Cold-Start Emissions testing of Snowmobiles Using Ethanol and Gasoline

Increasing prices for fuel with depletion and instability in foreign oil imports has driven the importance for using alternative and renewable fuels. The alternative fuels such as ethanol, methanol, butyl alcohol, and natural gas are of interest to be used to relieve some of the dependence on oil for transportation. The renewable fuel, ethanol which is made from the sugars of corn, has been used widely in fuel for vehicles in the United States because of its unique qualities. As with any renewable fuel, ethanol has many advantages but also has disadvantages. Cold startability of engines is one area of concern when using ethanol blended fuel. This research was focused on the cold startability of snowmobiles at ambient temperatures of 20 °F, 0 °F, and -20 °F. The tests were performed in a modified 48 foot refrigerated trailer which was retrofitted for the purpose of cold-start tests. Pure gasoline (E0) was used as a baseline test. A splash blended ethanol and gasoline mixture (E15, 15% ethanol and 85% gasoline by volume) was then tested and compared to the E0 fuel. Four different types of snowmobiles were used for the testing including a Yamaha FX Nytro RTX four-stroke, Ski-doo MX Z TNT 600 E-TEC direct injected two stroke, Polaris 800 Rush semi-direct injected two-stroke, and an Arctic Cat F570 carbureted two-stroke. All of the snowmobiles operate on open loop systems which means there was no compensation for the change in fuel properties. Emissions were sampled using a Sensors Inc. Semtech DS five gas emissions analyzer and engine data was recoded using AIM Racing Data Power EVO3 Pro and EVO4 systems. The recorded raw exhaust emissions included carbon monoxide (CO), carbon dioxide (CO2), total hydrocarbons (THC), and oxygen (O2). To help explain the trends in the emissions data, engine parameters were also recorded. The EVO equipment was installed on each vehicle to record the following parameters: engine speed, exhaust gas temperature, head temperature, coolant temperature, and test cell air temperature. At least three consistent tests to ensure repeatability were taken at each fuel and temperature combination so a total of 18 valid tests were taken on each snowmobile. The snowmobiles were run at operating temperature to clear any excess fuel in the engine crankcase before each cold-start test. The trends from switching from E0 to E15 were different for each snowmobile as they all employ different engine technologies. The Yamaha snowmobile (four-stroke EFI) achieved higher levels of CO2 with lower CO and THC emissions on E15. Engine speeds were fairly consistent between fuels but the average engine speeds were increased as the temperatures decreased. The average exhaust gas temperature increased from 1.3-1.8% for the E15 compared to E0 due to enleanment. For the Ski-doo snowmobile (direct injected two-stroke) only slight differences were noted when switching from E0 to E15. This could possibly be due to the lean of stoichiometric operation of the engine at idle. The CO2 emissions decreased slightly at 20 °F and 0 °F for E15 fuel with a small difference at -20 °F. Almost no change in CO or THC emissions was noted for all temperatures. The only significant difference in the engine data observed was the exhaust gas temperature which decreased with E15. The Polaris snowmobile (semi-direct injected two-stroke) had similar raw exhaust emissions for each of the two fuels. This was probably due to changing a resistor when using E15 which changed the fuel map for an ethanol mixture (E10 vs. E0). This snowmobile operates at a rich condition which caused the engine to emit higher values of CO than CO2 along with exceeding the THC analyzer range at idle. The engine parameters and emissions did not increase or decrease significantly with decreasing temperature. The average idle engine speed did increase as the ambient temperature decreased. The Arctic Cat snowmobile (carbureted two-stroke) was equipped with a choke lever to assist cold-starts. The choke was operated in the same manor for both fuels. Lower levels of CO emissions with E15 fuel were observed yet the THC emissions exceeded the analyzer range. The engine had a slightly lower speed with E15.

Wearable Computing und RFID in Produktion und Logistik – Ansätze zur bereichsübergreifenden Nutzung digitaler Informationen

Die voranschreitende Entwicklung von Konzepten und Systemen zur Nutzung digitaler Informationen im industriellen Umfeld eröffnet verschiedenste Möglichkeiten zur Optimierung der Informationsverarbeitung und damit der Prozesseffektivität und -effizienz. Werden die relevanten Daten zu Produkten oder Prozessen jedoch lediglich in digitaler Form zur Verfügung gestellt, fällt ein Eingriff des Menschen in die virtuelle Welt immer schwerer. Auf Grundlage dessen wird am Beispiel der RFIDTechnologie dargestellt, inwiefern digitale Informationen durch die Verwendung von in den Arbeitsablauf integrierten Systemen für den Menschen nutzbar werden. Durch die Entwicklung eines Systems zur papierlosen Produktion und Logistik werden exemplarisch Einsatzszenarien zur Unterstützung des Mitarbeiters in Montageprozessen sowie zur Vermeidung von Fehlern in der Kommissionierung aufgezeigt. Dazu findet neben einer am Kopf getragenen Datenbrille zur Visualisierung der Informationen ein mobiles RFID-Lesegerät Anwendung, mit Hilfe dessen die digitalen Transponderdaten ohne zusätzlichen Aufwand für den Anwender genutzt werden können.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum"

Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.

RTX toxins in Pasteurellaceae

RTX toxins (repeats in the structural toxin) are pore-forming protein toxins produced by a broad range of pathogenic Gram-negative bacteria. In vitro, RTX toxins mostly exhibit a cytotoxic and often also a hemolytic activity. They are particularly widespread in species of the family Pasteurellaceae which cause infectious diseases, most frequently in animals but also in humans. Most RTX toxins are proteins with a molecular mass of 100-200 kDa and are post-translationally activated by acylation via a specific activator protein. The repeated structure of RTX toxins, which gave them their name, is composed of iterative glycine-rich nonapeptides binding Ca2+ on the C-terminal half of the protein. Genetic analysis of RTX toxins of various species of Pasteurellaceae and of a few other Gram-negative bacteria gave evidence of horizontal transfer of genes encoding RTX toxins and led to speculations that RTX toxins might have originated from Pasteurellaceae. The toxic activities of RTX toxins in host cells may lead to necrosis and apoptosis and the underlying detailed mechanisms are currently under investigation. The impact of RTX toxins in pathogenicity and the immune responses of the host were described for several species of Pasteurellaceae. Neutralizing antibodies were shown to significantly reduce the cytotoxic activity of RTX toxins. They constitute a valuable strategy in the development of immuno-prophylactics against several animal diseases caused by pathogenic species of Pasteurellaceae. Although many RTX toxins possess cytotoxic and hemolytic activities toward a broad range of cells and erythrocytes, respectively, a few RTX toxins were shown to have cytotoxic activity only against cells of specific hosts and/or show cell-type specificity. Further evidence exists that RTX toxins play a potential role in host specificity of certain pathogens.

Characterization of Aqx and its operon: the hemolytic RTX determinant of Actinobacillus equuli

Actinobacillus equuli, a member of the family Pasteurellaceae is the etiologic agent of a frequently lethal septicemia in neonatal foals as well as other more chronic diseases like arthritis, pleuritis, pneumonia or peritonitis. It may also be isolated from the oral cavity of healthy horses. Hemolytic isolates of A. equuli are known but so far no virulence determinants have been described for this bacterial species. By screening hemolytic A. equuli strains with specific gene probes, a hemolysin, designated Aqx (A. equuli RTX (repeats in the structural toxin)) was identified. This hemolysin was shown to be an RTX type of toxin by characterization of the aqxCABD operon. All hemolytic A. equuli isolates contained a functional aqxCABD operon and expressed the Aqx hemolysin as shown by genetic and phenotypic assays. The structural toxin AqxA is the hemolysin of A. equuli as shown by expression of recombinant aqx constructs in E. coli. Its hemolytic activity can be inhibited by specific antibodies raised against AqxA. Sequence analysis of the 16S rRNA gene (rrs) of the taxonomically diffuse group of A. equuli and related strains defined two phylogenetically distinct groups. The presence of the Aqx operon is not correlated with this phylogenetic grouping. The operon is found in both groups of A. equuli strains where it specifies the hemolytic activity and is supposedly to be a determinative virulence factor. The aqx operon was not found in closely related members of the Pasteurellaceae family. The description of the Aqx hemolysin will open new ways for studying the pathogenesis of A. equuli.

Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxIVA. When expressed in Escherichia coli, recombinant ApxIVA showed a weak haemolytic activity and co-haemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxIVA. The apxIVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxIVA 14 d post-infection, indicating that apxIVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxIVA. The apxIVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxIVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxIVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.

Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.

AvxA, a composite serine-protease-RTX toxin of Avibacterium paragallinarum

Avibacterium paragallinarum, the etiological agent of infectious coryza in chicken, was found to encode a bivalent serine-protease - RTX-porin toxin named AvxA. This toxin is encoded on a classical RTX operon structure with the activator gene avxC, the structural serin-protease-RTX toxin gene avxA, and the genes for a proper type I secretion system avxBD. AvxA is activated by the product of the avxC gene, secreted by the avxBD specified type I secretion system and proteolytically processed leaving a 95 kDa RTX moiety that is found in culture supernatants of A. paragallinarum serovars A, B and C. The RTX moiety of AvxA (AvxA-RTX) is cytotoxic against the avian macrophage like cell line HD11 but not against bovine macrophage cell line BoMac. Purified IgG from hyper-immune rabbit anti-AvxA-RTX serum made by immunization with recombinant AvxA-RTX from a serotype A strain fully neutralizes the cytotoxic activity of recombinant active AvxA-RTX and of A. paragallinarum serotypes A, B and C. This indicates that AvxA is a common major virulence attribute of all A. paragallinarum serotypes.

Wearable schizophrenia treatment with real-time affective context awareness

The Personal Health Assistant Project (PHA) is a pilot system implementation sponsored by the Kozani Region Governors’ Association (KRGA) and installed in one of the two major public hospitals of the city of Kozani. PHA is intended to demonstrate how a secure, networked, multipurpose electronic health and food benefits digital signage system can transform common TV sets inside patient homes or hospital rooms into health care media players and facilitate information sharing and improve administrative efficiency among private doctors, public health care providers, informal caregivers, and nutrition program private companies, while placing individual patients firmly in control of the information at hand. This case evaluation of the PHA demonstration is intended to provide critical information to other decision makers considering implementing PHA or related digital signage technology at other institutions and public hospitals around the globe.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.