AbstractDryocoetes brownei from Northern India and Afghanistan, is described as a new to science and redescription of Scolytoplatypus kunala Strohmeyer, 1908, previously known only from the female holotype, is provided.A new bark beetle species,  Scolytinae in the genus Dryocoetes was discovered during study of materials kept in Natural History Museum, London (NHML), and several more examples of this species were found in the O.N. Kabakov collection from Afghanistan.One more new species of Scolytoplatypus species were recently reviewed by Scolytoplatypus kunala Strohmeyer, 1908 in most papers, including the cited one, is doubtful or erroneous. Schedl (1973) considered Scolytoplatypus kunala to be a senior synonym of Scolytoplatypus darjeelingi Stebbing, 1914, but Scolytoplatypus kunala as a junior synonym of Scolytoplatypus daimio Blandford, 1893 and Scolytoplatypus siomio Blandford, 1893, but as a distinct species from Scolytoplatypus darjeelingi. Importantly, all Scolytoplatypus species known from Himalaya, besides Scolytoplatypus kunala, were recorded only in Eastern Himalaya, whereas Scolytoplatypus kunala was discovered in Kashmir Province, Western Himalaya . We have found three males and two females of Scolytoplatypus species collected by O.N. Kabakov in Afghanistan in Russian collections. After comparison of these specimens with the female holotype of Scolytoplatypus kunala we consider these specimens from Afghanistan to be conspecific with Scolytoplatypus kunala and provide redescription of both male and female of this poorly known species. Scolytoplatypus kunala is reinstated here as separate good species. So far we extend the geographical range of Oriental Scolytoplatypus to the west and consider Scolytoplatypus kunala to be the most “western” Scolytoplatypus species from Asia.Oriental Mandelshtam & Petrov sp. n.urn:lsid:zoobank.org:act:6510C1A7-5D09-45D5-A994-2AD2CAE8522FDryocoetes autographus Ratz. All five studied specimens appear to be mature, not teneral.Holotype (NHML) body length 5.1 mm, width 2.0 mm, other specimens  4.8–5.0 mm in length. Body reddish brown, essentially as in Dryocoetes species. Hair-like setae significantly longer at lateral sides of pubescent frontal area. Epistoma with long dense yellowish setae directed downwards. Eyes rather large, emarginate anteriorly. Antennae typical for genus: funiculus 5-segmented and club obliquely truncate with recurved sutures on anterior face, and one suture near apex on posterior face.Frons is rather densely but not coarsely, uniformly punctured, with an enlarged puncture in center forming a small fovea. Frontal surface shining, without reticulation. Vertex more sparsely punctured and with smaller punctures. A median longitudinal black line (sulcus) on vertex probably indicates internal strengthening of the head. Frons with rather long, fairly sparse, yellowish hair-like setae, not forming tufts typical of females of Pronotum slightly longer than wide (1.9 vs. 1.8 mm); sides subparallel in basal three fourth and apex simply rounded, without teeth on anterior margin. Pronotal surface generally granulate, more coarsely anteriorly, punctured area very small and restricted to central portion of pronotal base around impunctate median line extending approximately one fifth of pronotal length only. Pronotal surface between granules shining, without any reticulation. Sides and anterior margin of pronotum with long, curved, yellow hair-like setae.PageBreakScutellum large, flat, flush with elytral surface, of same reddish color as elytra.Elytra slightly wider (2.0 vs. 1.9 mm) than pronotum, nearly cylindrical, only slightly widened towards apex, 3.2 mm long. Elytra extremely coarsely punctured on the disk, especially on striae 1–3  . JuxtasDeclivity slightly flattened, convex, not steep, dull. Ventrolateral sides of declivity not armed and lacking minute tubercles on declivital surface. First and second interstriae are widened at declivity. Strial punctures of first and second interstriae on declivity larger than punctures of other striae, but more than two times smaller than on disk. First and second striae slightly divergent towards elytral apex. Suture appearing only slightly convex on declivity because juxtasutural striae are not deepened. Elytra with rather long yellow interstrial hair-like setae, longer laterally and on the declivity; strial hair-like setae recumbent and approximately 5 times shorter that interstrial setae.Legs typical for the genus, protibia with 5 socketed teeth on outer surface, meso- and metatibiae each with 5 spines on outer margin.Underside of body reddish brown, abdomen covered with rather long setae on posterior margin of ventrites.Similar to male, but the frons with a dense tuft of long yellow hair-like setae.The new species is dedicated to the eminent British coleopterologist F.G. Browne, who first labeled the holotype as a possible new species.Dryocoetes sp.n. det. F.G.B. 1967 // Possibly one of Beeson’s undescr. spp. – deobanos himalahinorum [sic!] . Paratypes  : Afghan[istan], Nurestan N.Waygal 2500 m., 10.VII.1972, Kabakov [leg.], 1 spec. in Zoological Institute, St.Petersburg (ZISP) and 3 spec. in A.V. Petrov private collection.Holotype  is deposited in NHML. Labels of the holotype are as follows: Kashmir: Gulmarg. vi – 1931. Dr. M.Cameron. B.M. 931–452.// Dryocoetes by the exceptionally large punctures of the elytral discal striae and by the large size of the body.A new bark-beetle is described from Northern India that differs from all other Indian species of the genus Dryocoetes were previously reported to occur, namely Dryocoetes hewetti Stebbing, 1908, Dryocoetes himalayensis Strohmeyer, 1908, Dryocoetes quadrisulcatus Strohmeyer, 1908 and Dryocoetes indicus Stebbing, 1914. We were able to study all these four species in the Zoological Museum of Moscow University (ZMMU). The first of them has a spatulate antennal club, and extremely long declivital pubescence, and was correctly transferred to the genus Taphrorychus . Dryocoetes himalayensis and Dryocoetes quadrisulcatus differ from both Dryocoetes indicus and Dryocoetes brownei by the much smaller body size and by the strongly deepened first striae on the disk and the declivity. Although this feature is more evident in males of these species, it is also developed in the females. Dryocoetes indicus is also smaller in size (4.0 mm vs 5.1 mm) than Dryocoetes brownei, has equally punctured striae and interstriae on the disk, and minute but evident tubercles on the declivity.In India, four species of genus PageBreakStrohmeyer, 1908Holotype : Kashmir, Pir Panjal, Rost [leg.]// Female sign // Type // Spongocerus kunala Strohm. Determ. Strohmeyer // Coll. Strohmeyer // Holotypus (on red paper). Holotype is deposited in Senckenberg Deutsches Entomologisches Iinstitut (SDEI). Additional material examined: 2 males, Afghan[istan], Nurestan, N. Waygal, 2750 m., 9.7.1972, Kabakov [leg.] ; 1 more male and 2 females with labels: Afghan[istan] Nurestan N. Waygal 3500 m. 2.7.1972 Kabakov [leg.] (A.V. Petrov private collection).Length 2.7 mm, body stout, 2.1 times as long as wide , 3. GeneScolytoplatypus daimio with frontal tufts of hairs attaining epistomal margin . Upper and lateral edges of frons with long hairlike setae; those on upper part until middle of eyes very long and curved towards center of frons. Longest setae convergent in middle of frons, not extending beyond middle of frons; those on lateral edges of flattened lower frontal part become shorter ventrad and are not so curved ; frons il margin .PageBreakPronotum wide and short, 0.8 times as long as wide (1.9 mm vs 2.3 mm), widest in anterior third, vestiture of fine and short hair-like setae denser at anterior margin. Pronotal surface shining, minutely punctured, punctures very shallow and set 3–4 diameters apart from each other. Surface between punctures minutely reticulated. Posterior part of pronotum more lightly coloured than anterolateral angles. Lateral margins of pronotum sharply elevated and propleura strongly concave.Prosternum weakly convex, with an obscure triangular elevation between procoxae, its indistinct pointed apex oriented backwards, and base of triangle forming anterior prosternal margin. This margin is armed with two divergent translucent processes set far apart , 7.Scutellum small, triangular, flush with elytral surface.Elytra 1.38 times as long as wide, 2.0 times as long as pronotum, clearly widened posteriorly. Elytral surface minutely punctured, shining, without signs of reticulation. Elytral punctures not organized in rows and interstriae invisible besides at declivity where all interstriae with exception of the first one are finely carinate. First declivital interstriae at declivity are broadly elevated, not carinate and bear 9–10 tubercles of median size, towards elytral apex evidently divergent. Carina on all other interstriae very low and devoid of tubercles. Elytral declivity convex, not impressed. Posterior dark carinate portion of elytra has yellowish and dense recumbent hair-like setae, anterior light portion of elytra is glabrous.Underside of beetle is uniformly light yellow. Fourth and fifth abdominal sterna with abundant long yellowish hair-like setae protruding beyond abdominal apex.Front femora without tooth above apex. Front tibiae with three widely set spines at lateral edge and with apical mucro. Tarsi long, third segment not bilobed, fifth segment as long as all previous combined.Similar to male, but frons is not impressed and long pubescence at lateral and upper edges of frons is not developed . PronotuPageBreakLength 2.8 mm (HT – 2.96 mm), body stout, 2.15 times as long as wide. Body pale brown, elytral colour pattern essentially as in male, body surface faintly shining.Head brown, darker compared to other body parts. Frons faintly convex, dull. Frontal surface gently shagreened. Lateral frontal parts near eyes faintly shining. Middle part of frons with a sickle-shaped faintly shining impression above mandibles, upper part of impression near center of the frons with two symmetrically placed small tubercles that are seen with difficulty. Slightly elevated dark median line continues from vertex down to epistomal impression. Frontal surface covered by shallow round punctures. Median part of frons covered by sparse, short erect hair-like setae, with somewhat higher density in middle of frons. Short recumbent hair-like setae are concentrated around eyes. Vertex is covered with round shallow punctures, glabrous. Antennae brown with sand-coloured triangular flat club.Scolytoplatypus is acute. Prothorax has an impression for protibiae. Intercoxal piece flat, its lateral margins near apex with bunches of long golden hair-like setae.Pronotum subquadrate, surface faintly shining, covered by microscopic round punctures, with faintly developed flattened tubercles laterally from pronotum center. Slightly anteriorly to pronotum middle a well-developed mycangial pore with a bunch of pale fine hair-like setae is developed. Posterior part of pronotum up to the central pore is covered by pale microscopic hair-like setae; anteriorly to the pore, pronotum with more long pale erect hair-like setae. Lateral pronotal margin as in all Scutellum as in male.Elytra light brown, faintly shining. Elytral base 1.2 times as wide as pronotum base. Maximal elytral width at beginning of declivity. Declivital margins not evenly rounded toward elytral apex, elytral apex somewhat angular. Dorsum of declivity evenly arched towards apex. Elytra 1.4 times as long as wide. Elytral surface with circular punctures. Strial punctures not deepened and of similar size to interstrial punctures, so that striae are obscure, not clearly seen. Interstriae slightly elevated on declivity, first interstriae with minute tubercles, seen with difficulty. Elytral surface covered with microscopic light hair-like setae.PageBreakMetasternum light brown, covered by short pale hair-like setae. Metacoxal cavities with clearly marked raised margin.Abdomen light brown. Sternites covered by minute shallow round punctures and pale hair-like setae; these setae clearly longer on 3-rd, 4-th and 5-th sternites than on 1-st and 2-nd sternites.Legs light yellow. Denticles on outer lateral protibial surface rather strong in the specimen described, but probably intraspecifically variable.Scolytoplatypus daimio, but smaller in size and has another color pattern on elytra (with only poorly developed light dark pattern). Frontal pubescence essentially as in Scolytoplatypus zahradniki Knížek, 2008 or Scolytoplatypus tycon Blandford, 1893. Rather short frontal vestiture easily distinguishes the male Scolytoplatypus kunala from Scolytoplatypus daimio and Scolytoplatypus darjeelingi males in which the longest setae extend in a brush from the upper frontal parts up to the epistoma , by the uniformly dark reddish brown to blackish brown body color, by the presence of conspicuous interstrial ridges on the elytral disk both in males and in females and also by the very short frontal pubescence both in male and in female. Besides, sparse long, erect declivital hair-like setae present in Scolytoplatypus samsinghensis male are absent in Scolytoplatypus kunala. Both Scolytoplatypus lopchuensis and Scolytoplatypus gardneri are slightly smaller in size compared to Scolytoplatypus kunala (2.55–2.60 mm vs. 2.70- 3.00 mm). The female of Scolytoplatypus lopchuensis differs from that of Scolytoplatypus kunala by the absence of the mycetangium pore in the centre of the pronotum, and the female of Scolytoplatypus gardneri is unknown. Males of both species, Scolytoplatypus lopchuensis and Scolytoplatypus gardneri, are different from Scolytoplatypus kunala in having even frontal vestiture without a fringe of hair-like setae on the upper frontal rim with their apices converging towards the centre of the frons. Scolytoplatypus kunala is very closely related to Scolytoplatypus darjeelingi Stebbing sensu Scolytoplatypus darjeelingi has very long tufts of hair-like setae originating from the upper part of the frons (Unfortunately, the recently published key to Indian he frons  and extePageBreakScolytoplatypus species males by Scolytoplatypus kunala and recently described Scolytoplatypus zahradniki.The key to Oriental Scolytoplatypus species females by Scolytoplatypus daimio, and recently described Scolytoplatypus zahradniki Knížek.The key to Oriental


Cellulose waste biomass is the most abundant and attractive substrate for “biorefinery strategies” that are aimed to produce high-value products  by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass.Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation . Two paradigms have been applied for such, so far unsuccessful, attempts: a) “native cellulolytic strategies”, aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) “recombinant cellulolytic strategies”, aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers.By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product  biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates  with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms. Cellulose biomass is the largest waste produced by human activities and the most attractive substrate for “biorefinery strategies” to produce high-value products  , 2. The Clostridium spp. and Ruminococcus spp.) and are by far the most efficient biochemical systems for cellulose degradation ., 29.2 yi 25 fold –31. Rece studied , 32–34. rogenase –36. Howeutyricum.2 metabolic network in strict anaerobes is very complicated. A paradigm for this is the high diversity of clostridial hydrogenases and the existence of multiple forms, likely involved in different functions  within one species [Clostridium kluyveri couples NADH-dependent exergonic reduction of crotonyl-CoA to butyryl-CoA to endergonic reduction of Fd which can be used for H2 production [2 yield decrease in strains in which butyrate production was suppressed by Hdb inactivation. More detailed understanding of the metabolic networks involved in H2 production is definitely essential for successful engineering of H2 hyper producing strains.Actually, the H species . Recentl species , 38. Amo2/mole glucose yield was attained, still H2 production from cellulose would not be competitive with other cellulosic biofuels because of lower yields and higher waste by-product disposal [2/mole glucose are possible by redirecting glucose catabolism through the pentose phosphate pathway or by further acetyl-CoA oxidation through citric acid cycle [in vivo feasibility of such strategies.It is worth to note that, even if 4 mol Hdisposal –28. Theoid cycle –28. MoreMetabolic engineering strategies aimed at developing ethanol and/or butanol hyperproducing strains face two main challenges: a) the construction of solvent tolerant strains; b) the achievement of high solvent yield, titer and productivity.C. thermocellum strains able to tolerate added ethanol concentrations exceeding 50 g/l has been reported. However, the maximum ethanol titer produced by this organism remains at about 25 g/l [Termoanaerobacterium saccharolyticum, showed solvent production titers exceeding the same solvent tolerance exhibited in exogenous addition experiments [Distinct biochemical systems are generally involved in solvent resistance and biosynthesis. “Titer gap” is defined as the difference between the maximum concentration of a compound that is tolerated when it is added to a culture and the maximum concentration of that compound that is biosynthesized by a strain . The devt 25 g/l . A numbeeriments , 39. TheC. acetobutylicum, 50% growth inhibition occur for butanol concentration as high as 7–13 g/l and metabolism ceases once solvent reaches 20 g/l [The main solvent toxicity is attributed to chaotropic effects on biological membranes –41. Comps 20 g/l . Continus 20 g/l –40, 42. i.e. efflux pumps) and changes in cell size and shape [Solvent accumulation within biological membranes increases membrane fluidity and negatively affects membrane processes, e.g. energy generation and nutrient transport . Moreovend shape –41. Howend shape –41. The C. beijerinckii BA101, a butanol-tolerant mutant obtained by chemical methods, showed cell inhibition at 23 g/l butanol rather than 11 g/l typical of the wild-type (WT) strain as well as improved solvent production [C. acetobutylicum resulted in 85% less growth inhibition by butanol and 30% improved butanol production [C. acetobutylicum genomic library led to the identification of 16 genes contributing to butanol tolerance [C. acetobutylicum resulted in 81% increase in cell density in a butanol-challenged cultures. A more straightforward approach to achieve solvent tolerant phenotype is in vivo directed evolution under selective pressure. Cellulolytic C. thermocellum strains able to tolerate ethanol concentrations as high as 80 g/l were developed by adaptation approaches [in vivo evolution [C. acetobutylicum DSM 1731 mutants able to tolerate up to 19 g/l butanol were isolated, although butanol titers obtained by batch fermentations using this strain did not exceed 15.3 g/l [Development of solvent tolerant strains has been performed by different strategies. In some cases solvent tolerant mutants also showed increased solvent production. Utilization of random mutagenesis by chemical or physical methods or by using transposable genetic elements has been reported . C. beijoduction . An alteoduction –47. The oduction . Global oduction . The conolerance . The oveproaches –50. Wholvolution . Applicavolution –52. C. aThe same approaches could be successfully applied for developing other ethanol tolerant or butanol resistant cellulolytic strains.Clostridium sp. (e.g.C. thermocellum and C. cellulolyticum), and fungi, such as Rhizopus, Aspergillus, Neocallimastix, and Trichoderma, can synthesize ethanol although their yields and/or titers and /or productivities are insufficient for direct utilization in CBP [C. acetobutylicum[Several cellulolytic bacteria, such as n in CBP –7, 14. Eutylicum.Rational metabolic engineering to increase ethanol yield and purity has been performed by two main strategies: introduce heterologous gene, and disrupt genes involved in by-product formation that compete with ethanol synthesis .Zymomonas mobilis Pdc and Adh genes, was applied to C. cellulolyticum[C. cellulolyticum. As compared to the wild type strain, acetate production was improved by 93% whereas final ethanol concentration was increased by 53% only [The first strategy, by employing the expression of olyticum. Yet, achdb, involved in butyrate synthesis in C. acetobutylicum, led to a strain with impressive ethanol yield (i.e. 0.38 g/g of glucose) and productivity (i.e. 0.5 g/l/h) [C. acetobutylicum resulted in final ethanol amount of 33 g/l [hdb-deficient C. butyricum was 18-fold higher that in the WT strain [hdb deletion could be applied for improving ethanol production in butyrate producing cellulolytic clostridia, such as C. cellulovorans and C. thermopapyrolyticum[Significant ethanol yield improvement was recently obtained by targeted gene disruption. The inactivation of a single gene, i.e. 5 g/l/h) . Fed-batf 33 g/l . EthanolT strain . hdb delolyticum.pta gene in C. thermocellum did not increase final ethanol amounts and led to severe growth deficiency as concerns both growth rate and final biomass [pta or ack inactivated C. cellulolyticum strains were unsuccessful, so far [Repression of acetate biosynthesis by inactivation of Pta and/or Ack has been suggested as a key modification for driving pyruvate flux towards ethanol . However biomass . Actuall biomass , 35. Ind, so far .C. cellulolyticum H10 double mutant, where both Ldh paralogs, i.e. Ccel_2485 and Ccel_0137, were disrupted showed remarkable production of 0.27 g of ethanol per g of crystalline cellulose [C. thermocellum strain that was constructed by both ldh and pta disruption [Strategies employing Ldh disruption were more successful. A ellulose . Similarsruption . Howeversruption .ack, ldh and pta in the hemicellulolytic T. saccharolyticum[Impressive results were obtained by deletion of olyticum. The engolyticum, 30.Clostridium. The highest butanol amounts are synthesized by C. acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum[C. acetobutylicum or C. beijerinckii strains with improved butanol production (i.e. titers up to 19 g/l) by random mutagenesis or rational metabolic engineering was reported [All natural butanol producers belong to the genus etonicum. Developreported , 45. Howreported –55. By dC. acetobutylicum butanol pathway, i.e. thl, crt, bcd, etfB, etfA, bcd, and adhE genes (E. coli and Pseudomonas putida), but the highest reported butanol titers, i.e. by E. coli BUT2, were 1184 mg/l [C. acetobutilicum enzymes (i.e. thiolase and butyryl-CoA dehydrogenase), have been hypothesized as the main causes of such low butanol production [E. coli by assembling genes from three different organisms [Treponema denticola trans-enoyl-CoA reductase (Ter). Both enzymes catalyze crotonyl-CoA reduction to butyryl-CoA, but Ter reaction is more exoergonic since it does not involve concomitant Fd reduction. Anaerobic fed-batch cultures of recombinant E. coli resulted in the impressive production of 15 g/L of butanol [The expression of the clostridial butanol biosynthetic pathway in heterologous hosts, such as native cellulolytic bacteria, seems an alternative promising strategy. The whole hE genes , was int184 mg/l –57. Inefoduction . A chimerganisms . The clo butanol .C. acetobutylicum butanol biosynthetic pathway in other clostridium sp. hosts, such as C. tyrobutyricum, might be less complex [C. tyrobutyricum possesses its own butyrate biosynthetic pathway, the introduction of the C. acetobutylicum acetaldehyde/alcohol dehydrogenase AdhE2 and either ptb or ack inactivation, significantly diverted carbon flux from acetate and butyrate to butanol. About 10 g/l butanol was obtained by glucose fermentation [C. cellulovorans.Strategies for efficient expression of the  complex . Since Centation . This stin vivo construction of alternative pathways has been reported [C. cellulolyticum by the introduction of an engineered valine biosynthetic pathway [For a long time only the clostridial route to butanol synthesis has been known, but recently the reported , 39. Dir pathway  , an endoglucanase  and a β-glucosidase  . If RCSsSaccharomyces cerevisiae strain that was engineered by introduction of two heterologous cellobiohydrolases only was able to hydrolyze up to 6 g/l of crystalline cellulose in 168 hours [In both free-cellulase and cellulosome biosynthesizing native organisms, optimal biomass degradation is obtained by secretion of non-equimolar ratios of different protein components with Exgs generally among the most abundant enzymatic subunits . Indeed,68 hours . HoweverExgs generally have a tunnel-shaped active site which retains a single glucan chain and prevents it from re-adhering to the cellulose crystal, thus enabling them to catalyze processive crystalline cellulose degradation from either the reducing or non-reducing end , 63. EngC. cellulolyticum and C. thermocellum cohesin-dockerin interaction seems to be non-selective or characterized by slightly different dissociation constants, up to 100-fold differences in binding affinities have been observed in C. josui and C. cellulovorans[A number of experimental observations indicate that cellulosomes are more effective than free enzymes, with special regards to insoluble  cellulose hydrolysis, likely because the improved proximity enhances enzyme synergism –12. Howelovorans, 69.Recent studies showed that linker regions that connect cohesins in scaffoldins are crucial for cellulosome plasticity and catalytic efficiency –71. LinkC. thermocellum minicellulosomes assembled by three-cohesin bearing mini-scaffoldin CipA was about 38% as compared to complexes containing the full-length CipA, which consists of 9 cohesins [Which catalytic efficiency on native substrates can be expected for minicellulosomes with respect to natural complexes? Experimental evidences suggest an almost linear correlation between the number of cohesins that are beared by a scaffoldin and the activity on crystalline cellulose , 74. Thecohesins . Howevercohesins .Trichoderma reesei) usually secrete high amounts (i.e. >1 to 10 g/l) of GHs. Although cellulosome-forming microorganisms biosynthesize lower cellulase levels (i.e. 0.1 g/l), cellulase amount as high as 10 to 20% (w/w) of whole cellular proteins was estimated in C. thermocellum[Cellulolytic aerobic fungi  that maintain family 48 and 9 GHs in a competent state for translocation was hypothesized [C. cellulolyticum CipC scaffoldin prevented toxic effects and triggered enzyme secretion in C. acetobutylicum[S. cerevisiae secreting up 1 g/l of cellobiohydrolases could be engineered [S. cerevisiae, demonstrating that with sufficient efforts secretion of cellulase levels which are comparable to those observed in native cellulolytic strains is possible. Selected strains from Kluyveromyces spp. and S. cerevisiae expressing a library of cellulases were able to directly convert crystalline cellulose up to 0.4-0.5 g/l of ethanol without any externally added enzyme [The main concern of RCSs is to find efficient and reliable secretion methods. The products of genes coding for clostridial cellulosomal components including their original signal peptide, were efficiently secreted by lantarum, 78. In lantarum–80. Nowalantarum. We are revisiae, 78, 81.gineered . Secreted enzyme . Furtherd enzyme .B. subtilis WB800 and L. lactis HtrA mutants, may be required [In order to avoid the hydrolysis of the heterologously expressed cellulases, utilization of protease inactivated strains, such as required , 83.Microbial cell surface binding enhances cellulase activity , 79. HigS. cerevisae by means of agglutinin/flocculin display system [S. cerevisiae was able to ferment amorphous cellulose to ethanol with 62% of the theoretical yield [L. lactis[C. thermocellum have covalently been anchored at the surface of L. lactis by fusing them with the C-terminal anchor motif of the streptococcal M6 protein, a sortase substrate [C. thermocellum scaffoldins and cellulases to the B. subtilis cell wall [5 per cell, were estimated in engineered B. subtilis[S. cerevisiae to improve complex-display level [So far, designer cellulosomes binding up to 3 catalytic subunits have been functionally displayed on the surface of engineered microbial hosts. Such minicellulosomes have covalently been linked to the cell wall of the yeast y system , 84. Tri. lactis. Fragmenubstrate . A similell wall . Higher subtilis. Recentlay level . The recResearch on native cellulolytic strains, suggests that cellulose hydrolysis is not the only bottleneck of cellulose metabolism . As compHowever, improved conversion efficiencies by recombinant microorganisms developed by RCSs, could be obtained by introducing cellodextrin membrane transporters . The uptMetabolic flux analysis could be an essential tool to identifying further bottlenecks of cellulose catabolism in native cellulolytic microorganisms and improve recombinant strains by rational engineering of central metabolic pathways. Alternatively, evolutionary engineering strategies by continuous culture under selective pressure could be applied to optimize cellulose overall metabolism in recombinant microorganisms .C. cellulolyticum and C. thermocellum strains able to ferment crystalline cellulose to ethanol with yields close to 60% of the theoretical maximum and free cellulase-secreting or minicellulosome-displaying yeasts able to directly convert crystalline cellulose to ethanol [Both native recombinant strategies and recombinant cellulolytic strategies have made considerable progress. Outstanding results include the construction of  ethanol , 82, 85.in vivo enzyme catalytic parameters and metabolic fluxes; bioenergetics parameters . Furthermore, this information will enable synthetic biology strategies to design new metabolic pathways for the conversion of cellulosic biomass into a virtually unlimited number of valuable products [Rational engineering of biological systems so as to reach the high performances that are demanded by industrial processes will probably require the use of computational methods which can integrate: gene network regulation data; detailed information on products , 67.


Primula marginata, endemic to the southwestern Alps, includes both hexaploid and dodecaploid cytotypes that show no ecological or morphological differences. We used flow cytometry to determine variation and geographic distribution of cytotypes within and between populations and analyses of chloroplast (cp) and nuclear ribosomal (nr) DNA sequences from the Internal Transcribed Spacer (ITS) region to infer the evolutionary history of the two cytotypes and the auto- vs. allopolyploid origin of dodecaploid populations.Recent studies highlighted the role of Pleistocene climatic cycles in polyploid speciation and of southern Alpine refugia as reservoirs of diversity during glacial maxima. The polyploid P. marginata and P. latifolia, while the latter implies common origins between at least some ITS clones of P. marginata and P. allionii.We did not detect any intermediate cytotypes or variation of ploidy levels within populations. Hexaploids occur in the western and dodecaploids in the eastern part of the distributional range, respectively. The cpDNA and nrDNA topologies are in conflict, for the former supports shared ancestry between P. latifolia and P. marginata, followed by polyploidization between P. marginata-like and P. allionii-like lineages in a southern refugium of the Maritime Alps. The higher proportion of ITS polymorphisms in dodecaploid than in hexaploid accessions of P. marginata and higher total nucleotide diversity of ITS clones in dodecaploid vs. hexaploid individuals sequences are congruent with the allopolyploid hypothesis of dodecaploid origin.Our results suggest an initial episode of chloroplast capture involving ancestral lineages of  Polyploid speciation, characterized by a heritable increase in the copy number of the nuclear genome, has played a key role in plant evolution -7. IndeeThe effects of Pleistocene glacial cycles on alpine and arctic regions presumably played a key role in promoting polyploid speciation. According to the secondary contact model ,18, glacPrimula  suggests that glacial cycles likely influenced the origin of P. marginata. In the present study, we use multiple lines of evidence to reconstruct the likely evolutionary history of P. marginata in relation to Pleistocene climate-dynamics. Our combined results allow us to propose possible superimposed processes that left distinctive signatures on the distribution of cytotypes and phylogenetic structure of the P. marginata chloroplast and nuclear genomes, suggesting events that might have occurred at different points in the evolution of the lineage. The complementarity of information between cytotype variation and nuclear and organellar genomes has proven indispensible to elucidate complex evolutionary histories in other boreal taxa, e.g. Vaccinium [Artemisia [Nymphaea [The Pleistocene time frame for the evolution of hylogeny  and a Praccinium , Artemisrtemisia , and NymNymphaea .P. marginata. Failure to detect any intermediate cytotypes nor any variation of ploidy levels within populations  than to the clades supported by the cpDNA phylogeny  share more recent common ancestors with sequences from hexaploid or dodecaploid populations of P. marginata , which form a well-supported clade . Explanm [Viola -61. Recim [Viola  predictsm [Viola ,64. Convm [Viola ,65,66. Am [Viola ,67). Thes Figure B. The ins Figure A. The oc Maximum , might hs Figure . Howeveratifolia  implies atifolia .P. marginata .P. marginata does not warrant taxonomic distinction, as already observed in other species  and P. parryi and P. cusickiana (sect. Parryi). Therefore, a total of 47 accessions were used for cpDNA analysis. A total of 15 accessions belonging to selected populations of P. marginataP. allionii and P. latifolia were used for nrDNA analysis  supplemented with 1μg/μl DAPI  for 20 min at room temperature and analyzed with the flow cytometer Cell Lab Quanta™ SC MPL . Isolated nuclei were excited by a Mercury arc UV lamp and the fluorescence intensity of 20000 nuclei was recorded. Ploidy levels were estimated according to the principle that peaks vary proportionally to the DNA content [Flow-cytometric analyses were performed on fresh leaf tissue from a total of 100 individuals Table . The hap4.88 pg, ,75) was I buffer , modifie content .ndhF-rpl32psbD-trnTtrnD-trnT of the cpDNA were amplified using primers -R; trnT(GGU), trnD(GUC)F) and protocols designed by Shaw et al. [P. marginata detected no intra-population differences between the selected cpDNA target regions, therefore only one individual per population was included in subsequent cpDNA analyses.Total DNA was extracted from leaf tissue using the DNeasy Plant Mini Kit , following the manufacturer’s instructions. The spacers w et al. ,79. Ampl2), 2 mM MgCl2, 0.2 mM dNTP’s, 0.2 μM of each primer, 1 unit of Taq Polymerase. The final concentration of MgCl2 was 3 mM. To decrease the formation of secondary structures and the amplification of pseudogenes, 5% of dimethyl sulfoxide (DMSO) was added to the amplification mixture, as recommended by Álvarez & Wendel [The nuclear regions ITS1 and ITS2 were amplified using primers ITS-4 and ITS-Leu and protocols of Baum et al. . Amplifi& Wendel . AmplifiP. marginata, three populations of P. latifolia, and two populations of P. allionii  were used for transformation. The plasmid vector pJET was added to the cells, and these were shocked at 42°C for 30 seconds. After addition of 125 ml of LB medium, cells were incubated at 37°C for one hour and then spread on 50 μg/ml Ampicillin-agar plates. Twenty colonies per cloning reaction were selected to be PCR-amplified and then sequenced using the pJET1.2 primers.To check for intra-individual variation of ITS repeats, we cloned ITS from one individual each of ten populations of ii Table . To reduAll PCR products were cleaned by adding 1 μl of Calf Intestine Alcaline Phosphatase (CIAP) and 0.5 μl of Exonuclease I  to each sample at 37°C for 15 min, followed by 15 min at 94°C to denature the enzymes. Cycle sequencing reactions were performed with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit , using 1 μl of BigDye terminator per reaction. Cycle-sequencing products were purified on 96-well multiscreen filtration plates  to remove unincorporated BigDye terminator and then analyzed on an ABI 3100 Genetic Analyzer . The obtained sequences were edited and assembled using Sequencher 4.2 software .Rhododendron kanehirai . Sequen Z00044, ; the res Z00044, . Gaps we Z00044,  and addePhylogenies were inferred from the cpDNA and nrDNA matrices independently using both Maximum Parsimony (MP) and Bayesian Inference (BI). MP trees were inferred in PAUP 4.0b10 . A heuripsbD-trnT, the GTR + I + Γ model for the ndhF-rpl32, and the HKY + I + Γ model for the trnD-trnT. Since the models selected for the two last regions could lead to parameter interaction, which might cause difficulty in reaching stationarity [ndhF-rpl32R and HKY + Γ for trnD-trnT  in MrMoionarity , we testionarity  on the Cionarity  and consionarity , examiniionarity  convergeionarity . BFs werionarity . BFs witThe topologies inferred from analyses of the cpDNA and nrDNA matrices were not identical. Several methods have been devised to estimate whether incongruence between trees is significant or not ,96. HoweP. marginataP. latifolia, and P. allionii. A polymorphic site in one accession was identified as additive when different nucleotides at the site were also found at the same site in at least one other accession in the data set [A matrix including a total of 147 cloned nrDNA sequences was used to analyze the partitioning of infra-individual variation among nuclear ribotypes in data set . If the The structure of variation in the matrix of cloned nrDNA sequences was analyzed by Principal Coordinate Analysis (PCoA). We first calculated the pairwise distances in PAUP 4.0b10  using thThe possible occurrence of recombinant sequences among the nrDNA clones was determined using the program RDP3 , which dP. marginata/P. latifolia and P. allionii clades strongly supported by the cpDNA phylogeny; H2) current taxonomical classification ; H3) taxonomical classification with the subdivision of P. marginata in two cytotypes .We performed an analysis of molecular variance (AMOVA) based on the K2P model of nucleotide substitution and 1000 iterations , to tesP. marginata, the nucleotide diversity [In order to discriminate between the potential autopolyploid vs. allopolyploid origin of dodecaplois in iversity  of cloneiversity . Accordiiversity , a nucleiversity .The authors declare that they have no competing interests.GC, EC and LM conceived the study. LG performed all lab work and analyses. GC, EC and LG provided crucial samples and contributed to data interpretation. GC, EC and LM wrote the paper. All authors read and approved the final manuscript.Table S1. Sectional affiliations, species names, codes, and ploidy levels of species of sect. Auricula included in cpDNA phylogeny. Table S2. Species names, localities, codes and gene bank numbers of accessions of Primula sect. Auricula included in cpDNA phylogeny. Table S3. Species names, codes and GenBank/EBI accession numbers of Primula allionii, P. latifolia and P. marginata ITS clones included in nrDNA phylogeny. Table S4. Models of evolution, Base frequencies, and rates of substitutions estimated with MrModeltest for the different partitions used in this study. Table S5. Bayes factors across alternative models for two cpDNA data partitions. Table S6. Recombination analysis inferred among the nrDNA clones using the program RDP3. Figure S1. Maximum Parsimony 50% majority-rule consensus tree inferred from cpDNA sequences of Primula sect. Auricula accessions. Figure S2. Maximum Parsimony 50% majority-rule consensus tree of ITS clones.Click here for file


Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay. Listeria monocytogenes, a gram-positive pathogenic bacterium [L. monocytogenes is the only species in the genus Listeria that can infect humans [L. monocytogenes that is incapable of secreting LLO (Δhly) is not pathogenic [Listeriosis is a foodborne illness caused by infection with acterium . L. monot humans . After tt humans . L. monothogenic . Listerithogenic . LLO is thogenic . LLO alsthogenic , as wellthogenic   have many functions. They can be used to study structure/function of a protein, pathogenesis of an organism, and/or quantitative analysis of a protein , 12. To vailable –14. Sitehttp://www.biocompare.com/pfu/110627/soids/371711/Assay_Kit/listeriolysin_O and http://www.mybiosource.com/datasheet.php?products_id=705426) [There are two companies that manufacture ELISAs (BioCompare and MyBioSource) to detect LLO (=705426) , 16. In http://www.ncbi.nlm.nih.gov/protein/) to acquire the Listeria proteins listeriolysin O (LLO) (NP_463733) and ivanolysin O (P31831) [http://www.rcsb.org) at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi. Suilysin (pdb: 3HVN) [In this study we used the NCBI GenBank Protein database (P31831) . The acc31 . The http://www.ebi.ac.uk/Tools/msa/clustalw2/ [http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein [Pairwise sequence alignments of LLO were generated using the ClustalW2 Multiple Sequence Alignment at ustalw2/ . The pur=protein .http://zhanglab.ccmb.med.umich.edu/I-TASSER/ [To generate a molecular model of LLO, we used the molecular modeling program I-TASSER -TASSER/ , 22. We -TASSER/ , 23, 24.-TASSER/ . The C-shttp://polyview.cchmc.org/polyview3d.html) [Amino acids 60–525 of LLO were used to predict a three-dimensional model since these residues correspond to amino acids 32–242 and 245–499 in SLY. Molecular models were prepared in different orientations using POLYVIEW 3D  also uses physicochemical properties to predict B-cell epitopes similar to BepiPred [Important properties for predicting B-cell epitopes are flexibility, hydrophilicity, and linear epitope predictions. We analyzed the linear epitope predictions, flexibility, and hydrophilicity of LLO using the BepiPred linear epitope prediction , KarplusBepiPred .Listeria species revealed that it is highly conserved with only six polymorphic sites (L. seeligeri) and ivanolysin O (L. ivanovii), respectively. In contrast, NCBI-BLASTn searches against Listeria species L. grayi, L. innocua, L. marthii genomes (members of the genus Listeria that have their genomes sequenced) yielded no LLO homologs. The genomes of species L. fleischmannii and L. rocourtiae have not been sequenced.An alignment of all completely sequenced amino acid coding sequences of LLO from several ic sites . Howeveric sites . We usedSince there is no LLO crystal structure, we created a molecular model of LLO to visualize the locations of the predicted B-cell epitopes . The SLYWe used amino acids 60–525 of LLO to predict a three-dimensional model. In contrast, we used amino acids 28–529 to analyze the immunogenicity of LLO since the latter amino acids comprise the full-length mature protein . Three dWe also evaluated discontinuous epitopes in the LLO molecular model that we created. ElliPro predicted three discontinuous epitopes with PI scores higher than our cutoff: two in domain 4 and one in domain 1 . DiscontIn terms of antibody recognition, changing one amino acid in an antibody epitope can dramatically decrease the antigen-antibody interaction . Four ofImmunogenic epitopes are accessible on the protein surface, located in flexible regions, and often overlap . Our preIn some instances, when a protein has multiple overlapping B-cell epitopes it is referred to as immunodominant . We predBioinformatics has been used for many purposes, such as vaccine design, characterization of novel genes, and the discovery of novel viruses –39. ReceSeveral characteristics make domain 4 an attractive immunogenic candidate. Domain 4 is the only continuous domain in LLO . Domain L. seeligeri and ivanolysin O expressed by L. ivanovii. However, L. seeligeri does not infect humans, and L. ivanovii is not pathogenic [L. seeligeri, it is unlikely that the latter toxins would be present in contaminated food products consumed by humans. Thus, even if they readily cross-react with LLO L. seeligeri and ivanolysin O, antibodies capable of detecting LLO in an ELISA would not be likely to produce a false positive in food screening or clinical tests. Previous molecular models of LLO used PFO as a template [Bioinformatics of LLO demonstrates that its closest homologs are the LLO protein of thogenic . Therefotemplate , 42. OurL. monocytogenes as well as develop a diagnostic test for LLO detection. This analysis is important because it is focusing our antibody development efforts on domain 4 as an immunogen.Bioinformatics of LLO predicted that most of the epitopes deemed likely to be immunogenic were located in domain 4. Furthermore, since it is the only domain in LLO that is continuous, we believe that it has a high probability of eliciting antibodies that could be used to study the pathogenesis of


Helicobacter pylori (H. pylori) infection is confirmed to correlate with chronic gastritis, peptic ulcer disease, Mucosa Associated Lymphoid Tissue (MALT)-lymphoma, precancerous changes in the stomach , and gastric cancer. At the same time, H. pylori eludes the immunological response evoked by the host. This chronic infection has the local production and systemic diffusion of proinflammatory cytokines, which may influence the remote organic systems and result in extragastric manifestations [Currently,stations  , iron deficiency anemia (IDA), and vitamin B12 deficiency [ H. pylori might influence the clinical outcome of these diseases. However, many of these reports suffer from being case reports or case series without adequate controls.Several studies performed during the past years have supported the possible role forficiency . The eff H. pylori eradication resulting in increasing the platelet count in adult patients with primary immune thrombocytopenia (ITP) has been confirmed [ H. pylori infection as a cause of unexplained sideropenic anemia (refractory IDA) by several mechanisms [ H. pylori infection to be sought in IDA patients. Other hematological diseases possibly related with H. pylori included monoclonal gammopathy, megaloblastic anemia, and myelodysplastic syndrome [Theonfirmed , 4. Morechanisms . So, recsyndrome . H. pylori has significant interactions with the immune system. Recent epidemiological data suggest that aggressively eradicating H. pylori infection might be related to an increase in autoimmune diseases [ H. pylori have acquired several abilities that help them escape clearance through the host immune system. Then H. pylori interacts with the immune system and results in its downregulation. However, controversial results were reported in several studies. We need further research studies focusing on the possible impact of H. pylori on autoimmune diseases.Many previous studies stated that chronic infection withdiseases , but the H. pylori and outcome of cardiovascular disease is also an important issue. Previous studies have surveyed the association between H. pylori infection and coronary artery disease (CAD) [ H. pylori infection in the pathogenesis of CAD include persistent local or systemic inflammation and initiating autoimmune responses [ H. pylori eradication in these patients.The relationship between seropositivity forse (CAD) . The posesponses . However H. pylori infection is five to ten times stronger than with passive smoking exposure [ H. pylori might be a risk factor among non-smoking-related lung cancer. Many possible hypotheses have been proposed including the following: (a) the mechanisms may vary by both H. pylori strain and subtype of lung cancer; (b) H. pylori infection status/eradication should influence the clinical outcome of lung cancer; and (c) this association should be influenced by other factors [Some studies have disclosed that the association of lung cancer risk withexposure . It rais factors . However H. pylori in dermatological diseases is still a controversial subject. The association between chronic urticaria (CU) and H. pylori has been found by some research groups [ H. pylori eradication [ H. pylori eradication in patients with CU was weak [The role ofh groups . The evidication . But recwas weak . Other s H. pylori eradication [ H. pylori were noted in patients with type 1 diabetes (T1DM) and autoimmune thyroiditis (AT). In their results, H. pylori infection could be considered as an environmental trigger for development of AT in T1DM. They suggested that young patients with T1DM should be screened for H. pylori infection [One recent meta-analysis stated that Type 2 diabetes and insulin use in diabetic patients are significantly associated with a higher incidence ofdication . Previounfection . H. pylori and systemic disease needs more attention. The population-based strategies for H. pylori eradication in people with low prevalence are unlikely to be cost-effective, but this management might be necessary in people with high risk of developing systemic disease. The challenge we face is to investigate whether, at what magnitude, and in which direction, H. pylori may be linked to systemic diseases and in which populations. We hope that this special issue will be helpful in the possible pathogenesis of H. pylori related extragastric manifestations.When discussing medical economic policy, the relationship betweenChao-Hung KuoChao-Hung KuoYen-Hsu ChenYen-Hsu ChenKhean-Lee GohKhean-Lee GohLin-Li ChangLin-Li Chang


Hyalomma aegyptium is a hard-tick with a typical three-host life cycle. The main hosts are Palearctic tortoises of genus Testudo. However, other hosts can be used by immature ticks for feeding in natural conditions. Given this complex ecology and multiple host use, the circulation of pathogens by H. aegyptium between various hosts can be important from epidemiological point of view. The aim of this study was to evaluate the role of H. aegyptium as natural carrier of four important zoonotic pathogens.H. aegyptium ticks were collected from 45 Spur-thighed tortoises, Testudo graeca in Romania. DNA was extracted individually from each tick using a commercial kit. DNA was examined for the presence of specific sequences of Borrelia burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia canis and Coxiella burnetii by PCR, according to previously described protocols.From 2008 to 2011, 448 H. aegyptium revealed the presence of A. phagocytophilum (18.8%), E. canis (14.1%) and C. burnetii (10%). 32.4% of the ticks were infected with at least one pathogen and 9.8% had co-infections. The stages most frequently infected were nymphs (50%) followed by males (33.9%) and females (27%). The number of tortoises which harboured infected ticks was 27/45 examined (60%). From all tested T. graeca, 40% harboured ticks infected with A. phagocytophilum, 46.7% had ticks infected with E. canis and 33.3% had ticks with C. burnetii. This study reports for the first time the presence of A. phagocytophilum and E. canis in H. aegyptium.PCR analysis of H. aegyptium raises the question of their epidemiologic importance in disease ecology. As tortoises are unlikely to be reservoir hosts for A. phagocytophilum and E. canis and both these pathogens are common in H. aegyptium, this is an important indication for (1) a possible increased host-switching behaviour of these ticks to competent reservoir hosts (i.e. hedgehogs) and (2) transstadial transmission. Furthermore, if we consider also the presence of C. burnetii, we conclude that T. graeca and its ticks should be evaluated more seriously when assessing the eco-epidemiology of zoonotic diseases.The presence and relatively high prevalence of three important zoonotic pathogens in  Hyalomma aegyptium  is a hard-tick with a typical three-host life cycle [Testudo[H. aegyptium are less host-specific and feed on various vertebrates: tortoises, lizards, birds, small mammals and even humans [ [Testudo. Hence,  [Testudo-5. Howev [Testudo,7. Nevern humans .H. aegyptium between reservoir hosts in natural cycles can be important from an epidemiological point of view. Determining the vectorial capacity of a tick to a certain pathogen is questionable if based only on pathogen detection (mainly by PCR), without experimental trials [H. aegyptium: Theileria annulata[Borrelia turcica[Rickettsia spp. and Borrelia burgdorferi s.l. [H. aegyptium was shown for several pathogens: Hemolivia mauritanica[Hepatozoon kisrae[Rickettsia aeschlimannii[Coxiella burnetii[Given this complex ecology and multiple host use, the transmission of pathogens by l trials . Several annulata, Borrelia turcica, Ricketteri s.l. . Experimuritanica, Hepatozon kisrae, Ricketthlimannii and Coxi burnetii.H. aegyptium were found on only two hosts, the Spur-thighed tortoise, Testudo graeca and the Northern White-breasted hedgehog, Erinaceus roumanicus and its distribution matches the one of the tortoise host [H. aegyptium) studies regarding the presence of zoonotic agents are of particular interest because of the potential role of these ticks to maintain and cycle the pathogens in nature.In Romania, all stages of ise host . Reptileise host ,21. Partise host ,23. MoreA. phagocytophilum and E. canis and Majláthová et al. [Ixodes ricinus ticks infected with Borrelia burgdorferi s.l. Moreover, H. aegyptium was shown to be a competent vector for C. burnetii under laboratory conditions but the natural role of this tick in the ecology of Q fever was never assessed. Hence, the aim of the present study was to evaluate the role of H. aegyptium as natural carrier of four important zoonotic pathogens: Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Ehrlichia canis and Coxiella burnetii.In Romania, there are few studies on the epidemiology and distribution of zoonotic tick-borne pathogens. In the same geographical area (Tulcea and Constanța County), Mircean et al.  reportedá et al.  found IxH. aegyptium ticks  were collected from Spur-thighed tortoises, Testudo graeca ibera  forests  and examined for the presence of the specific fragment under UV light (Bio-Rad BioDoc-It™ Imagine System). DNA fragment size was compared with a standard molecular weight, 100 bp DNA ladder (Fermentas). Distilled water was used as negative control.DNA was extracted individually from each tick using a commercial DNA extraction kit , according to the manufacturer’s instructions. Extracted DNA was examined for the presence of specific sequences of B. burgdorferi s.l., A. phagocytophilum, E. canis and C. burnetii DNA and respective co-infections in H. aegyptium were established. These parameters were determined according to sex and developmental stage of the ticks  and geographic location. The difference of prevalence among groups was statistically analysed by chi-squared independence. A p value of <0.05 was statistically significant. All statistical analyses were performed using the EpiInfo 2000 software.Frequency, prevalence and its 95% confidence interval of H. aegyptium revealed the presence of three pathogens  from the four tested (no samples were positive for B. burgdorferi s.l.). From the total number of ticks examined, 145 were infected with at least one pathogen . The frequency and prevalence of each detected pathogen are shown in Table A. phagocytophilum (18.8%), followed by E. canis (14.1%) and C. burnetii (10%). The developmental stages most frequently infected with at least one pathogen were nymphs  followed by adult males  and females .PCR analysis of p < 0.00001). The only region where co-infected ticks were found was Măcin Mountains, with 10.9% from the total number of ticks . The most frequent co-infection type was A. phagocytophilum-E. canis , followed by A. phagocytophilum-C. burnetii .The prevalence of the co-infections was 9.8% . The prevalence of co-infections was higher in nymphs  and males  than in other developmental stages  harboured ticks infected with A. phagocytophilum, 21 (46.7%) had ticks infected with E. canis and 15 (33.3%) had ticks with C. burnetii. Interestingly, from the 27 tortoises which harboured infected ticks, 18 had ticks infected independently or co-infected with at least two pathogens (Table The number of tortoises which harboured infected ticks (regardless the pathogen) was 27 out of 45 examined . From all tested H. aegyptium, the trend is a decreasing geographical range [H. aegyptium is reported to alternatively feed on various other hosts, mainly during their pre-imaginal stages, evaluation of its zoonotic pathogen burden is of particular interest.Ticks are vectors of important pathogens of humans and animals and serve as indicators of infection in nature . The geoal range . Howeveral range . As H. aErinaceus spp.) are important mainly in synanthropic environments as reservoir hosts for important human pathogens like A. phagocytophilum, Babesia spp. [B. burgdorferi s.l. [H. aegyptium occasionally feeds on hedgehogs and it can potentially attack humans [Regarding their role in the ecology of zoonotic infectious diseases, tortoises and their ticks have received significantly less attention compared to mammals and birds. Among small mammals, hedgehogs  in fully engorged H. aegyptium collected from tortoises can be theoretically caused by two factors: (1) the infection was acquired by ticks during an earlier developmental stage feeding on competent reservoir hosts or (2) tortoises are competent reservoir hosts. The second hypothesis is less probable, as several studies showed that reptiles are not competent reservoir hosts for this pathogen [Anaplasma spp. in engorged Hyalomma lusitanicum and H. marginatum collected on domestic mammals in Sicily was much lower (~1%) [H. aegyptium is able to transstadially pass in A. phagocytophilum. However, the probable lack of transovarial transmission of A. phagocytophilum in ticks [d humans . This ispathogen ,43. Morepathogen . Prevaleer (~1%) . All thein ticks  confers Ehrlichia includes five species [E. canis is found in Europe. This study reports the presence of E. canis for the first time in H. aegyptium, with a noteworthy prevalence. The only recognized vector for E. canis is Rhipicephalus sanguineus. In Romania, the distribution range of H. aegyptium overlaps with the distribution of R. sanguineus[R. sanguineus feeds mainly on dogs [E. roumanicus) [E. canis from R. sanguineus to H. aegyptium using hedgehogs as bridging hosts.The genus  species , but onlanguineus. Althoug on dogs , in Romamanicus) . This isC. burnetii was present only in Măcin Mountains with a relatively high prevalence in ticks (37.9%). An interesting aspect in this area was that all tortoises harbouring C. burnetii-infected ticks had also A. phagocytophilum-infected ticks and 72.7% of these tortoises had ticks infected with E. canis, too. Additionally, out of the 11 tortoises infested with C. burnetii-infected ticks, 10 (90.9%) had co-infected ticks. Q fever affects a wide range of domestic and free living mammals, birds, reptiles, fish, and arthropods, as well as humans [C. burnetii, has been identified in over 40 tick species [Hyalomma aegyptium was shown to have an unquestionable potential in the epidemiology of Q fever natural foci [C. burnetii vertically  and horizontally  [The Q fever agent, s humans ,50. The  species . Hyalommral foci . Ticks t saliva)  but also saliva) . Sharing saliva)  to have  saliva) .Anaplasma phagocytophilum and E. canis being found in all three locations while C. burnetii was only present in one. This variance may be caused by the different habitat associations and its host-fauna composition. The two forested sample sites have high occurrence rates of small mammals, especially hedgehogs, while the third location is primarily used by small domestic ruminants [Coxiella burnetii is commonly reported in sheep and goats [We found a marked dissimilarity between the occurrences of individual pathogens in ticks among the different locations: uminants . Coxiellnd goats , hence ind goats .H. aegyptium raises the question of their epidemiologic importance in disease ecology. As tortoises are unlikely reservoir hosts for A. phagocytophilum and E. canis and both these pathogens are common in H. aegyptium, this is an important indication for (1) a possible increased host-switching behaviour of these ticks to competent reservoir hosts (i.e. hedgehogs) and (2) transstadial transmission. Furthermore, if considering also the presence of C. burnetii, it can be concluded that T. graeca and its ticks should be evaluated more seriously when assessing the eco-epidemiology of zoonotic diseases.The presence and relatively high prevalence of three important zoonotic pathogens in All authors have seen and approved the manuscript and declare that they have no competing interest.PAI wrote the manuscript and made the statistical analysis; MIA and DMO identified the ticks; PAI, MIA and KZ performed the DNA extraction and PCR; MAD study design, MS concept and wrote the discussion, MAD, DG and SAD collected the samples; LM performed DNA extraction; GCM research project coordinator; CV team coordinator. All authors read and approved the final manuscript.Origin of samples of Hyalomma aegyptium  used in this study.Click here for file


Plasmodium vivax threatens nearly half the world’s population and is a significant impediment to achievement of the millennium development goals. It is an important, but incompletely understood, cause of anaemia. This review synthesizes current evidence on the epidemiology, pathogenesis, treatment and consequences of vivax-associated anaemia. Young children are at high risk of clinically significant and potentially severe vivax-associated anaemia, particularly in countries where transmission is intense and relapses are frequent. Despite reaching lower densities than Plasmodium falciparum, Plasmodium vivax causes similar absolute reduction in red blood cell mass because it results in proportionately greater removal of uninfected red blood cells. Severe vivax anaemia is associated with substantial indirect mortality and morbidity through impaired resilience to co-morbidities, obstetric complications and requirement for blood transfusion. Anaemia can be averted by early and effective anti-malarial treatment. Plasmodium vivax threatens approximately 2.8 billion people globally and, because of its particular biological characteristics, will be more difficult to eradicate than Plasmodium falciparum[P. falciparum infection; observations that challenge the notion that vivax malaria is a benign disease. The causative role of P. vivax in some of these severe manifestations remains to be proven and many are sufficiently rare that they are unlikely to represent significant public health problems. Anaemia, on the other hand, is a common and frequently severe consequence of vivax infection [P. falciparum). The impact of anti-malarial treatment on haematological recovery is also described.alciparum-9. Over alciparum-13, survalciparum-17 and ralciparum,18-24 hanfection -16,25-27P. vivax is transmitted throughout the tropics in much the same geographical pattern as P. falciparum[P. vivax is largely absent. This has been attributed to selection over many millennia of individuals lacking the Duffy red blood cell surface antigen - an important mediator of P. vivax red cell invasion [P. vivax may have escaped its dependence on the Duffy antigen in certain regions [Endemic alciparum,8. The minvasion ,28. Mena regions .P. falciparum and P. vivax co-exist, both the incidence of infection and severity of haematological morbidity attributable to P. vivax tends to peak at a younger age than for falciparum malaria and in many regions, before the end of the second year of life [P. vivax transmission, approximately a quarter of infants hospitalized with vivax malaria have severe anaemia (haemoglobin less than 5 g/dL), whereas in individuals 45 years or older this proportion decreases to less than 5% [Where  of life ,27,30-32 of life ,34. In P than 5% . InfantsP. vivax malaria than males [P. vivax infection have a ~2-fold higher risk of moderate anaemia than uninfected pregnant women [Females are at greater risk of hospitalization with an males  and in ont women -37. WhetP. vivax parasitaemia in cross sectional studies but their effect on vivax anaemia is unknown [P. vivax parasitaemia and vivax-associated anaemia [P. vivax infections [P. vivax in Southern Papua compared with elsewhere in New Guinea [Several red blood cell and haemoglobin variants have been associated with reduced susceptibility to anaemia caused by falciparum malaria, the best known being sickle cell anaemia. Hypothesized mechanisms for this protection include reduced red blood cell invasion, relative inhibition of intracellular parasite replication, more efficient removal of infected red blood cells by the spleen and more efficient presentation of parasite antigens to the immune system -40. The  unknown -44. Prel anaemia ,46 where anaemia . Glucosefections ,49. The w Guinea .P. falciparum malaria coinfection has been shown to cause an additive reduction in haemoglobin in children and pregnant mothers when compared with monoinfection with either parasite alone [P. falciparum parasitaemia but evidence is conflicting [P. vivax infection and vivax-associated anaemia. Boel and colleagues showed a positive association between Ascaris lumbricoides infection during pregnancy and risk of vivax malaria [P. vivax infection in children between five and 14 years of age was attenuated by coinfection with hookworm, A. lumbricoides and Trichuris trichuria[Gastrointestinal helminth infections may cause anaemia through chronic blood loss, but the interaction with malarial anaemia is complex. In Africa, hookworm and te alone . Helmintflicting ,52. Few  malaria . Anothertrichuria.P. vivax malaria. Iron deficiency is protective against P. falciparum infection whereas iron supplementation increases the risk of falciparum malaria and high parasitaemia infections [P. vivax infection compared to those who did not receive supplementation [The haematological effects of chronic blood loss caused by intestinal helminthiasis are exacerbated by nutritional iron deficiency which in turn may interact with the haematological effects of fections ,56. The entation . A randoentation . In Papuentation . Large, Plasmodium species is the red blood cell. Plasmodium vivax has a very strong predilection for red blood cells that have emerged from the bone marrow within the last 14 days, in particular reticulocytes, whereas P. falciparum has only a moderate predilection for young red blood cells and significant ability to infect older cells [P. vivax infections is lower than in symptomatic P. falciparum malaria and parasitaemia >2% is rare [P. vivax infection should be sufficient to lead to extreme anaemia over a period of several months by choking the supply of mature red blood cells [P. falciparum malaria, these include increased removal of infected, and to a greater extent, uninfected red blood cells from circulation, compounded, in subacute and chronic forms, by impaired erythropoiesis [The primary target of human er cells -62. The  is rare . Despiteod cells -65. Direod cells . These oopoiesis -70. The opoiesis ,66-104).P. vivax malaria, approximately 34 non-infected cells are cleared for every one infected cell [P. falciparum malaria, this ratio is closer to 8 to 1 [Although parasitaemia is typically lower in vivax compared with falciparum infections, the absolute number of red blood cells removed from circulation, and hence the degree of anaemia resulting from infection by the two species, is often similar ,26,105. ted cell  whereas o 8 to 1 ,70. Theso 8 to 1 ,73 and TP. falciparum become less deformable than uninfected red cells and have reduced capacity to pass through narrow inter-endothelial slits in the wall of splenic sinuses (mean dimensions 1.89 × 0.65 μm) [P. falciparum and P. vivax have evolved two different means of escaping splenic filtration. In both vivax and falciparum malaria, parasitized, and possibly non-parasitized, red cells are hypothesized to be more fragile than red cells in non-infected individuals and more prone to damage from shear stresses [As in falciparum malaria, vivax-infected erythrocytes adhere to uninfected red blood cells (rosetting) ,107 but 0.65 μm) -87,110. 0.65 μm) ,110,111.stresses ,110,112.stresses .P. vivax antigens enhances the detection and removal of infected and abnormal uninfected red blood cells [P. vivax than P. falciparum and may partially explain the greater proportional removal of non-parasitized cells and lower fever threshold in vivax malaria [P. vivax infections [P. falciparum causes altered expression of complement components and deposition of parasite proteins on infected and uninfected red blood cells [In addition to these mechanical processes, activation of the innate, cell-mediated and humoral immune systems in response to the presence of od cells ,75,113.  malaria -116. Thi malaria ,97 and cfections . Macrophfections -121. To fections ,122. Infod cells ,92  is likely to be an important determinant of haematological impairment. Untreated primary sporozoite-induced infection in non-immune adult patients with neurosyphilis typically results in paroxysmal fever lasting 3–8 weeks followed by an extended period of increasing clinical ‘tolerance’ to persistent parasitaemia . Anti-papatients . There wpatients .P. vivax strain, whether due to reinfection, recrudescence or relapse, during the period of parasite tolerance may result in a transient rise in parasitaemia but rarely recurrent or exacerbated symptoms [Repeat infection with the homologous symptoms -144. Homsymptoms ) from unsymptoms ,145-148.P. vivax strains and the entomological inoculation rate, both of which are also likely to affect the rate of genetic recombination and hence parasite population diversity [Heterologous immunity however, is weak and exposure to a new strain following primary infection will usually result in clinical malaria with non-specific immune system activation, recurrent symptoms and presumably a repeat haematological insult ,149. If iversity .P. vivax relapses are known to be caused by heterologous strains (with regard to the previous vivax infection) [P. vivax transmission and/or extensive parasite strain diversity. In such regions, there will also be a greater chance of simultaneous inoculation of two different strains of P. vivax which has been shown to cause deficient homologous immunity and therefore presumably greater susceptibility to the haematological effects of future homologous infection [In endemic regions, a high proportion of fection) ,153. Thinfection .P. vivax strains have markedly different relapse patterns and in some instances may not even cause a primary infection [Different nfection ,141,155.nfection . In tempnfection . TropicaThe virulence of an individual parasite strain may also modulate its haematological effects. There is some evidence that the Chesson strain causes fever at lower parasitaemia than other strains and therefore that it may be more immunogenic . In 1947P. vivax on the island of New Guinea where the frequently relapsing Chesson strain is endemic and local populations experience particularly severe haematological impairment from vivax malaria [Given the aforementioned evidence, one might predict that the severest haematological impact of vivax malaria would be seen in tropical regions with high entomological inoculation rates and virulent circulating strains. These features are perhaps best exemplified by  malaria -16,27. I malaria .P. vivax infection are also exposed to P. falciparum and co-infection (not necessarily patent) with these two species is common [P. falciparum and P. vivax has been shown to attenuate the risk of severe anaemia associated with falciparum malaria – possibly due to some degree of cross-species immunity [P. vivax infection, however minor, is likely to have a protective effect. In New Guinea, where transmission is more intense, severe anaemia in those older than one year is more likely to be the result of repeated or continuous infections due to either species. In this situation, the haematological effects of infection with both species are more likely to be additive and any immunomodulatory effects relatively minor.Most populations at risk of s common ,157-159.immunity ,160-162.immunity ,16,27. TP. vivax parasite biomass. They also temporarily reduce red blood cell production [Early treatment of malaria can truncate the impending reduction in haemoglobin and accelerate haematological recovery ,163,164.oduction -168. In oduction ,170. Theoduction .P. vivax strains relapse as often as every three weeks.Complete removal of blood stage parasites following blood schizontocidal treatment allows faster haematological recovery Plasmodium vivax has developed high-grade resistance to chloroquine in parts of Oceania, Asia, Africa and Latin America [P. falciparum malaria [ America ,5 and su America . Clinica malaria ,164,177)P. vivax relapses and thus reduce the total haematological impact of a given infection. Unfortunately this medication has the potential to cause lysis of old red blood cells in all patients, but particularly those with glucose-6-phosphate dehydrogenase deficiency [Primaquine is a hypnozoitocidal drug that, if administered correctly, can prevent ficiency ,179. G6Pficiency . Althougficiency ,182. Genficiency ,179. Indficiency  whereas ficiency . Weekly,ficiency  whilst rficiency  though aficiency . Since tficiency ,187.Plasmodium vivax infection on haemoglobin concentration varies from negligible to dramatic [The impact of dramatic ,188,189.P. vivax parasitaemia was 8.7 g/dL, 0.3 g/dL lower than the equivalent value for those infected with P. falciparum[In Papua New Guinea, 1.6% of children under 5 years of age presenting to rural health clinics for treatment of vivax malaria were severely anaemic (haemoglobin <5 g/dL) . Across alciparum. In contalciparum.Although the spectrum of anaemia seen with vivax infection is reasonably well documented, the clinical, developmental, and socioeconomic consequences are largely unknown. Severe anaemia in isolation is associated with a ~2-fold increased risk of death in African children with falciparum malaria and has an even higher mortality when combined with other manifestations of severe disease such as cerebral malaria or respiratory distress . Severe Population-based estimates of mortality in severely anaemic individuals with vivax malaria have not been established but recent studies from Latin America, New Guinea and the Indian subcontinent have identified deaths in patients with severe vivax anaemia ,15-17. TAnaemia caused by vivax malaria is associated with requirement for blood transfusion ,189. ScrP. vivax parasitaemia at delivery is associated independently with an increased risk of moderate anaemia and a mean reduction in birth weight of 108 g [Pregnant women with haemoglobin concentrations under 8 g/dL in Papua New Guinea were at 2.4-fold higher risk of delivering a low birth weight baby than non-anaemic mothers . In thisof 108 g .P. vivax infections subsequently died; those with other comorbidities were at particularly great risk [Although evidence is lacking it seems plausible that severe vivax anaemia may reduce resilience to other infectious and non-infectious diseases and therefore may be associated with indirect mortality. In 1938, Swellengrebel and de Buck reported that 62 (7.7%) of a series of 807 patients with syphilis who were treated with induced eat risk .Plasmodium species have been associated with adverse effects on physical and cognitive development as well as school attendance; all of which may be exacerbated by concomitant malnutrition [Chronic or repeated episodes of malarial anaemia due to any utrition -202. AgaP. vivax infection is greatest in young children, especially in tropical countries such as Papua New Guinea and Eastern Indonesia where transmission is intense and local parasite strains relapse very frequently. In these regions, vivax malaria is commonly associated with severe anaemia both in the health care and community setting. The haematological effects of vivax malaria are likely to have complex interactions with gastrointestinal helminth infection, haemoglobin and red blood cell abnormalities and malnutrition.Haematological morbidity associated with Plasmodium vivax-infected red blood cells are minimally adherent and are more deformable than P. falciparum-infected erythrocytes resulting in relatively little red blood cell sequestration in the microvasculature and marrow sinuses and passage of a greater proportion of red cells through the spleen and other reticuloendothelial organs. The role of the spleen in the pathogenesis of vivax anaemia, particularly the removal of uninfected red blood cells, is an important area for future research (Figure Removal of uninfected red blood cells is a particularly important mechanism of anaemia in acute vivax malaria. h Figure .P. vivax is set to become the dominant Plasmodium species [P. vivax.As the global control and elimination of malaria progresses,  species ,203, yetAll authors declare they have no competing interests.NMD, PAB, NMA and RNP searched the relevant literature. NMD wrote the first draft of the manuscript. All authors appraised and revised the manuscript. All authors gave final approval for submission of the manuscript.


Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect.L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn's disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase.Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics. Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value S. thermophilus and L Brevis exhibit well documented probiotic properties and are considered probiotic bacteria. S. thermophilus is one of the components of the probiotic mixture VSL#3, which has been used in the treatment of pouchitis and ulcerative colitis, among other conditions L. brevis and S. thermophilus, was approximately ten-fold higher (p<0.01) than that of the two non-probiotic commensal bacteria E. coli and E. faecalis  both in control and IBD cells, the increase being significantly (p<0.05) greater for UC and CD compared to control LPMC , however, this increase was significant (p<0.05) only in UC and CD, but not control cultures  and unstimulated LPMC (p<0.05–0.01) (E. coli was unable to upregulate apoptosis of activated LPMC (L. brevis and S. thermophilus resembled that observed with LPMC (p<0.05–0.01)  Fig. 3a.Bacillus cereus-derived NSMAse (data not shown). The pro-apoptotic effect of NSMase required Mg2+ supplementation. Ceramide also induced apoptosis in activated LPMC and PBMC (p<0.05) compared to unstimulated cells (see below). Thus, these findings underscore that exogenous ceramide and NSMase reproduce the effects of probiotic sonicates in activated mucosal cells.Having detected marked differences in the NSMase activity between probiotic and non-probiotic bacterial sonicates, we next undertook a series of experiments aimed at ascertaining whether the addition of exogenous ceramide to immune cells could reproduce the pro-apoptotic effect observed with probiotic bacteria. Exposure of control, UC and CD LPMC to a pre-determined optimal ceramide concentration (30 µg/mL) resulted in a significant increase of the number of apoptotic cells in all groups (p<0.01–0.001) Fig. 4a.L. brevis and S. thermophilus (p<0.05–0.01). The pro-apoptotic effect of ceramide was also noted with activated PBMC (p<0.05–0.01) , suggesting that other nSMAse-mediated, signalling pathways are able to forward the pro-apoptotic signal.Because ceramide activates stress-activated protein kinases, like c-jun N-terminal kinase (JNK) ted LPMC Fig. 5a.L. brevis sonicates resulted in a 3-fold increase (p = 0.02) in ROS production compared to untreated cells . These results show that ROS are generated during LPMC apoptosis mediated by probiotic nSMAse, further supporting the involvement of the NSMAse/ceramide pathway in this process. Treatment of L. brevis sonicates with the phenolic antioxidant butylated hydroxyanisole (BHT), which prevents ROS production, did not influence the induction of LPMC apoptosis by L. brevis sonicates (data no shown), suggesting that other NSMAse-mediated, signalling pathways are able to forward the pro-apoptotic signal.Another hallmark of ceramide-mediated cell signalling is the production of reactive oxygen species (ROS) ed cells Fig. 6a.L. brevis or S. thermophilus previously preincubated with GSH, a complete abrogation of the sonicates' pro-apoptotic effect was observed (p<0.05)  (L. brevis sonicates a marked and significant (p<0.05–0.01) reduction of their capacity to induce LPMC apoptosis was observed, more so for UC and CD than control LPMC  Fig. 7a.L. brevis and S. thermophilus, two probiotic bacterial sonicates, can induce apoptosis of mucosal immune cells through the SMAse/ceramide pathway. This effect is not shared by E. coli and E. faecalis, the two non-probiotic enteric bacterial sonicates studied, suggesting that the ability to kill immunocytes is restricted to bacterial strains containing sufficient quantities of the key enzyme NSMase. We show that extracts of the probiotic L. brevis or S. thermophilus induced apoptosis of systemic and mucosal immune cells. Not only was this effect greater in magnitude than that of the non-probiotic E. coli or E. faecalis, but was also greater for LPMC from IBD than control non-IBD mucosa. Because IBD cells are in an enhanced state of activation, activation or differentiation may be a requirement to render immune cells susceptible to the pro-apoptotic effect of probiotics. This appears to be the case, as CD3/CD28-stimulated LPMC and PBMC displayed increased apoptosis upon exposure to L. brevis or S. thermophilus sonicates. The degree of cell activation, as determined by the duration of CD3/CD28 stimulation, correlated closely with the amount of mononuclear cell apoptosis, underscoring the importance of cell activation for probiotic-mediated cell death. These results are in keeping with previous evidence showing a tight relationship between immune cell activation and apoptosis. Activation-induced cell death is a complex physiological event aimed at deleting an excess of activated lymphocytes to prevent autoimmunity or uncontrolled immune reactivity The molecular mechanisms of the beneficial effects of probiotics remain to be fully elucidated. Recent observations showed that VSL#3, a probiotic containing a mixture of bacteria, stimulated mucosal alkaline sphingomyelinase and reduced inflammation L. brevis and S. thermophilus. To test this possibility a series of experiments was carried out.Because probiotics are live bacteria with a vast array of components and functions, the exact mechanisms explaining the beneficial effects are complex and difficult to dissect. If the capacity to induce immune cells apoptosis is an exclusive property of some probiotic bacteria, as found in the present study, certain products with the ability to induce apoptosis should be predominantly expressed by these probiotics. The striking quantitative difference in NSMAse activity between the two probiotic and the two commensal bacterial sonicates included in our study clearly points towards this enzyme as the likely culprit for the pro-apoptotic action of L. brevis and S. thermophilus but not E.coli or E. faecalis sonicates exhibited enhanced NSMase activity. Further, we demonstrated that exposure of resting and activated PBMC and LPMC, to either exogenous ceramide or recombinant NSMAse results in a marked enhancement of cell apoptosis, mimicking the effect seen with the probiotic strains L. brevis and S. thermophilus. As negative control, the inactive sphyngolipid dihydroceramide, failed to induce apoptosis. Corroborating evidence was obtained by showing that the level of ceramide-induced apoptosis was also dependent of the degree of immune cell activation.First, L. brevis, but not to the commensal E. faecalis. Of note, exposure of LPMC to recombinant NSMAse also induced JNK activation, and to a similar degree than L. brevis sonicates. Likewise, L. brevis, but not E. faecalis, induced a four-fold increase in ROS production by LPMC. The selectivity of L. brevis effects on JNK activation and ROS formation bolster the notion that NSMAse, present in large quantities in probiotics, and its product ceramide are intimately involved in the process of probiotic-induced immune cell apoptosis. The fact that neither JNK nor ROS production blockade separately were able to prevent probiotic sonicate-induced LPMC apoptosis reflects the complexity of signalling downstream of ceramide, suggesting the existence of redundant pathways to forward the pro-apoptotic signal. For instance, in addition to JNK/ROS, ceramide (independently of its mechanism of generation) can target other cell death regulators, such as cathepsin D or protein phosphatase PP1 or PP2, among others Second, we investigated some of the ceramide-triggered pathways during probiotic-induced mononuclear cell apoptosis. Signalling downstream of ceramide is extremely complex, but two of the best-known pathways involve JNK activation L. brevis and S. thermophilus sonicates with optimal concentrations of GSH, a potent inhibitor of NSMAse activity L.brevis, the strain with the highest SMAse activity. Additionally, pre-incubation of L. brevis sonicates with optimal doses of GW4869 obviously reduced L. brevis-induced LPMC apoptosis, an inhibitory effect more evident for IBD than control LPMC.Third, we investigated the consequences of NSMAse inhibition, which should result in a reduction of the probiotic pro-apoptotic capacity. Preincubation of L. brevis and S. thermophilus, were evaluated in our study. The fact that other probiotic microorganisms might have low or absent NSMAse activity, a key molecule for probiotic-induced immune cell apoptosis, as shown in our study, implies that the assessment of NSMAse activity may have clinical applications, helping the investigators to select the best probiotics to be tested in IBD.Our results cannot be extrapolated to all probiotic agents described to date, since only two of them, L. brevis and S. thermophilus to promote apoptosis may compensate the deficient cell death and thus contribute to re-establish mucosal immune homeostasis and decrease inflammation in IBD-involved intestine.The relevance of our findings mostly rests on the concept that resistance of mucosal immune cells to apoptosis is considered an essential component of IBD pathogenesis (17), probably in combination with an excessive cell proliferation L. brevis  and S. termophilus , and the non-probiotic non-pathogenic bacteria E. coli  and E. faecalis . Bacterial suspensions were prepared from cultures of each individual strain. L. brevis and S. thermophilus were grown in De Man Rogosa Sharp medium . E.coli and E. faecalis were grown in a 1% tryptone peptone medium (Difco), 0.5% NaCl, and 0.5% yeast extract (Difco). Bacteria were harvested at the end of logarithmic phase by centrifugation, suspended in four times their wet weight in desintegration buffer containing  and, after three freeze-thaw cycles, sonicated  with a Vibracell sonicator .Bacterial strains used in this study included the probiotic bacteria Lamina propria mononuclear cells (LPMC) were isolated from surgical specimens obtained from patients admitted for bowel resection for UC and CD, while malignant and nonmalignant conditions provided histologicaly normal control tissue. All diagnoses were confirmed by clinical, radiologic, endoscopic, and histologic criteria. LPMC were isolated as previously described 6 cells/ml, for 24 h. Apoptosis and cell cycle phase distribution were analyzed by propidium iodide staining followed by flow cytometry, as previously described L. brevis sonicates were preincubated for 30 min with 5 mM glutathione (GSH)  or various concentrations of the specific NSMAse Inhibitor GW4869 , before addition to the cell cultures. In an additional set of experiments, apoptosis was assessed by combined annexin V/propidium iodide staining , according to the manufacturer's instructions. In some cases, LPMC and PBMC were incubated with various concentrations of C2-ceramide , the inactive sphingolipid dihydroceramide , or nSMase from Bacillus cereus (Sigma) and examined for apoptosis as described above.A series of preliminary studies were carried out to ascertain the optimal concentration of bacterial sonicates and time of exposure of mononuclear cells to induce apoptosis. In subsequent experiments, bacterial sonicates were added to mononuclear cell cultures at a concentration of 1 mg protein of bacterial sonicate/102 and [N-methyl-14C]sphingomyelin , were used for determination of NSMase activity as described before Lyophilized bacteria (10 mg) were resuspended and sonicated as described above. Protein concentration was determined by the microbicinchoninic acid assay kit  with bovine serum albumin standards. Different amounts of bacterial sonicates  in 50 mM Tris·HCl buffer, pH 7.4, containing 5 mM MgClL. brevis sonicates were incubated with SP-600125 for 30 minutes, before its addition to LPMC cultures. LPMC apoptosis and cell cycle phase distribution were analyzed by PI staining followed by flow cytometry, as described above.At the end of 24 hr culture with bacterial sonicates, cells were washed, lysed and 40 µg of protein were run on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane at 140 mA in 25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 20% methanol. To study the activation of JNK, western blot analysis was performed using an anti-phosphorylated JNK antibody, dilution 1∶1000 , according to the manufacturer's instructions. In additional experiments, we also assessed the functional impact of JNK blockade on probiotic sonicates-induced LPMC apoptosis. The specific JNK inhibitor SP-600125 was purchased from Calbiochem  and dissolved in dimethyl sulfoxide . Stock solutions of at least 20 mM were made. Hydrogen peroxide was measured by cytometry using chloromethyl-2′-7′-dichlorofluorescein diacetate . LPMC were incubated with the fluorescent probe at 2 mM, in the absence or presence of bacterial sonicates. Fluorescence was determined at 529 nm for emission and 503 nm for excitation, with slit widths of 10 and 5 nm, respectively, after 30 and 60 minutes of LPMC exposure to bacterial sonicates t test. Values are expressed as the mean ± SEM. Statistical significance was set at a P value of less than 0.05.Data were analyzed by Stat View software  using the non-parametric Kruskall-Wallis and Mann-Whitney tests. Repeated measures for the same subject were analyzed by using the Student paired