Preliminary characterization of VmTPT2, a putative monoterpene indole alkaloid (MIA) transporter from Vinca minor L. (Apocynaceae)

The various steps of monoterpene indole alkaloid (MIA) biosynthesis are known to occur in specialized cell types and subcellular compartments. Numerous MIAs display powerful biological activities that have led to their use as pharmaceutical treatments for cancer, hypertension and malaria. Many of these compounds accumulate on the leaf surface of medicinally important Apocynaceae plants, which led to the recent discovery and characterization of an ABC transporter (CrTPT2) that was shown to mobilize catharanthine from its site of biosynthesis in epidermal cells to the leaf surface of Catharanthus roseus. Bioinformatic analysis of transcriptomes from several geographically distant MIA-producing species led to the identification of proteins with high amino acid sequence identity to CrTPT2. Molecular cloning of a similar transporter (VmTPT2) from Vinca minor was carried out and expressed in a yeast heterologous system for transport experiments and functional characterization. In planta studies involved transcript expression analysis of the early MIA biosynthetic gene VmTDC and putative transporter VmTPT2, and alkaloid profile analyses. RT-qPCR results showed that VmTPT2 expression increased 15-fold between the first two leaf pairs, and high levels were maintained across older leaves. The alkaloid accumulation profile on leaf surfaces matched that of VmTPT2 expression, especially for the MIAs vincadifformine and vincamine. Gene expression and alkaloid profile analyses suggest that the functional protein may act as a similar transporter to CrTPT2. However, although VmTPT2 had 88.4% identity at the amino acid level to CrTPT2, it displayed an altered expression pattern in planta across developing leaves, and functional characterization using a previously developed yeast heterologous system was unsuccessful due to difficulties with reproducibility of transport assays.

Populationsgenetik und Phylogeographie des Archäophyten und Kulturreliktzeigers Vinca minor L. (Apocynaceae)

Das Kleine Immergrün (Vinca minor L.) aus der Familie der Apocynaceae ist in der Krautschicht sommergrüner Wälder Südeuropas heimisch, während es in weiten Teilen Mitteleuropas als wahrscheinlich von den Römern eingeführter, altetablierter Archäophyt gilt. Noch heute ist die Art als Kulturreliktzeiger häufig in der Umgebung ehemaliger römischer Tempel und mittelalterlicher Burgruinen zu finden. Zudem wird V. minor in zahlreichen Gartenformen kultiviert. In Teilen Nordamerikas wird der Chamaephyt hingegen als eingeführte, invasive Art eingestuft, die die einheimische Flora und Fauna bedroht. Da V. minor Stolonen bilden kann und in Mitteleuropa selten reife Samen beobachtet werden, wurde bislang vermutet, dass V. minor Bestände in Mitteleuropa sich rein asexuell erhalten. Diese Hypothese wurde aber bisher nie mit molekularen Methoden überprüft. Auch zur Populationsgenetik der Art ist bisher nichts bekannt. Aus diesen Gegebenheiten resultieren folgende Fragen: Wie hoch ist die genetische Diversität von V. minor im submediterranen Ursprungsgebiet im Vergleich zu Mitteleuropa und Nordamerika und wie ist sie in den Großregionen jeweils strukturiert? Korreliert die anthropogen bedingte Einführung mit einer genetischen Verarmung in Mitteleuropa? Gibt es in mitteleuropäischen und nordamerikanischen Populationen Hinweise auf sexuelle Reproduktion, oder erfolgt eine rein vegetative Vermehrung? Gibt es genetische Hinweise für Auswilderungen aus Gärten? Lassen sich die historischen Ausbreitungswege der Art von Süd- nach Mitteleuropa, innerhalb Mitteleuropas sowie nach Nordamerika rekonstruieren? Mikrosatellitenmarker stellen für populationsgenetische Analysen heute die weitaus gängigste Technik dar. Als codominante, locusspezifische Marker erlauben sie die präzise Erfassung populationsgenetischer Parameter zur Quantifizierung der genetischen Diversität und Struktur, die Abschätzung von Genfluss, und die Detektion von Klonen. Mikrosatelliten sind mit Hilfe neuer DNA-Sequenziertechniken (NGS) unproblematisch und kosteneffektiv isolierbar. Im Rahmen der hier vorliegenden Arbeit wurden daher zunächst nukleäre und plastidäre Mikrosatellitenmarker über NGS-454-Sequenzierung entwickelt. Etablierung von nukleären und plastidären Mikrosatellitenmarkern Zur Etablierung artspezifischer nukleärer Mikrosatellitenmarker wurden zwei Verfahren angewendet. Zum einen wurde in einer öffentlich zugänglichen, über 454-Sequenzierung der cDNA von V. minor gewonnene und im 'sequence read archive' von NCBI hinterlegte Datenbank (Akzessionsnummer SRX039641) nach Mikrosatelliten gesucht. Zum anderen wurde die 454-Technologie eingesetzt, um in Kooperation mit Dr. Bruno Huettel vom Max-Planck-Institut für Pflanzenzüchtung in Köln genomische Sequenzdaten anhand einer V. minor-Akzession zu generieren und aus diesen Mikrosatelliten zu etablieren. Eine Assemblierung der 723.230 cDNA-Sequenzen mit insgesamt 387 Mbp erzielte eine Reduzierung auf 267.199 Unigenes (267 Mbp), die der genomischen Sequenzen eine Reduzierung von 43.565 (18 Mbp) auf 24.886 Sequenzen (13,7 Mbp). Die assemblierten Datensätze enthielten 25.253 bzw. 1.371 Mikrosatellitenloci aus Mono- bis Hexa-Nukleotidmotiven. Die Effizienz der Assemblierung war somit v. a. bei den cDNA-Sequenzen gering. Da die Etablierung von Mikrosatellitenloci aber auch auf Basis redundanter Sequenzen möglich ist, sofern ein manueller Abgleich der selektierten Sequenzen erfolgt, wurde auf eine weitere Optimierung der Assemblierung verzichtet. Aus den so identifizierten Loci wurden 60 (cDNA) bzw. 35 (genomische DNA) Di-, Tri- und Tetranukleotidmotive selektiert, flankierende Primer synthetisiert und in umfangreichen Pilotstudien getestet. Jeweils neun der Loci erwiesen sich als robuste, polymorphe Marker. Die sieben vielversprechendsten Marker wurden schließlich für die populationsgenetische Untersuchung ausgewählt. Auch die Etablierung plastidärer Mikrosatellitenmarker erfolgte über zwei Ansätze. Zum einen wurde das Plastom von V. minor aus dem genomischen 454-Sequenzdatensatz rekonstruiert und auf das Vorhandensein von (A)n/(T)n-Wiederholungseinheiten hin untersucht. Für 14 der 17 dabei detektierten Loci konnten Primer entworfen werden. In einer Pilotstudie erwiesen sich vier der Loci als funktionelle, polymorphe Marker. Zusätzlich wurden die zehn universellen (ccmp) Primerpaare zur Amplifikation plastidärer Mikrosatellitenloci aus Weising & Gardner (1999) getestet, von denen zwei als funktionelle, polymorphe Marker für die Hauptstudie geeignet waren. Populationsgenetische und phylogeographische Analyse Ein Probenset aus insgesamt 967 Pflanzenproben aus 70 Populationen aus Mitteleuropa inkl. der Alpen, den Regionen südlich und westlich der Alpen sowie aus Kanada und 18 Cultivaren wurde mittels der sieben neu etablierten, artspezifischen nukleären Mikrosatellitenmarker populationsgenetisch untersucht. Dabei erwiesen sich 21 der 31 untersuchten Populationen südlich und westlich der Alpen als genetisch hoch divers, die übrigen 10 zeigten vor allem klonales Wachstum und wiesen jeweils ein bis drei Multilocus-Genotypen (MLGs) auf. In 30 der 36 mitteleuropäischen Vorkommen (inkl. der Alpen) sowie den kanadischen Beständen war jeweils nur ein einziger MLG präsent. Drei der Vorkommen zeigten mit einem Heterozygotendefizit einzelner Stichproben Hinweise auf Geitonogamie, an drei weiteren Vorkommen traten jeweils zwei sowohl hinsichtlich der Blütenfarbe und -architektur als auch des MLG unterschiedliche Linien auf. An einem dieser Vorkommen wurde ein Hybrid-Genotyp detektiert, bisher der einzige molekulare Hinweis auf sexuelle Reproduktion im engeren Sinn in Mitteleuropa. Die 967 Stichproben konnten insgesamt 310 individuellen Multilocus-Genotypen (MLGs) zugeordnet werden. Davon traten 233 MLGs nur in jeweils einer einzigen Probe auf, die 77 verbleibenden wurden in mehreren Akzessionen detektiert. Aus einer Simulation ging hervor, dass diese wiederholten MLGs auf rein asexuelle Reproduktion zurückzuführen sind. In Mitteleuropa waren lediglich 18 MLGs vertreten, von denen sieben an bis zu zehn, mehrere hundert Kilometer entfernten Fundorten auftraten. In Nordamerika gehören gar alle drei untersuchten Populationen dem gleichen Klon an. In Mitteleuropa traten in zwei Fällen somatische Mutationen zwischen zwei MLGs auf, sodass diese zu klonalen Linien (Multilocus-Linien; MLL) zusammengefasst werden konnten. Sieben der 18 Cultivare weisen einen zu diversen Freilandvorkommen identischen Genotypen auf. Die Ergebnisse reflektieren den durch die anthropogene Selektion bedingten genetischen Flaschenhalseffekt, in dessen Folge der Genpool von Vinca minor in Mitteleuropa gegenüber der südeuropäischen Heimat der Art stark reduziert wurde. Sexuelle Reproduktion in Mitteleuropa zwischen zwei genetisch unterschiedlichen Individuen ist nur an wenigen Standorten überhaupt möglich und da meist nur ein Klon am gleichen Fundort auftritt, sehr selten. Die Ausbreitung erfolgt zudem rein anthropogen und über erhebliche Strecken, wie die identischen MLGs an unterschiedlichen, weit auseinander liegenden Fundorten belegen. Südlich und westlich der Alpen hingegen ist sexuelle Reproduktion über Samen häufig. Aus den kalkulierten Neighbour-Joining Phenogrammen, Neighbour-Nets und der Bayes'schen Analyse ergibt sich prinzipiell eine Abtrennung der in Norditalien und Slowenien gelegenen Vorkommen von den übrigen Regionen, wohingegen mehrere mittelitalienische Populationen mit denen westlich der Alpen und den mitteleuropäischen Vorkommen in einer engeren genetischen Beziehung stehen. Da die mittelitalienischen Vorkommen jedoch Anzeichen anthropogenen Ursprungs aufweisen (Monoklonalität, Lage an Wegrändern oder Burgen), lassen sich diese Populationen nur bedingt als potentielle Ursprungspopulationen ableiten. Die genetisch diversen norditalienischen und slowenischen Populationen sind trotz der Fragmentierung der norditalienischen Waldvegetation insgesamt nur moderat voneinander differenziert (FST=0,14, GST=0,17, RST=0,19). Die AMOVA ergab, dass über 80 % der genetischen Variation auf Variation innerhalb der Populationen zurückzuführen ist. Dennoch ergab sich aus einem Mantel-Test eine zunehmende genetische Differenzierung mit zunehmender geographischer Distanz (r=0,59). Die phylogeographische Analyse wurde mit Hilfe von vier plastidären Mikrosatellitenmarkern aus der 454-Sequenzierung und zwei universellen plastidären ccmp-Mikrosatellitenloci durchgeführt. Untersucht wurden jeweils eine bis sechs Stichproben aus den o. g. 70 Populationen, die 18 Cultivare sowie zusätzliche Einzelproben aus mehreren Ländern, deren DNA aus Herbarbelegen isoliert wurde. Insgesamt wurden 297 Proben untersucht. Unter diesen wurden in der phylogeographischen Analyse sieben plastidäre Haplotypen detektiert. In der Region südlich der Alpen traten sechs Haplotypen auf (H1 bis H5, H7), in Mitteleuropa vier Haplotypen (H1 bis H3, H6), in Nordamerika, Großbritannien, Schweden und Nordamerika trat hingegen nur ein einziger Haplotyp H1 auf. Die beiden häufigsten Haplotypen nahmen im berechneten Haplotypen-Netzwerk periphere Positionen ein und waren durch sieben Mutationschritte voneinander getrennt. Südlich der Alpen ergab sich jedoch keine klare geographische Verteilung der Haplotypen. Auch die plastidären Daten indizieren somit eine geringere genetische Diversität in den Gebieten, wo V. minor eingeführt wurde. Der geographische Ursprung der mitteleuropäischen Vorkommen in Südeuropa konnte nicht abschließend geklärt werden, jedoch lässt das Vorkommen von zwei weit entfernten Haplotypen den Schluss zu, dass Vinca minor mindestens zweimal (und vermutlich mehrfach) unabhängig in Mitteleuropa eingeführt wurde.

Development of 18 Polymorphic Microsatellite Markers for Vinca minor (Apocynaceae) via 454 Pyrosequencing

• Premise of the study: Polymorphic microsatellite markers were developed in Vinca minor (Apocynaceae) to evaluate the level of clonality, population structure, and genetic diversity of the species within its native and introduced range. • Methods and Results: A total of 1371 microsatellites were found in 43,565 reads from 454 pyrosequencing of genomic V. minor DNA. Additional microsatellite loci were mined from publicly available cDNA sequences. After several rounds of screening, 18 primer pairs flanking di-, tri-, or tetranucleotide repeats were identified that revealed high levels of genetic diversity in two native Italian populations, with two to 11 alleles per locus. Clonal growth predominated in two populations from the introduced range in Germany. Five loci successfully cross-amplified in three additional Vinca species. • Conclusions: The novel polymorphic microsatellite markers are promising tools for studying clonality and population genetics of V. minor and for assessing the historical origin of Central European populations.

Localization of monoterpenoid indole alkaloid (MIA) biosynthesis by in situ hybridization (ISH)

Monoterpenoid indole alkaloids (MIA) are among the largest and most complex group of nitrogen containing secondary metabolites that are characteristic of the Apocynaceae plant family including the most notable Catharanthus roseus. These compounds have demonstrated activity as successful drugs for treating various cancers, neurological disorders and cardiovascular conditions. Due to the low yields of these compounds and high pharmacological value, their biosynthesis is a major topic of study. Previous work highlighting the leaf epidermis and leaf surface as a highly active area in MIA biosynthesis and MIA accumulation has made the epidermis a major focus of this thesis. This thesis provides an in-depth analysis of the valuable technique of RNA in situ hybridization (ISH) and demonstrates the application of the technique to analyze the location of the biosynthetic steps involved in the production of MIAs. The work presented in this thesis demonstrates that most of the MIAs of Eurasian Vinca minor, African Tabernaemontana e/egans and five Amsonia species, including North American Amsonia hubrichitii and Mediterranean A. orienta/is, accumulate in leaf wax exudates, while the rest of the leaf is almost devoid of alkaloids. Biochemical studies on Vinca minor displayed high tryptophan decarboxylase (TOe) enzyme activity and protein expression in the leaf epidermis compared to whole leaves. ISH studies aimed at localizing TOe and strictosidine synthase suggest the upper and lower epidermis of V. minor and T. e/egans as probable significant production sites for MIAs that will accumulate on the leaf surface, however the results don't eliminate the possibility of the involvement of other cell types. The monoterpenoid precursor to all MIAs, secologanin, is produced through the MEP pathway occurring in two cell types, the IPAP cells (Gl0H) and epidermal cells (LAMT and SLS). The work presented in this thesis, localizes a novel enzymatic step, UDPG-7-deoxyloganetic acid glucosyltransferase (UGT8) to the IPAP cells of Catharanthus longifolius. These results enable the suggestion that all steps from Gl0H up to and including UGT8 occur in the IPAP cells of the leaf, making the IPAP cells the main site for the majority of secologanin biosynthesis. It also makes the IPAP cells a likely cell type to begin searching for the gene of the uncharacterized steps between Gl0H and UGT8. It also narrows the compound to be transported from the IPAP cells to either 7-deoxyloganic acid or loganic acid, which aids in the identification of the transportation mechanism.

The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.

Anatomical Study Of Minor Alterations In Neonate Vocal Folds.

Minor structural alterations of the vocal fold cover are frequent causes of voice abnormalities. They may be difficult to diagnose, and are expressed in different manners. Cases of intracordal cysts, sulcus vocalis, mucosal bridge, and laryngeal micro-diaphragm form the group of minor structural alterations of the vocal fold cover investigated in the present study. The etiopathogenesis and epidemiology of these alterations are poorly known. To evaluate the existence and anatomical characterization of minor structural alterations in the vocal folds of newborns. 56 larynxes excised from neonates of both genders were studied. They were examined fresh, or defrosted after conservation via freezing, under a microscope at magnifications of 25× and 40×. The vocal folds were inspected and palpated by two examiners, with the aim of finding minor structural alterations similar to those described classically, and other undetermined minor structural alterations. Larynges presenting abnormalities were submitted to histological examination. Six cases of abnormalities were found in different larynges: one (1.79%) compatible with a sulcus vocalis and five (8.93%) compatible with a laryngeal micro-diaphragm. No cases of cysts or mucosal bridges were found. The observed abnormalities had characteristics similar to those described in other age groups. Abnormalities similar to sulcus vocalis or micro-diaphragm may be present at birth.

On-line identification of minor flavones from sugarcane juice by LC/UV/MS and post-column derivatization

This work describes the on-line characterization of minor flavones from sugarcane (Saccharum officinarum) juice by high-performance liquid chromatography coupled to diode array UV detection and mass spectrometry (LC/UV/MS) using atmospheric pressure chemical ionization-collision-induced dissociation (APCI-CID-MS/MS) and post-column derivatization using UV shift reagents. HPLC-UV analysis with shift reagents provided information about the substitution pattern in the flavonoid skeleton and, combined with MS data, these techniques allowed for the on-line identification of five "garapa" flavones: luteolin-8-C-glucosyl-7-O-glucuronide; tricin-7-O-neohesperoside-4'-O-rhamnoside; tricin-7-O-methylglucuronate-4'-O-rhamnoside; tricin-7-O-methylglucuronide; swertisin, while four other compounds were partially identified as glycosylflavones. Only swertisin (7-O-methylapigenin-6-C-glucoside) was reported previously in sugarcane molasses.

Minor and Unsystematic Cortical Topographic Changes of Attention Correlates between Modalities

In this study we analyzed the topography of induced cortical oscillations in 20 healthy individuals performing simple attention tasks. We were interested in qualitatively replicating our recent findings on the localization of attention-induced beta bands during a visual task [1], and verifying whether significant topographic changes would follow the change of attention to the auditory modality. We computed corrected latency averaging of each induced frequency bands, and modeled their generators by current density reconstruction with Lp-norm minimization. We quantified topographic similarity between conditions by an analysis of correlations, whereas the inter-modality significant differences in attention correlates were illustrated in each individual case. We replicated the qualitative result of highly idiosyncratic topography of attention-related activity to individuals, manifested both in the beta bands, and previously studied slow potential distributions [2]. Visual inspection of both scalp potentials and distribution of cortical currents showed minor changes in attention-related bands with respect to modality, as compared to the theta and delta bands, known to be major contributors to the sensory-related potentials. Quantitative results agreed with visual inspection, supporting to the conclusion that attention-related activity does not change much between modalities, and whatever individual changes do occur, they are not systematic in cortical localization across subjects. We discuss our results, combined with results from other studies that present individual data, with respect to the function of cortical association areas.

Compton suppression instrumental neutron activation analysis performance in determining trace- and minor-element contents in foodstuff

In 2003-2004, several food items were purchased from large commercial outlets in Coimbra, Portugal. Such items included meats (chicken, pork, beef), eggs, rice, beans and vegetables (tomato, carrot, potato, cabbage, broccoli, lettuce). Elemental analysis was carried out through INAA at the Technological and Nuclear Institute (ITN, Portugal), the Nuclear Energy Centre for Agriculture (CENA, Brazil), and the Nuclear Engineering Teaching Lab of the University of Texas at Austin (NETL, USA). At the latter two, INAA was also associated to Compton suppression. It can be concluded that by applying Compton suppression (1) the detection limits for arsenic, copper and potassium improved; (2) the counting-statistics error for molybdenum diminished; and (3) the long-lived zinc had its 1115-keV photopeak better defined. In general, the improvement sought by introducing Compton suppression in foodstuff analysis was not significant. Lettuce, cabbage and chicken (liver, stomach, heart) are the richest diets in terms of human nutrients.

Distribution of major, minor, and trace metals in lake environments of Antarctica

The concentrations of major, minor and trace metals were measured in water samples collected from five shallow Antarctic lakes (Carezza, Edmonson Point (No 14 and 15a), Inexpressible Island and Tarn Flat) found in Terra Nova Bay (northern Victoria Land, Antarctica) during the Italian Expeditions of 1993-2001. The total concentrations of a large suite of elements (Al, As, Ba, Ca, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, Gd, K, La, Li, Mg, Mn, Mo, Na, Nd, Ni, Pb, Pr, Rb, Sc, Si, Sr, Ta, Ti, U, V, Y, W, Zn and Zr) were determined using spectroscopic techniques (ICP-AES, GF-AAS and ICP-MS). The results are similar to those obtained for the freshwater lakes of the Larsemann Hills, East Antarctica, and for the McMurdo Dry Valleys. Principal Component Analysis (PCA) and Cluster Analysis (CA) were performed to identify groups of samples with similar characteristics and to find correlations between the variables. The variability observed within the water samples is closely connected to the sea spray input; hence, it is primarily a consequence of geographical and meteorological factors, such as distance from the ocean and time of year. The trace element levels, in particular those of heavy metals, are very low, suggesting an origin from natural sources rather than from anthropogenic contamination.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

Synchronous adenocarcinoma of the major and minor duodenal papilla

A 50-year-old woman presented with pancreatitis, fluctuant jaundice, weight loss, and abdominal pain. Contrast-enhanced computed tomography and abdominal ultrasound showed slight dilatation of the biliary tree and gallbladder without calculi. Endoscopy demonstrated a tumor protruding from the papilla of Vater. First endoscopically biopsy diagnosed no tumor, and a second biopsy diagnosed as papillary adenocarcinoma. The patient underwent duodenopancreatectomy. The specimen was fixed in formalin (10%). The tissue was processed routinely, and paraffin sections were stained with hematoxylin-eosin and periodic acid Schiff. Gross examination showed two tumors seen as prolapsed nodules growing isolated from the minor and major duodenal papillae measuring 1.5 and 1.0 cm, respectively, both covered by duodenal mucosa and the histologic study of both lesions demonstrated a moderately differentiated tubular adenocarcinoma, which invaded duodenal wall. After surgery, she is alive 24 months without evidence of recurrence.

Digital vasculitis in systemic lupus erythematosus: a minor manifestation of disease activity?

The objective of this study is to determine if digital vasculitis (DV), a clinical manifestation with a high systemic lupus erythematosus disease activity index (SLEDAI) score, is associated with lupus severity. DV and other clinical manifestations defined according to the SLEDAI were evaluated in 168 consecutive patients with systemic lupus erythematosus (SLE). Two groups were defined according to presence (DV+, n = 27) or absence of DV (DV-, n = 141) at the time of evaluation. The exclusion criterion was the presence of antiphospholipid syndrome (Sapporo`s criteria). The two groups were comparable with regard to age (P = 0.09), gender (P = 1.00), white race (P = 0.81), and disease duration (P = 0.78). Compared to the DV-group, the DV+ group had a significantly higher frequency of mucocutaneous manifestations (66.7 vs. 39.0%, P = 0.01), haematological abnormalities (22.2 vs. 6.4%, P = 0.02) and constitutional symptoms (11.1 vs. 0.7%, P = 0.01). Renal and neurological involvements were similar in both groups ( P = 0.57 and P = 1.00, respectively). The evaluation of each SLEDAI parameter confirmed that the DV+ group had higher frequencies of mild manifestations, such as new rash (P = 0.02), alopecia (P = 0.02), oral ulcers (P = 0.045), fever (P = 0.01) and leucopenia (P = 0.005). In contrast, both groups had similarly increased anti-dsDNA (P = 0.78) and decreased complement levels (P = 0.29). In conclusion, DV in patients with SLE identifies a subgroup of a mild disease. The high `weighted` index attributed to this alteration in the SLEDAI score should therefore be revised. Lupus (2009) 18, 990-993.