm the significant abundance of immunoglobulin-G (IgG) and distribution within the cortex (CTX), brainstem (BS) and hippocampal formation (HPF) in SFA fed wild-type (WT) Figure . The SFAThe longer duration of SFA feeding significantly increased the apo B distribution in the parenchyma  treatment. Treatment with pravastatin or ibuprofen, although less potent than atorvastatin, nonetheless resulted in a uniform reduction of IgG and apo B abundance within HPF, CTX and BS. Ibuprofen showed evidence of efficacy by 2 weeks in HPF and CTX, but pravastatin had no significant effect within these regions at 2 weeks. Collectively, these findings indicate that atorvastatin was most effective in regression of hippocampal plasma protein abundance independent of duration of intervention. Longer-term intake of pravastatin and ibuprofen was required to ameliorate accumulation of plasma proteins within the HPF.The HPF is the vital brain region involved in learning, memory and some suggest, particularly vulnerable to oxidative damage induced by inflammation,41. PathThe mechanisms by which atorvastatin, pravastatin and ibuprofen influenced BBB integrity were not specifically explored in this study. However, the data show that the improvements in BBB function were not associated with plasma lipid homeostasis. These findings suggest that the diets were well tolerated in our mouse model. Consistent with our previous observations where SFA induced BBB dysfunction was independent of hypercholesterolemia. Many stWe previously demonstrated that SFA induced BBB leakage was associated with significant reduction in the tight junction protein occludin. FurtherA paradoxical finding was that abundance of apo B lipoproteins within brain parenchyme required a longer duration of treatment with statins or ibuprofen to be reduced compared IgG, yet apo B lipoproteins have a molecular weight of up to 100 times greater than IgG. Notionally, penetrance into brain parenchyme of macromolecules such as apo B lipoproteins would become evident more quickly than large proteins such as IgG. However, previous findings have shown significant retention of apo B lipoproteins associated with extracellular matrices including heparin-sulphate proteoglycans, biglycan and decorin. The exaDysfunction of the BBB is increasingly recognised in neurodegenerative diseases with cerebral capillary disturbances including AD. AlthougEvidence showing cerebral capillary dysfunction preceding amyloid deposition is growing. This study provides novel insight into the effects of atorvastatin, pravastatin and ibuprofen on regression and prevention of SFA-induced BBB permeability and preceding amyloidosis. Further studies are required to ascertain the underlying mechanisms of how statins and ibuprofen modulate BBB integrity.The Curtin University Animal Experimentation and Ethics Committee approved housing, handling and experimental procedures described for this study. Six-week-old female WT mice (C57BL/6 J) were housed in groups and randomised into the diet or drug treatment groups (6 mice per group). All mice were maintained in a 12 h light and dark cycle room, at 22°C and with free access to water and food. Mice were weighed weekly and average daily diet consumption was recorded.The LF control group of mice were fed a semi-purified diet  containing 4% (w/w) total fat (derived from canola oil) and <1% of total digestible energy from lipids. As previously demonstrated, mice were fed SFA diet containing 20% (w/w) cocoa butter  to induce BBB damage. The SFA→ SFA + At), pravastatin (SFA → SFA + Pr) or ibuprofen (SFA → SFA + Ib) to determine the effect of drugs with insult (SFA diet). Mice switched to LF diets containing identical doses of atorvastatin (SFA → LF + At), pravastatin (SFA → LF + Pr) or ibuprofen (SFA → LF + Ib) to determine the effects of drugs in the absence of dietary insult. Animals that were initially fed SFA diet for 12 weeks were switched to the drug containing diets (LF + drug or SFA + drug). They were then sacrificed at two time points, at 2 weeks and 8 weeks after drug intervention, to observe any progressive effects of the drugs on BBB restoration. Mice given LF or SFA alone were run parallel with all experiments and sacrificed at each end point.To determine the putative restorative effects of atorvastatin (At), pravastatin (Pr) and ibuprofen (Ib) on BBB damage, WT mice were initially fed with SFA diet for a period of 12 weeks to induce damage to the BBB. The SFA fed mice were then switched to SFA diets containing atorvastatin (SFA The agents were incorporated into either LF or SFA chow at a concentration of 20 mg/kg (w/w) atorvastatin , 23.4 mg/kg (w/w) pravastatin sodium  and 333.3 mg/kg (w/w) ibuprofen . Based on measured consumption rates, the daily ingested dose for each agent approximated three times the highest recommended dose for human studies per unit body. However, the bioavailability of atorvastatin and pravastatin has been reported to be reduced when consumed with food,57.Mice were maintained on the indicated diets and weighed weekly. Tissue samples were collected as previously described by Takechi et al.. Mice weBrain tissues were carefully isolated, washed with chilled phosphate buffered saline , and the right hemispheres were separated and fixed in 4% paraformaldehyde for 24 h. The tissues were then cryoprotected with 20% sucrose solution at 4°C for 72 h, frozen in isopentane with dry ice and stored at −80°C. For histology and fluorescence microscopy, serial cryo-sections of 18 μm were cut from the right cerebral hemispheres for each mouse and mounted on Polysine slides.Cerebrovascular leakage of IgG and apo B were detected as previously described. Brain cFor IgG staining, tissues were incubated with goat anti-mouse IgG-Alexa 488 flurochrome conjugated antibody (Invitrogen) at 1:100 dilution, overnight at 4°C. The sections were then washed with PBS and nuclei were counterstained with DAPI (1:1000) for 5 min at room temperature. Thereafter, the stained sections were mounted with anti-fade mounting medium.Cerebral apo B was detected by overnight incubation with polyclonal rabbit anti-apo B as the primary antibody  at 1:500 dilution, at 4°C. Post-overnight incubation, primary antibody was labelled at room temperature with the secondary goat anti-rabbit IgG-Alexa 488 conjugate (Invitrogen) for 2 h. The tissues were then counterstained with DAPI and mounted as per IgG staining method.Digital images for photomicroscopy were acquired through AxioCam HRm camera (Zeiss Germany) with an AxioVert 200 M inverted microscope by Zeiss (Germany) at × 200 magnification . Three-dimensional (3-D) images were captured through ApoTome optical sectioning methodology (Carl Zeiss),58. EachCerebrovascular leakage of plasma proteins IgG and apo B were quantified within the CTX, BS and HPF. The pixel intensity of protein of interest surrounding the blood vessels for each 3-D image was quantitated utilising the automated optical intensity measurement tool in Volocity  and expressed as per unit volume. The investigator was blinded during image capturing and quantitative analysis.Plasma Cholesterol and triglycerides were determined in duplicate by enzymatic assays . Non-esterified fatty acids were determined with NEFA-C .This study utilised 6 mice per group and minimum of nine 3-D images were captured per mouse for detection of IgG and apo B leakage within the CTX, BS and HPF. In each group, 324–540 two-dimensional images were generated for adequate statistical comparison.P-values < 0.05 were considered to be statistically significant.Normally distributed data were analysed by parametric one-way analysis of variance to assess the main effects of the dietary SFA, atorvastatin, pravastatin and ibuprofen treatment. The Kruskall-Wallis test was utilised if data was not-normally distributed. Post-hoc comparison of means was done if the associated main effect or interaction was statistically significant within the analysis of variance procedure. (AD): Alzheimer’s disease; (Aβ): Amyloid-β; (apo B): Apolipoprotein B; (At): Atorvastatin; (BBB): Blood–brain barrier; (BS): Brainstem; (CTX): Cortex; (COX): Cyclooxygenase; (HPF): Hippocampal formation; (Ib): Ibuprofen; (IgG): Immunoglobulin; (LF): Low fat; (SFA): Saturated fatty acid; (NEFA): Non-esterified fatty acids; (NSAIDs): Non-steroidal anti-inflammatory drugs; (OR): Odds ratio; (PBS): Phosphate buffered saline; (Pr): Pravastatin; (WT): Wild-type.The authors declare that they have no competing interests.MPG carried out the design of project, data collection, immunofluorescence, statistical analysis and drafting of the manuscript. VL and RT assisted in tissue collection and interpretation of data. SG helped in the collection of tissues. KC assisted in critically analysing and drafting of the manuscript. JM conceived the study, helped in data interpretation, drafting of the manuscript, obtaining funding and general supervision of the research group. All authors have approved manuscript for submission.


Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil.Multiple isolates of  Mycobacterium abscessus is an environmental organism found in soil, water, and other ecological niches, and it acts as an opportunistic pathogen causing diseases ranging from cutaneous to pulmonary infections (M. abscessus subsp. bolletii (formerly referred to as M. abscessus subsp. massiliense) (The nontuberculous mycobacterium (NTM) fections . Bacteriiliense)  were liniliense) –5. The eiliense) , 4, 6.M. abscessus subsp. bolletii GO-06, from the Brazilian state of Goiás, was previously released  at 130× coverage and with Roche 454 GS-FLX Titanium  at 10× coverage. MiSeq reads were assembled into contigs with the software packages A5  and VelvCC 19977 , and genM. abscessus subsp. bolletii plasmids, pMAB01  were predicted, including 3,378 CDSs (71.1%) with functional annotations and 1,372 CDSs (28.9%) that were annotated as hypothetical proteins. The genome contains 46 tRNAs and one rRNA cistron. A comparative analysis between CRM-0020 and ATCC 19977 revealed that 4,189 CDSs (88.2%) are shared between the two strains, while 561 (11.8%) CDSs are unique to CRM-0020. A single contig  aligned with 99% identity to the , pMAB01  and BRA1M. abscessus subsp. bolletii CCUG 48898  (1.28 SNPs per Kb) between CRM-0020 and GO-06, showing a low level of divergence among the regional outbreak strains from Brazil. In contrast, 49,354 SNPs (10.2 SNPs per Kb) were observed between CRM-0020 and the type strain of UG 48898 , revealiATFQ00000000. The version described in this paper is version ATFQ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


One of the major aims of contemporary evolutionary biology is the understanding of the current pattern of biological diversity. This involves, first, the description of character distribution at various nodes of the phylogenetic tree of life and, second, the functional explanation of such changes. The analysis of character distribution is a powerful tool at both the morphological and molecular levels. Recent high-throughput sequencing approaches provide new opportunities to study the genetic architecture of organisms at the genome-wide level. In eukaryotes, one overarching finding is the absence of simple correlations of gene count and biological complexity. Instead, the domain architecture of proteins is becoming a central focus for large-scale evolutionary innovations. Here, we review examples of the evolution of novelty in conserved gene families in insects and nematodes. We highlight how in the absence of whole-genome duplications molecular novelty can arise, how members of gene families have diversified at distinct mechanistic levels, and how gene expression can be maintained in the context of multiple innovations in regulatory mechanisms. Drosophila sechellia  To understand the structure of my new Cirripede, I had to examine and dissect many of the common forms: and this gradually led me on to take up the whole group.'' This illustrates perfectly well what lies on the agenda of today's evolutionary biologists. What has changed since Darwin's time is that now we have the tools required to describe natural variation from the molecular level to the ecological one. It follows that, for a given node of a phylogenetic tree, variation and repartition of characters can and need to be addressed at all these levels. What we have to understand is the connection between the various layers of biological complexity, combining and integrating the results of laboratory and fieldwork approaches [In his autobiography, Darwin  wrote thproaches .a priori more potentials than others to contribute to novelty at a higher phenotypic level. Uncoupling and buffering of natural variation at various integration scales has been clearly demonstrated, implying that the number of molecular events that can be directly correlated with a phenotypic change at the organismal level is probably very low, and that they are the results of exceptional contingency and structural constraints [de novo such interesting changes in nonmodel organisms is thus also probably very low, and it decreases quickly with an increase of the phylogenetic distance. Additionally, one should not forget that to really understand the link between genetic variation and phenotypic diversity, it is necessary to be able to explain cases where a molecular change triggers novelty, and those where phenotypic traits are maintained in spite of molecular innovations in the genes that specify them. We argue that the partial data on genomic variation are already sufficient to indicate that no specific attribute of a given molecular structure can indicate straints . The pos


Although potentiostats are the foundation of modern electrochemical research, they have seen relatively little application in resource poor settings, such as undergraduate laboratory courses and the developing world. One reason for the low penetration of potentiostats is their cost, as even the least expensive commercially available laboratory potentiostats sell for more than one thousand dollars. An inexpensive electrochemical workstation could thus prove useful in educational labs, and increase access to electrochemistry-based analytical techniques for food, drug and environmental monitoring. With these motivations in mind, we describe here the CheapStat, an inexpensive (<$80), open-source (software and hardware), hand-held potentiostat that can be constructed by anyone who is proficient at assembling circuits. This device supports a number of potential waveforms necessary to perform cyclic, square wave, linear sweep and anodic stripping voltammetry. As we demonstrate, it is suitable for a wide range of applications ranging from food- and drug-quality testing to environmental monitoring, rapid DNA detection, and educational exercises. The device's schematics, parts lists, circuit board layout files, sample experiments, and detailed assembly instructions are available in the supporting information and are released under an open hardware license. An inexpensive instrument that is versatile enough to generate standard waveforms could thus significantly broaden the use of electrochemistry in resource-poor environments. Such a device would enable, for example, quantitative measurements in food and drug quality control Potentiostats, the cornerstone of electrochemistry research, have seen little adoption in resource poor environments such as the developing world and teaching laboratories. An important factor in this is cost, with academic labs and industrial groups typically paying upwards of US $10,000 for a general-purpose, research quality workstation. Indeed, even the least expensive commercially available laboratory potentiostats .With the exception of the printed circuit boards, which were custom ordered from PCBex , all of the components required to build the CheapStat were obtained from Mouser Electronics  at a total cost of ∼$80 per device in small numbers. The schematics for this device , and insAs a simple demonstration of the educational applications of the CheapStat, we have used it to measure the ascorbate concentration in orange juice. Because ascorbic acid is redox active its level in orange juice can be measured via cyclic voltammetry In this experiment, four orange juice samples were prepared, one of which was unmodified and the remaining three of which were modified via the addition of exogenous ascorbic acid at 0.1, 0.2 or 0.3 M. To increase their conductivity potassium chloride was added to each of these to 1 M. A graphite pencil “lead” taken from a mechanical pencil was used as the working electrode. A standard Ag/AgCl reference and platinum counter electrode were used. Measurements were made in 5 mL of the various orange juice samples . Cyclic voltammetry measurements were taken from 200 to 900 mV, and the current at 550 mV was used for quantification of the orange juice. Of note, although it is the most abundant redox active species in orange juice, ascorbate is not the only redox active compound present. Specifically, other substances interfere with these measurements at potentials above 600 mV and thus ascorbate should not be determined using readings collected above 600 mV.To determine the initial ascorbate we make a linear plot of current at 550 mV versus added ascorbate Figure 2The voltammetric response and redox cycling of the reversible ferricyanide-ferrocyanide couple is a commonly used educational tool to introduce electrochemistry, and here we demonstrate this reaction using the CheapStat. In addition to its pedagogical roles, ferricyanide is a common oxidant in organic chemistry and has been used as a probe for a variety of organic and biological reactions, as the reduction of ferricyanide to ferrocyanide is easily monitored.In this experiment, we dissolved potassium ferricyanide in 50 mL of 1 M potassium nitrate to final concentrations of 1 and 2 mM. As our working electrode we employed a simple platinum disk electrode. To further highlight the low cost and self-assembled nature of the CheapStat, these experiments were performed independently with either a standard, commercial Ag/AgCl electrode or a hand-fabricated glass pipette-based Ag/AgCl electrode  in 250 mL of 2 M sulfuric acid to a final theoretical concentration of 13.33 mM. Splitting this into four samples we adulterated three with increasing amounts of exogenous, pure acetaminophen at 0.002, 0.005 and 0.01 M. As our working electrode we employed a Teflon-coated gold wire of diameter 0.08 mm, a 1 cm length of which was heated to exposed the bare gold surface. A standard Ag/AgCl electrode and a length of platinum wire were employed as the reference and counter electrode respectively. We conducted linear sweep voltammetry on these samples over the potential range from 500 to 1000 mV at a rate of 10 mV/sec.To calculate the acetaminophen concentration in our unadulterated sample we linearly extrapolate the current observed at 850 mV Figure 4In some rural parts of India and Bangladesh, arsenic in drinking water is a major health concern. We have used the CheapStat to measure arsenic Samples of lake water were collected from Lake Cachuma  and stored in Nalgene polyethylene bottles. The lake water was acidified by mixing it with concentrated hydrochloric acid in an 11 to 1 ratio. Gold disk electrodes  were polished in a slurry of 0.5 micron alumina on a polishing cloth and then rinsed thoroughly with deionized water. A sealed ampoule of arsenic oxide  was diluted 1000 fold with acidified lake water. Aliquots of the diluted standard were added to 5 mL samples of acidified lake water. The concentration of added arsenic in the samples was 5 ppb and 20 ppb.In each lake water sample, a gold disk, platinum counter, and silver/silver chloride reference electrode were immersed. The current at the working electrode was held at −0.500 mV for 120 seconds while the solution was mixed by pipetting. And then a square wave measurement at 40 Hz was run from −270 mV to 600 mV. The CheapStat was easily able to make measurements at spiked 5 and 20 ppb arsenic concentrations Figure 5salmonella genome.Electrochemical DNA (E-DNA) biosensors are inexpensive, reusable DNA detectors The sensing element in these devices is a synthetic 17-base oligonucleotide commercially synthesized with a thiol on its 5′-terminus and a methylene blue reporter on the opposite terminus. The thiol readily anchors the oligonucleotide to a gold electrode (via very facile self assembled monolayer chemistry), leaving the other end free to approach the electrode and transfer electrons Figure 6The DNA probe employed in this sensor was synthesized by Biosearch Technologies , and the target by Sigma Genosys, a division of Sigma Aldrich . The sequences of the oligonucleotides were:2)6−TGGATCGGCGTTTTATT−(CH2)7−NH−MB-3′5′-HS− overnight. Before use the sensors were rinsed in deionized water.To fabricate the sensor we polished gold disk electrodes using 0.05 µm alumina before cleaning them electrochemically in 0.5 M HThe sensors were immersed in 5 mL of phosphate buffered saline. An initial square wave voltammetry scan was made from 0 to −0.5 V at 100 Hz. We then challenged the sensor by adding 100 µL of a 10 µM stock of a 34-base analyte DNA. At 30 min and 60 min after mixing we recorded new square wave scans, observing the expected 60% decrease in signal after 1 hour. These results were in good agreement with our earlier work, which used a CH Instruments 630B  potentiostat The heart of the CheapStat is a closed-loop analog control circuit, capable of regulating electrode voltage with sub-millivolt precision as it measures electrode current with nanoamp precision. This control circuit is driven by an Atmel XMEGA microcontroller containing a Digital to Analog Converter and an Analog to Digital Converter of sufficient precision to support the relevant voltage waveform generation and current quantification. Coupled to this microcontroller a Universal Asynchronous Receiver-Transmitter to Universal Serial Bus (USB) chip provides a convenient interface between this microcontroller and a data analysis computer via a USB port. An operational amplifier feedback system sets the voltage across the electrochemical cell and supplies the current needed to drive the electrochemical reaction. We employed Texas Instruments TLC2262CP operational amplifiers (Mouser Electronics) in the CheapStat because they are low power and require little input current. Specifically, these amplifiers have an input bias current of only 1pA, which permits reliable sensing of low nanoamp currents.Inherent in the specific device choices described above is the overarching design theme of the CheapStat: ease of fabrication, modification and use. For example, using the above-described components and an inexpensive, custom-built (and easily obtained) PC board we have hand fabricated a number of CheapStats without the use of a reflow oven, wave soldering, or other sophisticated circuit assembly tools. The CheapStat firmware is likewise easily updated with the aid of a simple microcontroller programming kit. Finally, the designs of the CheapStat are available under a Creative Commons license, and the attached documentation is detailed enough to support efforts to build the CheapStat or expand upon its design.As currently implemented the CheapStat supports square wave, linear sweep, stripping, and cyclic voltammetry over potentials from −990 mV to 990 mV, frequencies from 1 to 1000 Hz, and currents from ∼100 nA to 50 µA. These operation ranges can be expanded with simple hardware adjustments. In the event that a given experiment produces currents greater than 50 µA, for example, a simple resistor can be added in series with the working electrode to bring the current back into the useful range of the device. The CheapStat is easy to use: it includes a three-line LCD display and a joystick by which the operator selects the specific protocol, defines its parameters , and initiates the experiment. Copying data from the CheapStat onto a computer is similarly easy, requiring only a single mouse click. Finally, the CheapStat is hand-held, weighs only 115 grams, is powered via its USB port, and it can be controlled by a simple laptop or netbook computer, rendering the device portable and field ready.To demonstrate the utility of the CheapStat we have conducted a set of simple experiments spanning a range of representative applications. These include the use of cyclic voltammetry and an inexpensive, pencil “lead” working electrode to measure vitamin C (ascorbic acid) in orange juice Figure 2The CheapStat may prove of particular value as an educational tool. In each of the above experiments, for example, we obtained results of sufficient quality and reproducibility for facile interpretation by people who have little experience with chemical benchwork. Moreover, from an educational standpoint, the difficulty of using the CheapStat is quite low, rendering it quite suitable for college freshmen, or even high school students. With the increasingly significant role that electrochemistry plays in alternative energy and analytical technologies, the importance of accessible access to electrochemistry continues to grow. Electrochemistry likewise provides a powerful vehicle for teaching students about a broad range of topics within chemistry, including thermodynamics, redox reactions, reaction kinetics and titrations. Given these observations, we believe the availability of an inexpensive potentiostat such as that described here could have a positive impact on chemical education.2, an all too common environmental contaminant across broad areas of South Asia, where groundwater contamination is a pressing problem Beyond educational use, the CheapStat may also prove useful for analytical applications in the developing world. As we have shown, for example, the CheapStat supports the determination of arsenic in lake waterThe CheapStat represents one example of a growing trend towards low-cost, easily fabricated analytical devices. Recent years have seen, for example, the development of inexpensive microfluidic devices made from polystyrene sheets Appendix S1Instructions for building a CheapStat, parts list, circuit diagrams and user notes.(DOCX)Click here for additional data file.Layout S1Schematics that can be used to view or order the necessary printed circuit board.(GBL)Click here for additional data file.Layout S2Schematics that can be used to view or order the necessary printed circuit board.(GBS)Click here for additional data file.Layout S3Schematics that can be used to view or order the necessary printed circuit board.(GTL)Click here for additional data file.Layout S4Schematics that can be used to view or order the necessary printed circuit board.(GTO)Click here for additional data file.Layout S5Schematics that can be used to view or order the necessary printed circuit board.(GTS)Click here for additional data file.Layout S6Schematics that can be used to view or order the necessary printed circuit board.(OLN)Click here for additional data file.Layout S7Schematics that can be used to view or order the necessary printed circuit board.(TXT)Click here for additional data file.


To clarify the impact of the choice of vasopressor support on mortality in pediatric septic shock (SS).A retrospective study based on the institutional database analyzing 1,050 patients admitted from October 1999 to January 2005. We studied children with SS after the neonatal period admitted to the pediatric intensive care (PICU) and we assessed the vasopressor support in the first 24 hours, PICU and hospital (HSP) length of stay (LOS), number of vasoactive drugs used, association between drugs and HSP mortality.P = 0.047), hypoxemia  and mechanical ventilation utilization .There were 101 consecutive patients with SS, mean age 41 months (95% CI = 30 to 52 months); mean of PICU LOS 16.73 days (95% CI = 11.18 to 22.28) and hospital LOS 55.46 days (95% CI = 43.16 to 67.75). PICU mortality was 32% and HSP mortality after PICU discharge was 10.8%. Of these, 33% patients received dobutamine and 26% patients dopamine as the only vasoactive drug. Dopamine plus dobutamine was used in 17.8%; dobutamine plus norepinephrine in 18% and dopamine plus norephinephrine in 3.9%. The HSP mortality associated with dobutamine was 29.4%; dopamine 53.8%; dopamine plus dobutamine 50%; dopamine plus norepinephrine 25%. The dopamine and dopamine plus dobutamine groups had higher hospital mortality (66% vs. 34%). Dopamine was associated with hypertensive state (odds ratio, 0.433; 95% CI = 0.192 to 0.976; Adrenergic support for pediatric patients with SS remains controversial. A prospective randomized controlled trial will be important to determine which subgroups of SS patients will benefit most with each drug.


Implicit in this formulation is that a system that drives appetitive pursuit toward needed resources is essential for survival. Dopamine is the go-and-get-it neurotransmitter. In it's association with addiction and compulsive behavior— a “hijacked reward system”— dopamine has become neuroscience's version of Freud's id, driving appetitive pursuit without regard to consequences.If dopamine were a politician, it might have an image problem. The organizing metaphor for dopamine function is modulatory. That is, dopamine adjusts these functions, which begs the question to what end? The impulse behind this research topic is that the “root” function of dopamine is not to drive reward behavior, but rather to adjust reward oriented behavior in order to achieve adaptive behavioral flexibility. That is, dopamine evolved to adapt reward pursuit, not blindly drive it like a catecholaminergic id. From this perspective, compulsive pathology arises from the loss of dopamine's capacities to adapt appetitive behavior to the environment. Instead of thinking of dopamine as a “reward neurotransmitter,” we might just as well consider it a “flexibility neurotransmitter.” This is not merely semantic but can reframe the questions we ask, how experiments are designed and how data are interpreted. However, conceptual frameworks for thinking about dopamine and behavioral flexibility are not as well developed as those that emphasize dopamine and reward.However, some evidence suggests that dopamine is not essential for basic reward related behaviors. Even with diminished dopamine transmission, animals still like food, still eat, and can still learn about rewards. Dopamine is not at the root of these functions; rather, it is activation of D2 receptors facilitates flexible behavioral responding and appears necessary for restoring sensitivity to changes in outcome value; that is, necessary for updating value associated with actions and subsequent behavior. Similar to Nelson, Barker found, conversely, that antagonism of D1 facilitated flexibility. These studies, together, suggest that D1 may facilitate exploitation of prior learning while D2 may promote behavioral flexibility. Klanker et al. (The papers in this research topic highlight several themes relevant to viewing dopamine as a neurotransmitter that has evolved to promote flexibility in the service of appetitive pursuit. Fundamental to behavioral flexibility is the ability to strike the right balance between exploiting acquired knowledge and exploring to update one's knowledge, the so-called explore-exploit dilemma, which dopamine may regulate (Beeler et al., r et al.  systematr et al.  report aOf course, the dopamine system is not monolithic. Although we may abstract general themes, as above, other papers in this topic remind us that dopamine contributes to a mosaic of functions. In a review of its role in prefrontal function, Floresco  challengBaudonnat et al.  take a sfear to signaling safety, once the animal learns to avoid the aversive stimulus, engaging dopamine incentive learning.Associated primarily with signaling positive, rewarding outcomes, what role does dopamine play in altering behavior in response to deleterious outcomes? Oleson and Cheer  review rFinally, returning to pathology in the dopamine system, Cepeda and Levine (Chen et al., id of neurotransmitters, dopamine emerges more as the brain's chief comptroller in energy allocation (Beeler et al., The traditional view of dopamine as the “reward neurotransmitter” has, in recent years, been gradually evolving. Through the work of many, dopamine is increasingly framed in neuroeconomic, decision-making terms (e.g., Gan et al.,


There was an error in affiliations 1, 3, and 4 for authors Jonathan Maelfait, Kenny Roose, Pieter Bogaert, Mozes Sze, Xavier Saelens, Isabelle Carpentier, Geert van Loo, and Rudi Beyaert. Affiliation 1 should be: Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB, Ghent, Belgium. Affiliation 3 should be: Unit of Molecular Virology, Department for Molecular Biomedical Research, VIB, Ghent, Belgium. Affiliation 4 should be: Cell Culture and Sorting Core Facility, Department for Molecular Biomedical Research, VIB, Ghent, Belgium.


High Content Screening (HCS) has become an important tool for toxicity assessment, partly due to its advantage of handling multiple measurements simultaneously. This approach has provided insight and contributed to the understanding of systems biology at cellular level. To fully realize this potential, the simultaneously measured multiple endpoints from a live cell should be considered in a probabilistic relationship to assess the cell's condition to response stress from a treatment, which poses a great challenge to extract hidden knowledge and relationships from these measurements.in vitro HCS assays and related to the findings to in vivo histopathological observations. We measured multiple HCS assay endpoints for 122 drugs. Since LDA requires the data to be represented in document-term format, we first converted the continuous value of the measurements to the word frequency that can processed by the text mining tool. For each of the drugs, we generated a document for each of the 4 time points. Thus, we ended with 488 documents (drug-hour) each having different values for the 10 endpoints which are treated as words. We extracted three topics using LDA and examined these to identify diagnostic topics for 45 common drugs located in vivo experiments from the Japanese Toxicogenomics Project (TGP) observing their necrosis findings at 6 and 24 hours after treatment.In this work, we applied a text mining method of Latent Dirichlet Allocation (LDA) to analyze cellular endpoints from We found that assay endpoints assigned to particular topics were in concordance with the histopathology observed. Drugs showing necrosis at 6 hour were linked to severe damage events such as Steatosis, DNA Fragmentation, Mitochondrial Potential, and Lysosome Mass. DNA Damage and Apoptosis were associated with drugs causing necrosis at 24 hours, suggesting an interplay of the two pathways in these drugs. Drugs with no sign of necrosis we related to the Cell Loss and Nuclear Size assays, which is suggestive of hepatocyte regeneration.in vitro assays along with an in vivo histological finding, necrosis. Effectiveness of this approach may add substantially to our understanding of systems biology.The evidence from this study suggests that topic modeling with LDA can enable us to interpret relationships of endpoints of  In vitro assays, such as High Content Screening (HCS) methods, have become an important tool for safety screening. HCS has been actively evaluated for use in drug discovery due to the advantages of being high-throughput and requiring less physical material for testing.Toxicity screening is an essential step in drug development since safety concerns have been one of the main causes of bottlenecks before drug approval ,2. In viUnlike conventional cytotoxicity assays, HCS offers the promise of understanding the biological functions underlying toxicity by simultaneously testing various cellular activities in live cells . Providiet al. reported an HCS assay based on the HepG2 cell line and paired the HCS endpoints with the conventional in vitro cytotoxicity assay in a one-to-one comparison to assess the human hepatotoxicity of the tested drugs [et al. generated eight cellular measurements in an HCS assay based on rat primary hepatocytes and employed a Boolean logical OR to indentify individual endpoints with high predictivity for clinical drug-induced liver injury [HCS has notable advantages over traditional cytotoxicity assays because it measures multiple cellular endpoints simultaneously so that it captures a more complete and dynamic picture of cellular response to an insult. We hypothesized that these endpoints together indicate the cell's condition under stress responding to a treatment in a probabilistic relationship. Such a characteristic can not be accurately described by most of the common approaches such as clustering or PCA and should be modeled with a Bayesian relationship. Unfortunately, most, if not all, post-experiment analysis often involves building discriminative models that use each read-out assay  as an independent feature. Specifically, these data analyses treat individual endpoints as independent features rather than observing their interdependencies in a probabilistic relationship . For exaed drugs . Likewisr injury . As promIn order to best use the multi-parameter measurements of live cells that HCS assays provide, a statistical analysis method must have the capability to extract hidden knowledge and relationships from these measurements. The best way to address this issue is to adapt a systems approach that would not only model relations between endpoints, but also link such a relationship to elucidate the cellular events leading to toxicity . For thiWe used Latent Dirichlet Allocation (LDA)  for topiin vivo) study. Our study demonstrated the utility of topic modeling, including the innate properties of the assay, to interpret the HCS results and thus reach a better understanding of the toxic response. The results indicate that endpoints under significant topics corresponded to the cellular mechanisms involved in the progression of hepatocellular necrosis in vivo as well as recovery from liver injury. This proof-of-concept study demonstrates that topic modeling has the potential to model biological data beyond simply text documents to exploit the relationships of assay endpoints.In this work, we built a topic model using LDA for rat primary hepatocyte-based HCS assays to investigate the relationship of the cellular level response to the drug treatment observed in this assay and the liver injury related necrosis observed in the whole animal (® profile). Eight endpoints, Cell loss, Nuclear Size, DNA Damage, Apoptosis, Lysosomal Mass, DNA Fragmentation, Mitochondrial Potential, and Steatosis were measured simultaneously in populations of cultured rat primary hepatocytes at multiple time points to profile both the potency and specificity of the cellular toxicological responses [et al. [A set of compounds with a wide range of known mechanisms of action was chosen to test the range of detection of the mechanistic profiling assays applying the cellular systems biology (CSB™) approach .The necrosis data used in this study was obtained from the Japanese Toxicogenomics Project (TGP). Details regarding the animal study protocol are available elsewhere ,26. BrieThe HCS assays included 8 endpoints  that were observed for 1, 6, 24, and 48 hours after treatment of 122 drugs  in that document. The analogy we carried out here allowed us to use 10 endpoints as a vocabulary and construct different profiles (documents) for every time step of each compound  with parameter θ. To make the likelihood estimations computationally tractable, θ is assumed to follow a Dirichlet distribution which is the conjugate prior of multinomial distribution. Therefore a hyperparameter, α, is introduced and θ is sampled from Dir(α). Similarly, word distributions are also controlled by another multinomial distribution for given topics and parameters having Dirichlet distribution which takes β as another hyperparameter. For a document which has N words, this generative process can be concisely presented as follows [In this study, we used Latent Dirichlet Allocation (LDA) as a topic modeling tool, which is an improved version of earlier models ,29 and a follows :θ ~ Dir(α).1. Choose n wwhere 2. For each of the words n ~ Mult(θ)a. Choose a topic zn wfrom another multinomial distribution that is conditioned on the topic zn and a prior β. i.e., pb. Choose a word A distinguishing feature of LDA is that it can assign an unseen document to discovered topics. Furthermore, it provides interpretable conditional probability tables (CPT) for aforementioned associations such as document-topic (p(topic|document)) and word-topic (p(word|topic)). CPTs not only give the mixture weights of topics for given documents, but also tell how likely it is that a word comes from a given topic.diagnostic topics [k. Secondly, each class (c) mean for topic k, k. If there are C categories, the following expression gives the significance score of topic k, for a class c.CPTs can be used to measure the document-topic associations. These probabilistic values can also be used to rank the most probable topics. However, there is a need to measure c topics  for a clc topics . The metCxK matrix in which rows are the scores for topics and we declare k* as the diagnostic topic for a group c if Finally, we have a in vitro findings with in vivo study in order to better understand the mechanisms underlying toxic insult. Importantly, the concordance of the in vivo and in vitro results is a key indicator of potential to replace and reduce the animal uses in assessing risk of a broad range of medical and pharmaceutical products. Implementation of the model on HCS data was followed by analyzing 45 compounds whose histopathological data and HCA data available for this study, where the histopathological data was obtained from the TGP database while the HCS study was conducted by a commercial vendor , we set the number of topics equal to three for the vocabulary of 10 endpoints (terms). Next, using the implementation by Blei et al. , we devet) not only grouped the endpoints based on their distribution across drugs, but also provided an importance measure with the probability scores of endpoints (e) for given topics, p(e|t). More specifically, p(e|t) indicates which endpoints are more important for that particular topic. In Table The topics (p(e|t) values. Namely, we compared three probabilities p(e|Topic 1), p(e|Topic 2), and p(e|Topic 3) for each e and assigned it to the topic with the highest probability. In doing so, we identified groups of terms with the highest association to their topics and underlined them in Table Each topic contains a ranked list of 10 endpoints, but these can be separated into disjoint groups by comparing pe|Topic , and p to multiple topics where they can be sorted based on probabilities. In other words, every topic consists of the same endpoints with different orders and endpoints are not forced to be assigned to a single cluster as it happens in Topic 1 was assigned as the diagnostic topic for necrosis at 6 hours, and was associated with the endpoints Steatosis, DNA Fragmentation, Mitochondrial Potential, and Lysosome Mass. Changes in DNA Fragmentation are characteristic of necrotic cell death, where the presence of 5' overhangs are seen, and changes in Lysosome Mass and Mitochondrial Potential are consistent with the changes to cell ion permeability that eventually lead to cell rupture ,35. SteaTopic 2 was associated with DNA Damage and Apoptosis, which were diagnostic for necrosis at 24 hours. The longer time after exposure reveals the difference between more and less rapid-acting compounds. These results are somewhat perplexing given the differing pathological mechanisms underlying necrosis and apoptosis, although they do share common features such as membrane potential dysregulation ,37. HoweCell Loss and Nuclear Size showed a highly significant connection with Topic 3, which was an indicator of non-necrosis drugs as shown in Table in vitro measurement in some pre-lethal endpoints including Steatosis, DNA Fragmentation, Mitochondrial Potential and Lysosome Mass, while the drugs that caused necrosis after 24 hours were associated with the cellular events in DNA Damage and Apoptosis. Obviously, the former observation in the cellular assay seems to reveal more acute injury, while the latter one reflects the cell death that might correlate the necrosis in rats observed even after 24 hour treatment. In other words, our incorporation of in vivo histopathology data over time with data from HCS agreed with the conventional wisdom regarding the cause of toxic necrosis.All endpoints measured in the HCS assay here are essential for toxicity assessments. In that sense, besides an independent analysis or a pair-wise comparison, it is important to interpret how these events lead to toxicity. For this reason, we not only used LDA to retrieve probabilistic associations of each endpoint to topics, but also incorporated a histopathological assessment of necrosis to test whether there is any biological meaning hidden behind these topics. For example, the drugs that caused necrosis in rats after 6 hours of treatment were also observed with significant changes of in vitro and in vivo data should be considered complements of each other. One of the goals here was to make use of these two data types to reveal biological facts. Besides using a novel computational tool to analyze HCS, we provide an example of a way to efficiently bridge in vivo and in vitro data by means of topic. By using this intermediate variable, we were able to correlate histopathological findings with the results from the HCS assays. The ability of the model to discover patterns with an unsupervised nature indicates its potential to be an alternative approach for analyzing HCS data. Hence, one direct application of this methodology for us is the early detection of drug-induced liver injury by interpreting the HCS content under probabilistic measures for drugs and endpoints. We intend to apply this method to predict the DILI potential of drugs by not only considering a single endpoint, but relying on the full set of data generated by HCS.Although they involve different practices and meanings, in vitro data. Further, discovered relations were analyzed along with corresponding in vivo data. The results showed that Latent Dirichlet Allocation (LDA) could improve the interpretation of HCS data for use in systems biology. The agreement we observed between in vitro and in vivo data through topics obtained by LDA provide early evidence for the effectiveness of this strategy.We have presented here a systems approach that is capable of integration of multiple measurements from High Content Screening (HCS) by considering the interdependencies across endpoints. By analogy with text mining, endpoint distributions and proportions across topics were used to gain insight into the content of The findings and conclusions in this article have not been formally disseminated by the US Food and Drug Administration (FDA) and should not be construed to represent the FDA determination or policy.The authors declare that they have no competing interests.HB performed all calculations and data analysis, and wrote the first draft of the manuscript. WT and XX had the original idea, developed the methods, and guided the data analysis and presentation of results. MC, YW, RK, and HF contributed to the data analysis, verified the calculations, and assisted with writing the manuscript. All authors read and approved the final manuscript.Click here for fileClick here for fileClick here for file


Reactive hyperplastic lesions of the oral connective tissue are associated with injuries of soft tissue and have high prevalence rates and different involvement patterns in different parts of the world. The aim of this study was to analyze demographic data of a university department.Patient records of the Department of Oral Pathology during a four-year period were reviewed for diagnosis of oral connective tissue reactive hyperplastic lesion. Data including the area involved and the type of lesion were collected and analyzed using descriptive statistical methods and t-test with SPSS 15 statistical software.197 cases  matched study criterion. The most common affected site was gingiva (83.9%) and the most common lesion was fibroma (45.2%). Giant cell granuloma and pyogenic granuloma were more common in the maxilla than in the mandible. Pyogenic granuloma was seen to be equally distributed in males and females. The results of the present study are overall consistent with the findings of previous studies. The results of a study carried out by Katsikeris et al4 on 224 new peripheral giant cell granulomas and 959 reported instances in papers indicated that the incidence of this lesion is more common in males and in the mandible and is often detected in 40-60 year-olds. Auclair et al5 studied 25 central giant cell granuloma cases and indicated that this lesion occurred most in females with an average age of 21. In the study of Jalayeri et al,6 peripheral giant cell granuloma was more prevalent in the forth decade of life and in the mandible, and the central type was more common in females, in the second decade, and in the mandible.It seems the prevalence of reactive hyperplastic lesions of oral connective tissue differs according to type of lesion, age, gender, and affected site. In a study carried out by Gungormus & AkgulReview of the literature reveals that there are controversies among the findings of previous studies, and that all reactive hyperplastic lesions have rarely been evaluated in one study. The aim of this survey was to evaluate oral connective tissue reactive hyperplastic lesions referring to a university department in the north-west of Iran during a four-year period and to compare the results with those of similar studies.This retrospective cross-sectional study was performed on the archives of the Department of Oral Pathology at Tabriz University of Medical Sciences during the period between 2004 and 2008. Patient records were assessed to select those with the diagnosis of reactive hyperplastic lesions. Data including the type of the lesion, age, gender, and the affected site were collected using prepared forms. Descriptive statistical methods  were applied to data, and t-test was employed to assess mean differences using SPSS 15 statistical software.From a total of 412 records evaluated, 197 (48%) of the lesions were reactive hyperplasia. Of these, 124 (62.8%) cases were females  and 73 (37.2%) cases were males , with a total mean age of 37.68 ± 18.97 years . There was no statistically significant difference in mean age between genders (p=0.13). The most common lesion was fibroma with 80 cases (40.7%) . Gingiva7 reactive hyperplastic lesions were more common in females , and the mean age of females was higher than that in males. In addition, in a study carried out by Al-khatten,8 the mean age of females was higher than that of males, and the most commonly affected site was gingiva (83.6%). The lesions in the order of most prevalent were as follow: giant cell granuloma (64%), fibroma (40.7%), peripheral ossifying fibroma (40%), pyogenic granuloma (21.3%), and epulis fissuratum (1%); generally, these lesions were more common in the maxilla than in the mandible.8 The most common site of pyogenic granuloma is gingiva, which is more prevalent in the maxilla than in the mandible.2 This is consistent with our results and the results of studies carried out by Saravan7 and Zarei.9This survey included all types of oral connective tissue reactive hyperplastic lesions in an oral pathology department in north-west of Iran. In most areas evaluated, our results were similar to the results of others studies. However, it is remarkable that, giant cell granuloma was more common in the maxilla and pyogenic granuloma was equally distributed in males and females. Oral connective tissue reactive hyperplastic lesions were more prevalent in females than males . In a study carried out by Zarei et al,1 In the current study, giant cell granuloma was a little more common in the maxilla than in the mandible, which is consistent with the results of a study carried out by Shamim et al.10Giant cell granuloma is a more prevalent in the mandible than in the maxilla.10 fibroma was also more commonly found in the mandible, consistent with the results of our study.In the current study, fibroma was more common in the mandible than in the maxilla. In the study carried out by Shamim et al,13 In the present study, male-to-female ratio was 1:1.4. In a study carried out by Motamedi et al14 the prevalence of peripheral giant cell granuloma was the same in both genders.Peripheral giant cell granuloma is often more common in females than in males.9 oral pyogenic granuloma was more common in females than in males. Zhang et al6 found the most common gingival reactive lesion to be peripheral fibroma. In the present study, fibroma was more common in females than in males, which is consistent with a previous study.13In a study carried out by SaravnaReactive hyperplastic lesions of the oral connective tissue are more common in females and the majority of the lesions occur in gingiva. Giant cell granuloma and pyogenic granuloma are more common in the maxilla whereas irritation fibroma is more commonly found in the mandible. The mean age of female patients is higher than that of males.


Adaptive motivated behavior requires rapid discrimination between beneficial and harmful stimuli. Such discrimination leads to the generation of either an approach or rejection response, as appropriate, and enables organisms to maximize reward and minimize punishment. Classically, the nucleus accumbens (NAc) and the dopamine projection to it are considered an integral part of the brain’s reward circuit, i.e., they direct approach and consumption behaviors and underlie positive reinforcement. This reward-centered framing ignores important evidence about the role of this system in encoding aversive events. One reason for bias toward reward is the difficulty in designing experiments in which animals repeatedly experience punishments; another is the challenge in dissociating the response to an aversive stimulus itself from the reward/relief experienced when an aversive stimulus is terminated. Here, we review studies that employ techniques with sufficient time resolution to measure responses in ventral tegmental area and NAc to aversive stimuli as they are delivered. We also present novel findings showing that the same stimulus – intra-oral infusion of sucrose – has differing effects on NAc shell dopamine release depending on the prior experience. Here, for some rats, sucrose was rendered aversive by explicitly pairing it with malaise in a conditioned taste aversion paradigm. Thereafter, sucrose infusions led to a suppression of dopamine with a similar magnitude and time course to intra-oral infusions of a bitter quinine solution. The results are discussed in the context of regional differences in dopamine signaling and the implications of a pause in phasic dopamine release within the NAc shell. Together with our data, the emerging literature suggests an important role for differential phasic dopamine signaling in aversion vs. reward. Since Olds and Milner’s  seminal Our behavior is potently modified by both beneficial and harmful outcomes. Inappropriate affective responses are hallmarks of many psychiatric disorders including depression, bipolar, and other mood disorders. For example, in animal models drug-addicted rats will continue to respond for drug even if they must simultaneously endure a foot shock that would normally be considered aversive  and suppression of a behavior that would lead to an aversive event (absence of punishment). Additionally, omission of an expected reward, disappointment, and subsequent extinction of behavior, can also be dissociated from aversion as defined here, and these events may invoke a different set of learning mechanisms , we will not discuss these data. Instead, we refer the interested reader to excellent reviews on stress, dopamine, and NAc  and VTA project to dorsal and ventral striatum, respectively. In the vast majority of studies made in primate and rodent subjects, during reward-related stimuli – e.g., presentation of primary reward, reward-predictive cues, and during reward-directed actions Schultz,  – these Relative to the reward literature, there are far fewer examinations of the dopamine neuron response to aversive events. In the studies that have been conducted with aversive stimuli, outcomes are much less uniform than seen when reward-related stimuli are used. As such, aversive events are commonly shown to have both excitatory and inhibitory effects on the firing of midbrain dopamine neurons. These studies are reviewed in Table Fluctuations in dopamine concentration in dopamine terminal regions overcome some of the limitations of recording neural activity in the ventral midbrain. There is no controversy surrounding the identity of the compound studied when microdialysis or fast-scan cyclic voltammetry are used. Microdialysis, though, lacks the sampling resolution required to resolve changes in dopamine evoked by discrete aversive stimuli. Only a handful of studies have employed fast-scan cyclic voltammetry to measure fluctuations in dopamine concentration evoked by aversion Table  Roitman and are n = 15) were used. Two cohorts were dedicated to the CTA experiment and were divided into Paired (n = 5) vs. Unpaired (n = 5) groups. A third group received intra-oral infusions of quinine as a comparison (n = 5). All rats were singly housed under standard housing conditions. Food and water were available ad libitum throughout the experiment. Surgical procedures were identical to Roitman et al. . The data clearly demonstrate that while both groups of rats had equal exposure to sucrose and LiCl, a CTA was established only in Paired rats. Importantly, taken together with dopamine concentration fluctuations, the data establish that in this paradigm, the NAc shell dopamine response matches the hedonic value of the stimulus and, when aversive, the taste stimulus suppresses phasic dopamine signaling.Conditioned taste aversion rats received a session of sucrose infusions and their orofacial responses were analyzed. In Paired rats, sucrose infusions evoked predominantly negative orofacial movements whilst sucrose evoked predominantly positive orofacial movements in Unpaired rats Figure . These dElectrophysiological recordings from dopamine neurons suggest a heterogeneous response to aversive stimuli – with some studies supporting mostly inhibitory responses . D2 receptors are high affinity (Richfield et al., Future work must address the mechanisms by which aversive stimuli in general, and taste stimuli specifically, suppress phasic dopamine signaling. Recent publications have focused on this question. Local GABA neurons that suppress the firing rate of VTA dopamine neurons are excited by foot shock in anesthetized rats (Tan et al., Here, we have reviewed literature and presented novel findings detailing the effect of brief aversive stimuli on the neuronal responses of midbrain dopamine neurons and dopamine release in terminal regions. Our data show that in one of these projection sites, NAc shell, the response to aversive stimuli is uniformly a suppression of spontaneous dopamine release. Importantly, the stimuli used were presented in the same modality as rewarding stimuli, which evoke increases in dopamine release. Future work will determine whether these patterns hold true for other projection regions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular imaging emerging in the early 21st century has been rapidly developed. Over more than 10 years, optical molecular imaging has become an important tool in preclinical research and has been translated into the clinical for clinical diagnosis and therapy. Computational methodology has shown its indispensable position in developing molecular probes, building imaging instruments, researching reconstruction algorithms, and studying signal and image processing. Relevant research not only adapts existing computational methods but also develops novel strategies to tackle with challenging problems in optical molecular imaging.This special issue is devoted to the topic of computational algorithms and strategies in optical molecular imaging. Five exciting papers assembled in this special issue present a common theme, that is, improving the imaging and image processing performance in optical molecular imaging by using computational techniques. The papers address several problems in optical molecular imaging, including optical tomographic reconstruction for bioluminescence tomography and image processing such as image segmentation, 3D rendering, and cell morphological measurements. In detail, J. Yu et al. make use of a hybrid multilevel adaptive finite element reconstruction scheme and sparse regularization to acquire quantitative information of bioluminescent sources. The work of Q. Wu et al. is to take advantage of a high-order photon propagation model and Bregman optimization algorithm to improve bioluminescence tomography. The morphology information of living cells and 3D tissue images at the micrometer level can be obtained by using existing computational algorithms from the developed digital holographic microscopy and multichannel spectral imaging laser scanning confocal microscope in the research progress of Y. Wang et al. and Y. Zhang et al., respectively. J. Zheng et al. report that retinal vessels are also efficiently extracted with multiscale hessian-enhancement-based nonlocal mean filter.


Laricobius spp. (Coleoptera: Derodontidae) predators use to locate suitable habitats and prey, which limits our ability to collect and monitor them for classical biological control of adelgids (Hemiptera: Adelgidae). The aim of this study was to examine the visual ability and the searching behavior of newly emerged L. nigrinus Fender, a host-specific predator of the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Phylloxeroidea: Adelgidae). In a laboratory bioassay, individual adults attempting to locate an uninfested eastern hemlock seedling under either light or dark conditions were observed in an arena. In another bioassay, individual adults searching for prey on hemlock seedlings (infested or uninfested) were continuously video-recorded. Beetles located and began climbing the seedling stem in light significantly more than in dark, indicating that vision is an important sensory modality. Our primary finding was that searching behavior of L. nigrinus, as in most species, was related to food abundance. Beetles did not fly in the presence of high A. tsugae densities and flew when A. tsugae was absent, which agrees with observed aggregations of beetles on heavily infested trees in the field. At close range of prey, slow crawling and frequent turning suggest the use of non-visual cues such as olfaction and contact chemoreception. Based on the beetles' visual ability to locate tree stems and their climbing behavior, a bole trap may be an effective collection and monitoring tool.Very little is known about the searching behavior and sensory cues that  Laricobius Rosenhauer are arboreal predators of adelgids (Hemiptera: Adelgidae), a prey that feeds on sap or stored starches of trees in the Pinaceae family  in the genus e family . The DerL. nigrinus is a host-specific predator of A. tsugae (Tsuga heterophylla (Rafinesque) Sargent   Carrière. To evaluate L. nigrinus as a biological control agent, life-history studies were conducted showing that they could be effective  . In the troduced  and has ffective . L. nigrablished .L. nigrinus adults feed in fall, winter, and early spring on their primary prey, A. tsugae, which are sessile at the base of needles. Females lay eggs in adelgid ovisacs (egg clusters) from late winter to early spring, and eclosing beetle larvae are encircled by eggs, their preferred prey stage. Late instars may search twigs for eggs or nymphs if all the eggs in an ovisac are eaten, as is the case for L. erichsonii  in late winter. Rearing conditions followed standardized protocols (Adult rotocols .A. tsugae (75% of needle cushions infested) were collected from locally infested forests in Giles Co., Virginia.The following fall, after aestival diapause, newly emerged adults were individually held in Petri dishes on wet filter paper and held at 15° C and 12:12 L:D for up to 24 h before each bioassay. Eastern hemlock seedlings with high densities of 2 surface area (15 cm diameter) in a climate controlled room at 9.7–12.9° C and 42–78% RH. The arena size was deemed adequate given the small size of the beetles (2–3 mm length). An uninfested hemlock seedling  was positioned in the middle of the arena. Although beetles also encounter saplings and mature trees in the field, we did not attempt to mimic these trees in the laboratory. A border of white paper covered in fluon was placed along the perimeter of the arena and the arena was covered in beige-colored sand. Individual L. nigrinus adults were placed in the arena (7 cm from the seedling) and recorded as locating the seedling or not during an arbitrary 10-minute time period under either light (25W soft-white light bulb to mimic daylight) or dark (25W red light bulb to mimic night) conditions (n = 20). Beetles were randomly assigned to the light treatment and not reused. Beetle sex was not determined. The same bioassay was repeated in winter to confirm results of the fall experiment. The beetles in winter had fed on A. tsugae for two months in rearing containers.The experimental arena was a soil filled pot with a 177 cmA. tsugae-infested and uninfested hemlock seedlings (n = 10) in the laboratory using the same experimental arena, similarly sized seedlings, and environmental conditions described previously. First, an ad libitum observation of 13 beetles on the seedlings was conducted to catalogue behaviors. Nine behaviors were defined for examination during the bioassay  that was linked to a desktop computer to transfer, archive, and watch the video , flew off the seedling, or 30 min had expired. From the footage, the frequency of each behavior (bouts), elapsed time until each behavior began (latency) and the total time length of each behavior, when applicable, were recorded in seconds. Beetles were randomly assigned the seedling treatment and not reused. Beetle sex was determined after each bioassay by protraction of the genitals.Prey searching behavior was studied on bioassay . Individhe video . The camThe visual cue bioassay data were arranged in a 2 × 2 contingency table to test if the proportion of beetles locating trees in either light or dark was similar (using Yate's correction for continuity). To determine if results from the fall and winter bioassays were homogenous, a heterogeneity chi-square analysis was conducted (using Pearson's Chisquare).A. tsugae-infested and uninfested seedlings by non-parametric Mann-Whitney U tests (0.05 significance level). All data are presented as standard error of the mean. In addition, the number of times a beetle switched from one behavior to another was tallied and behavioral sequence diagrams were created from the pooled activity of ten individual beetles on each treatment using Microsoft PowerPoint. Arrows were used in the diagrams to indicate the sequence of behaviors; their font size indicates the relative frequency of behavioral flow.For the prey searching behavior bioassay, the bouts, latency, and total time length for each behavior, when applicable, were compared between L. nigrinus adults locating trees .In fall, light had an effect on the probability of L. nigrinus behavioral footage on hemlock seedlings were analyzed to the nearest second. Each treatment had 1:1  sex ratios. On the A. tsugae-infested seedlings, 100% of the beetles ultimately fed . Use of vision by L. nigrinus, at least at close range, was not a surprising result in the visual cue bioassay as insectivores typically rely on more than one sensory modality . Likewise, of trees . Whether of prey . Crawlin of prey , Syrphid of prey . General of prey . LaricobA. tsugae” on infested trees is analogous to “local convoluted search” (L. nigrinus study. Flowers et al. (A. tsugae. When beetles were among A. tsugae they crawled slowly, turned frequently, and appeared to antennate and probe the white flocculence with their maxillary and/or labial palps (A. tsugae generations onto branches, boles, and the forest floor and could cue beetles that prey may be present nearby. Other close range nonvisual cues, such as olfaction, may be relied upon to locate A. tsugae. Mechanosensory cues are doubtful, because A. tsugae is sessile, although nymphs and adults fidget slightly when disturbed.In the prey searching behavior bioassay, “evaluating  search”  and “ints et al.  found thal palps . Contactal palps , palps, L. nigrinus searching behavior, like most species, is related to food abundance. Beetles did not fly when A. tsugae is abundant and flew readily when A. tsugae was absent. This dispersal is consistent with field studies that show a positive correlation between A. tsugae density and adult beetle density (The primary finding of this study is that  density , resulti density  may be e


In addition, IPSIM could also be used to help make diagnoses on commercial fields. It is important to point out that the presented concepts are not crop- or pest-specific and that IPSIM can be used on any crop.The limitation of damage caused by pests  in any agricultural crop requires integrated management strategies. Although significant efforts have been made to i) develop, and to a lesser extent ii) combine genetic, biological, cultural, physical and chemical control methods in Integrated Pest Management (IPM) strategies , there is a need for tools to help manage Injury Profiles . Farmers design cropping systems according to their goals, knowledge, cognition and perception of socio-economic and technological drivers as well as their physical, biological, and chemical environment. In return, a given cropping system, in a given production situation will exhibit a unique injury profile, defined as a dynamic vector of the main injuries affecting the crop. This simple description of agroecosystems has been used to develop IPSIM (Injury Profile SIMulator), a modelling framework to predict injury profiles as a function of cropping practices, abiotic and biotic environment. Due to the tremendous complexity of agroecosystems, a simple holistic aggregative approach was chosen instead of attempting to couple detailed models. This paper describes the conceptual bases of IPSIM, an aggregative hierarchical framework and a method to help specify IPSIM for a given crop. A companion paper presents a proof of concept of the proposed approach for a single disease of a major crop (eyespot on wheat). In the future, IPSIM could be used as a tool to help design  Third millennium agriculture must reconcile environmental protection and productivity. The world population is projected to reach 8.7–10 billion by 2050 and annual_production will need to increase by 200 million tons by then to meet the projected 470 million ton demand True IPM is quite different from the practices recommended up to now sensu lato have been developed to represent pest dynamics, often to help decision making for pesticide treatments. However, these models usually take into account rather poorly the critical effects of cropping practices In order to help design cropping systems, modelling is a key tool ex-ante or ex-postIPSIM is embedded in http://www-ai.ijs.si/MarkoBohanec/dexi.html). DEX is a method for qualitative hierarchical multi-attribute decision modelling and support based on a breakdown of a complex decision problem into smaller and less complex sub-problems. This tool is generally used to evaluate and analyse decision problems IPSIM was developed using the DEX method, and is implemented with the DEXi software ; escape strategies through the choice of the sowing date  and mitigation through crop status . The genetic control represents the level of resistance of the cultivar (or the cultivar mixture) to the considered pest. For some pests, biological control can be applied using living organisms released at the field or greenhouse scale. Physical control consists of using any mechanical, thermal, or electromagnetic actions to limit the pest population. Finally, the attribute “Chemical control” describes the efficacy of pesticide treatments and/or use of non-lethal chemicals such as pheromones or repellents. The effect of soil and climate are described independently and later aggregated in a “Soil and climate” attribute. Finally, the effects of elements  at the territory level are taken into account by describing sources of primary inoculum and beneficials at the territory level, as well as the presence of physical barriers that might limit these interactions between the considered field and its surrounding environment. Harmless living organisms  are not specifically represented in the model.The input attributes of IPSIM describe cropping practices, soil and climate , and biological interactions at the territory level . Figure The scales and the aggregating tables used for the attributes presented in sensu lato in a given field and its dispersal ability. Pests with a high level of persistence and low dispersal ability are highly endocyclic. Pests with a low level of persistence are slightly endocyclic, regardless their dispersal ability. Pests with a high level of persistence and a high dispersal ability are moderately endocyclic. The inoculum produced by an endocyclic pest in one season can be carried over to the next, thus building up a cumulative inoculum reservoir over the years. Endocyclic organisms are thus highly dependent on field history. The categorisation of pests into two groups (high/medium and low levels of endocyclism) can help identify the main level to address to control them: the field or territory level.So far, IPSIM was presented as a simulator of the severity levels for single pests interacting in an injury profile . This deMeloidogyne spp.) on horticultural crops, wireworms on potato (Agriotes spp.), wheat common bunt (Tilletia spp.), take-all on wheat (Gaeumannomyces graminis var. tritici), dicotyledonous weeds such as Chenopodium album and Fallopia convolvulus are highly endocyclic pests. However, highly endocyclic pests can sometimes be spread to other fields by anthropic activities . This dispersal mechanism will not be taken into account in the model. Aphids on several crops (e.g. Brevicoryne brassicae), powdery mildew on grapevine (Erysiphe necator), rusts on cereals (e.g. Puccinia recondita), codling moth on apple tree (Cydia pomonella), and weeds such as some Asteraceae (e.g. Taraxacum dens leonis) or grassy weeds (e.g. Bromus sterilis) are slightly endocyclic pests.For example, root-knot nematodes  highly/moderately and ii) slightly endocyclic pests . ConsideOculimacula yallundae and O. acuformis, anamorph Pseudocercosporella herpotrichoides is considered to be the most important stem-base disease of cereals in temperate countries. In France, sharp eyespot, another soil-borne fungus caused by Rhizoctonia cerealis, is one of the minor diseases of the foot disease complex of winter wheat, but is thought to interact strongly with eyespot. The two pathogens show distinct antagonistic behaviour within the infected stem base, which translates into a negative correlation between sharp eyespot and eyespot incidence Puccinia triticina, is the most common rust disease of wheat and is now recognised as an important pathogen in wheat production worldwide, causing significant yield losses over large geographical areas This article aims to present the whole modelling process: i) development of a conceptual framework; ii) implementation of this conceptual scheme into a simulation model for a simple case; iii) simulation to exemplify potential uses of IPSIM models. The specification of IPSIM will be performed for a simple injury profile on wheat: two highly endocyclic diseases (eyespot and sharp eyespot) and a slightly endocyclic disease (brown rust). Eyespot, caused by the necrotrophic and soil-borne fungi The design of IPSIM-Wheat-Eyespot and the evaluation of its predictive quality is described in a companion paper The use of the model presented in the first sub-section of the “results” section is exemplified for three contrasting cropping practices in a given production situation . The thrThe DEXi software computed the aggregated attribute values of the model presented in the first sub-section of the “results”. In the same production situation, the three cropping practices led to contrasting injury profiles. In the absence of estimates of potential yield losses caused by these injury profiles, it is difficult to provide direct recommendations for cropping practices adaptations. However, these simulations enable a diagnosis in terms of pest development for the three simulated systems. The intensive system led to IP4, i.e. a medium final injury level for highly endocyclic pests associated with a low final injury level for slightly endocyclic pests . For thiex-ante the performance of various cropping systems with regard to the control of pest injury on a given crop. This information is useful when designing innovative cropping systems, either by prototyping, e.g. in silico for two major reasons. First, crop damage is not simulated by IPSIM, which makes it difficult to rank pests with respect to the crop losses they cause. Second, the social, economic and environmental performance of the simulated cropping systems are not calculated. To tackle this problem, IPSIM could be coupled to a damage model  can be built into IPSIM, offering a framework for these various communities to interact and combine their knowledge.IPSIM could also be used in an i.e. cropping practices, soil and climate, field environment, injury profiles), along with the social, economic and environmental performances of the monitored agroecosystems. It is important to also add measurements of state variables characterising the crop status  in order to better describe important state variables of the agroecosystem for other possible future analyses of the created datasets. However, due to the lack of datasets containing a description of injury profiles, the confidence that users may have in IPSIM models could also be enhanced by comparing simulation outputs with their own expertise to identify any mismatches. All the information contained within IPSIM models is held in the hierarchical trees and the associated aggregating tables. One of the consequences of this specificity of models developed with the IPSIM framework is that, once developed, the predictive quality of the models can be enhanced easily using experimental datasets by modifying aggregating tables, and, if need be, the structure of the model.Like any other model, the predictive quality of IPSIM should be assessed prior to its use. This highlights the urgent need to collect data in commercial fields describing the input and output variables of IPSIM  or indirectly (though modification of the biotope). This implies that not all potential theoretical injury levels could occur simultaneously. This is a limitation of IPSIM which does not account for the impact of injuries on crop growth. Secondly, the soil, climate, cropping practices and landscape occurring in a given territory might not be diverse enough to lead to all theoretical injury levels . Another limitation of IPSIM is the way that interactions between pests are represented. If n pests are considered, n (n-1) interactions are to be described. This is similar to the three-body (or n-body) problem in physics, which has a global analytical solution in the form of convergent power series From the conceptual viewpoint, it could be asked why the crop which is entered in the field biological component  does notWe recommend to develop models with no more than 7 final injury levels for a single pest. The lack of precision of IPSIM models could be seen as a drawback as compared to quantitative epidemiological models. Firstly this is because these latter models address a much simpler system: a single pest, rather than an injury profile. Secondly, when developing models of complex systems, accuracy should be sought rather than precision. Searching for better precision would certainly lead to an increase in the model’s complexity and possibly to a dead end. We believe that the proposed precision of the models that will be developed with the IPSIM framework is more than enough for the main ultimate purpose of the model: helping the design of innovative cropping systems less vulnerable to pests.The presented structure of IPSIM is not exhaustive in terms of control methods that can be undertaken. However, developers of models within the IPSIM framework can always easily modify its structure in order to take into account the effects of control measures not present in The main breakthrough of IPSIM is to be able to handle complexity in a simple way. Input variables of the IPSIM models should be simple to provide. Most of these input variables will be static variables, except for weather variables that will be dynamic. The price to pay to handle the level of ecological complexity , with regard to the main pests that have to be managed. IPSIM will generate new knowledge by combining various sources of information from experiments, diagnoses of commercial field, models, and expert panels in a simple way, despite the high ecological complexity of the system addressed. The associated companion paper provides a proof of concept of the proposed method for a single pest.


Mesenchymal stem cells (MSC) are emerging as a therapeutic option for a plethora of immunological and degenerative diseases. Preclinical research is focusing on the mechanisms of MSC-mediated immunomodulation and regeneration. This Research Topic shines light on mechanistic and regulatory aspects that are of importance for understanding the full potential of MSC for clinical use in transplantation and tissue regeneration.A first consideration to make when studying MSC that is addressed in this Research Topic is the source of the cells. MSC are present in bone marrow and adipose tissue, amongst other tissues, and can be used in an autologous or allogeneic way. A commercially available allogeneic “off the shelf” cell product as described by Vaes et al.  has the Functionally, MSC are influenced by environmental factors. Leijs et al.  demonstrin vitro and in vivo after infusion. Although MSC and regulatory T cells target the same cell types, there is no evidence that they impede each other's function. The induction of regulatory T cells by MSC may be crucial for the long-term effects of MSC after infusion. Eggenhofer et al. , and National Competent Authorities (NCA) guidance documents, initiatives, and interaction platforms are available to make the regulatory framework more understandable and accessible for investigators both in the public and private sectors Ancans, . Good unThe tremendous therapeutic potential of MSC has once again been highlighted in this Research Topic. MSC can alleviate alloreactivity, but also reduce inflammatory responses involved in ischemia/reperfusion injury. We are encouraged by the results of the first clinical trials, however, as we have seen in this topic, there are still several questions to be resolved before MSC can be broadly applied in transplantation. The knowledge obtained from this Research Topic may aid to further development of MSC-based therapies in transplantation and tissue regeneration.


Kaposi’s Sarcoma is a vascular tumour caused by the Kaposi’s sarcoma-associated herpesvirus (KSHV). Although KSHV and HIV co-infection is often observed in HIV disease, the majority of patients nevertheless do not develop KS. Innate immunity, and in particular human leukocyte antigen alleles (HLA) and killer cell immunoglobulin-like receptor (KIR) are suspected to play a role in the development of viral associated carcinomas. We verified possible correlations between KIR/HLA complexes and KS by analyzing 82 HIV subjects: 15 with KS (KSpos), 32 KSHV-infected without KS (KSHVpos/KSneg) and 35 KSHV-uninfected (KSHVneg/KSneg); results were compared to those of 103 age-and sex-matched KHSV-seronegative individuals (HC).Molecular genotyped was performed by Single Specific Primer (SSP) method for HLA class I, and KIR alleles. Chi-square analysis Yates corrected (py) and Fisher exact test (pf) were evaluated when appropriated. Results showed a statistically higher frequency of the activating KIR2DS2 allele in KSpos subjects (93.3%) than either in KSHVpos/KSneg , or KSHVneg/KSneg  patients, and HC . Moreover, the homozygous genotype KIR2DS2+/2DL2- was present in 3/15 KSpos, but in none KSHVpos/KSneg (pf=0.03) or KSHVneg/KSneg (pf=0.02) and only in 1/103 HC (pf=0.006).Finally there was an higher frequency of KIR2DS2/HLAC-1 functional complex in KSpos (66.7%) than in all the other groups , though not statistically significant.Results herein, although stemming from analyses performed on a small number of individuals, suggest that HIV and KSHV co-infected subjects in whom activating KIRs and activating KIR-HLA ligands functional complexes are detected, are more prone to develop KS.


Both antibody mediated (AMR) and T-cell mediated (TCMR) rejections either acute or chronic represent the main reason for late graft dysfunction. In this study we aimed to evaluate differences in the intrarenal expression patterns of immune system related genes in acute and chronic rejections. Graft biopsies were performed and evaluated according to Banff classification. Using the TaqMan Low Density Array, the intrarenal expressions of 376 genes relating to immune response  were analyzed in the four rejection categories: chronic AMR, chronic TCMR, acute AMR, and acute TCMR. The set of genes significantly upregulated in acute TCMR as compared to acute AMR was identified, while no difference in gene expressions between chronic rejections groups was found. In comparison with functioning grafts, grafts that failed within the next 24 months after the chronic rejection morphological confirmation presented at biopsy already established severe graft injury , longer followup, higher expression of CDC20, CXCL6, DIABLO, GABRP, KIAA0101, ME2, MMP7, NFATC4, and TGFB3 mRNA, and lower expression of CCL19 and TRADD mRNA. In conclusion, both Banff 2007 chronic rejection categories did not differ in intrarenal expression of 376 selected genes associated with immune response. Both acute and chronic rejections have been shown to affect the long-term outcome of kidney transplantation. Chronic rejection is thought to be associated with both cellular and humoral alloimmune responses . ChronicBeside conventional morphological evaluation, molecular histology offers better insight into rejection pathogenesis and prognosis. Moreover, molecular phenotype may better predict the graft outcome , 4.In this study we aimed for evaluation of molecular signatures of acute and chronic rejections categories and for evaluation of association of gene transcripts with kidney graft loss due to chronic rejection. n = 9), early acute T-cell mediated rejection (TCMR) (n = 10), chronic AMR (n = 13), and chronic TCMR (n = 9) performed in 2007–2009 were evaluated. Basic demographic parameters of patients are shown in For the purpose of this study, 41 case biopsies revealing early acute AMR  guided by ultrasound . Small portions of renal tissue from the cortical or juxtamedullary zone were immediately stored in preserve solution  for expression analysis, while the majority of renal tissue taken by core biopsy was used for routine histology performed by the standard method. Samples were routinely stained according to the protocol of our laboratory . Immunofluorescence detection of C4d was performed in all cases. Biopsy tissue was scored on the basis of the Banff '07 working classification [http://dx.doi.org/10.1155/2013/509259). Specific gene expression was calculated relative to that of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the calibrator sample  by comparative threshold cycle method (2T−ΔΔC). RQ Manager 1.2 software for automated data analysis (Applied Biosystems) was used and results were expressed as relative quantity (RQ). The renal tissue was homogenized. Total RNA was extracted by RNA Blue (Top-Bio) and reversely transcribed into cDNA, using the SuperScript II Reverse Transcriptase (Invitrogen). Complementary DNA samples from each biopsy were analyzed on TaqMan Low Density Array Cards containing primers and probe sets for targets by 7900HT Fast Real-Time PCR System (Applied Biosystems). The set of targets was chosen on the basis of potential relevance to the study of renal allograft rejection according to the existing literature data . Significantly more patients received ATG induction therapy in AMR group as compared to other groups. Followup to biopsy was longer in acute AMR as compared to acute TCMR (P < 0.05); however, no differences were observed between chronic AMR and chronic TCMR. The clinical characteristics of patients are listed in We compared the molecular profile among all rejection groups. In hierarchical clustering, samples with AMR were clustered in one cluster, except for two samples in which the AMR was combined with TCMR . The cluThe effect of gene expression patterns on the chronic rejection outcome was analyzed. There were no differences in expressions of evaluated genes between chronic AMR and chronic TCMR. In 7 cases, graft function deteriorated during the 24-month followup after biopsy to CKD5T and dialysis therapy was initiated. These patients exhibited significantly worse renal function and proteinuria at the biopsy. Similarly, they had longer followup after transplantation, higher expression of CDC20, CXCL6, DIABLO, GABRP, KIAA0101, ME2, MMP7, NFATC4, and TGFB3 mRNA, and lower expression of CCL19 and TRADD mRNA . All patIn this study, both acute and chronic rejections were shown to be associated with several patterns of immune system related gene transcripts. Recently, despite different pathologies, both T-cell mediated and antibody mediated rejections were shown to be associated with similar molecular features , 5. In bIn conclusion, in this study, beside transcripts differences observed between acute T-cell mediated and antibody mediated rejections, both chronic rejections did not differ in 305 analyzed genes. The list of 345 genes evaluated by real-time quantitative PCR. The set of targets was chosen on the basis of potential relevance to the study of renal allograft rejection according the existing literature data.Click here for additional data file.