AbstractStegodoncf.orientalis) and locally  extinct taxa were also present. The identification of Axisaxis in Khok Sung, a chital currently restricted to the Indian Subcontinent, represents the first record of the species in Southeast Asia. Three reptilian taxa: Crocodyluscf.siamensis, Python sp., and Varanus sp., are also identified. Faunal correlations with other Southeast Asian sites suggest a late Middle to early Late Pleistocene age for the Khok Sung assemblage. However, the Khok Sung mammalian fauna is most similar to that of Thum Wiman Nakin, dated to older than 169 ka. The Khok Sung large mammal assemblage mostly comprises mainland Southeast Asian taxa that migrated to Java during the latest Middle Pleistocene, supporting the hypothesis that Thailand was a biogeographic pathway for the Sino-Malayan migration event from South China to Java.The fluviatile terrace deposits of Khok Sung, Nakhon Ratchasima province, have yielded more than one thousand fossils, making this the richest Pleistocene vertebrate fauna of Thailand. The excellent preservation of the specimens allows precise characterization of the faunal composition. The mammalian fauna consists of fifteen species in thirteen genera, including a primate, a canid, a hyaenid, proboscideans, rhinoceroses, a suid, cervids, and bovids. Most species correspond to living taxa but globally (PageBreak Ailuropoda (giant panda) and/or Stegodon (extinct proboscidean), also called “Ailuropoda–Stegodon faunal complex”. This faunal association is a characteristic of the long period ranging from the Early to Late Pleistocene  and islands (Sundaic) Fig. . The fosMalaysia , and TriMalaysia , all regMalaysia , 2001. SHomo sp.  was excavated Fig.  for the. The Khoaultima . The Khoaultima . Howeveraultima . Some remyda sp. , and a gawanicus . The mamly known . In thisPageBreakfossils were additionally searched, using the water spraying technique to remove covered sediments on the surface of the fossiliferous layer  Fig. . Fossilsyer Fig. . UnfortuDepartment of Mineral Resources(DMR) (Bangkok). Individual fossils are catalogued with the collection (DMR), locality (KS), and unique specimen number, respectively. The comparative material is from the recent and fossil vertebrate collections housed at the following natural history museums and institutes:All fossil specimens are housed at the Khok Sung local museum (Nakhon Ratchasima) and at the Université de Poitiers  Institut International de Paléoprimatologie et de Paléontologie Humaine: Evolution et Paléoenvironnements, iPHEPInstitute of Vertebrate Paleontology and Paleoanthropology IVPPNatural History Museum Prague NHMPMuseum Wien  Naturhistorisches NMWMuséum National d’Histoire Naturelle  Zoological collection of mammals and birds, MNHN-ZMO Dubois collection, Rijksmuseum van Natuurlijke Histoire RMNH DUBNatural History Museum  Mammal collection, Thailand THNHM-MZoological Institute, Russian Academy of Sciences ZINZoologische Staatssammlung München ZSMThe dental nomenclature follows PageBreakIn the case of measurements of stegodontid cheek teeth, the methods and parameters used for molar and ridge dimensions were given in Fig. laminar frequency (LF) were calculated, using the formula proposed by All specimens were measured using digital callipers to the nearest 0.01 mm. The tooth dimensions for all mammals were measured at the base of the crown along the anterior-posterior margins for the maximum length (L) and from the labial (incisors and canines)/buccal (premolars/molars) to lingual margins for the maximum width (W). 2> 0.93) and cervids (r2> 0.95)  because it has the smallest influence of sample sizes and emphasizes faunal resemblances  = (Nc / N1) × 100, where Nc is the number of identified taxa shared by two faunas and N1 is the number PageBreakof identified taxa in the smaller of the two faunas (Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) as cluster algorithms for our analysis because the dendrogram represents higher values of cophenetic correlation coefficient than the others.We compared differences in species compostion of Southeast Asian large mammal fauna during the Middle Pleistocene, using an analysis of the faunal similarity. According to unequal sampling conditions for our data, we applied two criteria for undertaking this analysis: localities are disqualified when they have fewer than 10 taxa identified at the species level and taxa are excluded when their appearances are still doubtful (i.e. poor taxonomic description or identification). We therefore selected Simpson’s mblances , 1960. W indices . The Simds e.g., . The foro faunas . A higheo faunas . We seleTaxon classificationAnimaliaPrimatesCercopithecidaeA right tibia, DMR-KS-05-04-04-1.The right tibia is complete Fig.  and elonHylobates and all arboreal cercopithecoids) is characterized by more rounded borders of the trochlear surface and a convex proximal border of the medial malleolus joining the trochlear surface (Hylobates (gibbon), Presbytis (surili), and Macaca (macaque). We suggest here to make a distinction between these genera based on the ratios of the greatest length of the tibia to the length or width of the proximal tibia (GL/Bp or GL/Dp). Based on these indices, the Khok Sung tibia falls within the range of recent Macaca , the caput femoris is round, and the upper border of the neck is long and flat , DMR-KS-05-03-28-14; a left DP4 (anterior part), DMR-KS-05-03-19-7; a left M2, DMR-KS-05-03-29-1 (posterior part); a right M3, DMR-KS-05-03-22-19 (posterior part); a fragmentary tusk, DMR-KS-05-03-15-2; a left dp3 (anterior part), DMR-KS-05-04-01-8; two mandibles with m3—DMR-KS-05-03-08-1 (right) and DMR-KS-05-03-08-2 (left); a right humerus fragment , DMR-KS-05-03-10-5; a left humerus, DMR-KS-05-03-10-6; two ulna fragments —DMR-KS-05-03-09-7 and DMR-KS-05-03-10-2; a femoral head fragment, DMR-KS-05-03-10-3; a right femur, DMR-KS-05-03-10-4; a right tibia fragment , DMR-KS-05-03-10-3; a right fibula, DMR-KS-05-03-00-124; two pelvis fragments—DMR-KS-05-03-10-11 (right) and DMR-KS-05-03-10-12 (left); five vertebrae—DMR-KS-05-03-17-11, DMR-KS-05-03-10-7, DMR-KS-05-03-09-18, DMR-KS-05-03-10-1, and DMR-KS-05-03-28-20; a sacrum fragment, DMR-KS-05-03-10-8; two ribs—DMR-KS-05-03-10-13 and DMR-KS-05-03-10-14; three rib fragments—DMR-KS-05-03-09-6 (body), DMR-KS-05-03-09-45 (body), and DMR-KS-05-03-09-4 (head and neck).Upper dentition: both fragments of DP4  preserves three posterior ridges with a small cingulum Fig. . Two antDMR-KS-05-03-22-19 (M3) preserves only three posterior ridges with a cingulum Fig. . The ridPageBreakStegodontrigonocephalus in its more medial-laterally than the dorso-ventrally compressed cross-section. The macroscopic distinctive features in cross-section are similar to Stegodonsompoensis (A fragmentary tusk (DMR-KS-05-03-15-2) contains dentine (outer and inner layers), cementum, and a pulp cavity Fig. . It is smpoensis  but showPageBreakLower dentition: DMR-KS-05-04-01-8 (dp3) is heavily worn and comprises three preserved ridges and an anterior cingulum  are moderately well-preserved Tab. . The comStegodon . The antPostcranial remains: postcranial elements include two humeri s on molars, V-shaped valleys between ridges on molars , and step-like worn surface reliefs on the enamel layer  represent a total ridge number of ten , different from the ridge formula (×11× for this species) given by Stegodonorientalis therefore ranges from ten to eleven. The m3 of Stegodonorientalis commonly has a total number of twelve ridges . According to the fact that only a few comparative specimens of the m3 of Stegodonorientalis are complete with the total ridge number of twelve, some of them  display a total of 11 ridges . In Stegodonorientalis, the number of ridges on the m3 thus ranges from eleven to twelve. Stegodoninsignis has a total number of ridges ranging from eleven to thirteen  . But it differs from Stegodoninsignis in having more delicately folded enamel, more pronounced curvature of the crown, and V-shaped valleys (between the two ridges) slightly less filled by cements. The ridge sizes of Khok Sung lower third molar are almost comparable to those of Stegodonorientalis and Stegodoninsignis, but are distinctly larger than other derived Stegodon species from Indonesia  . Anothersia Tab. . We thusTaxon classificationAnimaliaProboscideaElephantidaeA fragmentary tusk, DMR-KS-05-03-22-1; a posterior fragment of a right lower molar, DMR-KS-05-03-17-12.Upper tusk: DMR-KS-05-03-22-1 is a short fragmentary tusk. The dorsal side is partially broken away  by a more rounded cross-section, a larger diameter, and a radiate fracture pattern with the development of concentric incremental lines  is distinguished from DMR-KS-05-03-15-2 . The lo and left (p3–m3) tooth rows, DMR-KS-05-03-00-126; a partial mandible, DMR-KS-05-03-31-28; a fragmentary nasal bone, DMR-KS-05-03-00-56; a left scapula, DMR-KS-05-03-00-58; a left humerus, DMR-KS-05-03-31-3; a right metacarpus II, DMR-KS-05-03-28-29; a metacarpus III, DMR-KS-05-03-22-49; a right metacarpus IV, DMR-KS-05-04-05-15; a left tibia, DMR-KS-05-03-00-52; a right calcaneus, DMR-KS-05-04-27-19; a left astragalus, DMR-KS-05-03-26-23.A left P2, DMR-KS-05-03-00-128; a left P3, DMR-KS-05-03-22-17; a left M1, DMR-KS-05-03-00-129; a left M3, DMR-KS-05-03-00-127; a mandible Upper dentition: P2  preserves both sides of cheek tooth rows (right p2–m3 and left p3–m3), but most of its symphysis and entire ramus are broken off  is short and robust, bending downward and narrowing anteriorly towards the tip , because its anterior part is pointed rather than rounded  assigned to ondaicus .Rhinoceros  and Indian (Rhinocerosunicornis) rhinoceroses  are notably typical of Asian rhinoceroses. The two small alveoli corresponding to the lost central incisors are autapomorphic of inoceros . Our obsoceroses . This fed as far . In the  p2 Fig. . The lat are notaRhinocerosunicornis or Rhinocerossondaicus due to heavy wear. In addition, there is a significant size overlap between these two species (PageBreakmolar rows provide a better distinction (little overlap in size) than those of isolated teeth. The lengths and widths of the cheek teeth on the mandible DMR-KS-05-03-00-126 fall almost within the range of Rhinocerossondaicus, with the exception of some specimens  that fit well with the larger-sized Rhinocerosunicornis  and outside of the ranges for Rhinocerosunicornis  is nearly square in outline and displays a flattened ectoloph and a well developed crochet, medifossette, and posterior fossette  is strongly compressed laterally and preserves a partial mandibular ramus and body with worn cheek teeth, except for the m3 which is unbroken , as well as its larger size than that of Rhinocerossondaicus. These upper molar features are characteristic of Rhinocerosunicornis   to nicornis . For the-13 Tab.  are compTaxon classificationAnimaliaArtiodactylaSuidaeMüller, 1838A left maxillary fragment with P3–M2, DMR-KS-05-04-19-2; two left M2—DMR-KS-05-04-19-5 and DMR-KS-05-03-18-23 (posterior portion); two right M3—DMR-KS-05-04-03-4 and DMR-KS-05-04-19-4 (anterior portion); two mandible with two tooth rows—DMR-KS-05-03-15-1  and DMR-KS-05-04-19-1 ; a left posterior fragment of m3, DMR-KS-05-04-19-3; a right humerus, DMR-KS-05-03-26-8.Upper dentition: DMR-KS-05-04-19-2 is a maxillary tooth row preserving a slightly worn P3 to M2  are distinct and the protofossa is present. Upper molars are unworn to slightly worn and exhibit distinct main  and accessory  cusps. The posterior cingulum on the M2 is more developed than on the M1   Tab. . The Khobarbatus . The lowteriorly .Susscrofa from Susbarbatus only based on the cheek teeth because both species overlap in size ) or “verrucosic” (Susbarbatus) type  on the talonid. The pentapreconulid on the m3 is small or absent in Susbarbatus  type . Similarus) type , 1997. Tbarbatus . For othsscrofa . HoweverSusbarbatus according to those described features. We also suggest that Pleistocene Susbarbatus probably shows evidence of sexual size dimorphism because the female specimen DMR-KS-05-04-19-1 is markedly smaller than the male specimen DMR-KS-05-03-15-1, as seen in the recent population.The female mandible (DMR-KS-05-04-19-1) and other isolated teeth are assigned to PageBreakTaxon classificationAnimaliaArtiodactylaCervidaeFour crania—DMR-KS-05-04-18-50 (with two antlers), DMR-KS-05-03-00-30 , DMR-KS-05-03-18-X9 (with pedicles), and DMR-KS-05-03-27-1 (with pedicles); two right complete antlers—DMR-KS-05-03-31-30 and DMR-KS-05-03-22-4; a nearly complete left antler, DMR-KS-05-04-4-1; five right fragmentary antlers—DMR-KS-05-03-18-21, DMR-KS-05-03-19-82, DMR-KS-05-03-28-22, DMR-KS-05-06-22-2, and DMR-KS-05-03-28-1; eight left fragmentary antlers—DMR-KS-05-03-00-12, DMR-KS-05-03-19-81, DMR-KS-05-03-22-2, DMR-KS-05-03-24-1, DMR-KS-05-04-09-1, DMR-KS-05-03-19-13, DMR-KS-05-03-26-21, and DMR-KS-05-03-08-17; two left fragmentary maxilla—DMR-KS-05-03-28-6 (with M1–M3) and DMR-KS-05-03-08-31 ; a right P4, DMR-KS-05-04-01-3; a left M1, DMR-KS-05-04-28-5; a left M2, DMR-KS-05-03-14-5; thirteen right mandibles—DMR-KS-05-03-14-2 (with m3), DMR-KS-05-03-20-1 (with p4–m3), DMR-KS-05-03-20-2 (with m2 and m3), DMR-KS-05-03-22-7 (with m2 and m3), DMR-KS-05-04-03-1 (with p2–m3), and DMR-KS-05-03-27-3 (with m2 and m3), DMR-KS-05-03-19-1 (with p2–m3), DMR-KS-05-03-22-8 (with m2 and m3), DMR-KS-05-04-01-1 (with p2–m3), DMR-KS-05-03-24-4 (with m2), DMR-KS-05-03-26-12 (with m2 and m3), DMR-KS-05-04-7-10 , and DMR-KS-05-03-26-10 (with p2–m1); eight left mandibles—DMR-KS-05-03-18-22 (with p2), DMR-KS-05-03-22-6 (with m1–m3), DMR-KS-05-03-27-22 (with p3-m2 sockets and broken m3), DMR-KS-05-04-09-2 , DMR-KS-05-03-00-102 (with p4 and m1), DMR-KS-05-03-19-2 (with m1–m3), DMR-KS-05-03-23-1 (with p2 and p3 roots and p4–m3), and DMR-KS-05-03-29-1 (with p2-m3); a left m1, DMR-KS-05-04-28-6; three m2—DMR-KS-05-03-25-4 (right), DMR-KS-05-03-00-104 (left), and DMR-KS-05-03-22-11 (left); four left m3—DMR-KS-05-04-9-4, DMR-KS-05-03-22-9, DMR-KS-05-04-01-2, and DMR-KS-05-03-08-33; three right fragmentary humeri —DMR-KS-05-03-13-4, DMR-KS-05-04-11-32, and DMR-KS-05-03-17-17; six metacarpi—DMR-KS-05-03-18-2 (right), DMR-KS-05-03-19-3 (right), DMR-KS-05-03-22-28 (right), DMR-KS-05-03-08-2 (right), DMR-KS-05-04-30-20 , and DMR-KS-05-03-19-37 (left); a right fragmentary femur, DMR-KS-05-03-27-4 ; three metatarsi—DMR-KS-05-03-26-3 (right), DMR-KS-05-03-29-30 (left), and DMR-KS-05-03-15-14 (left).Crania and upper dentition: four crania are almost complete, lacking only the anterior portions   between the main beam and the brow tine, and a well-developed burr   Fig. . The spela) Fig. . The craeam Fig. . The speeam Fig.  and DMR-eam Fig.  preserveles Fig. . The basles Fig. . The laturr Fig. . A smallurr Fig. . The maiurr Fig. . The skuAxis, characterized by well-developed styles, medial cristae (more distinct on the P4), and posterolingual fossettes  to nearly complete (lacking only the ascending ramus and coronoid process) individuals   Fig. . But the-27 Fig. . The bac-27 Fig. , but abs-27 Fig. . The pos-27 Fig. .PageBreakPostcranial remains: postcranial bones include isolated humeri , we thus demonstrate some dental morphological variation within species. The m3 of Axisaxis appears more morphologically variable than the other molars, such as the more or less developed posterior talonids and the presence/absence of PageBreakback fossae. The cheek teeth of extant Axisaxis are relatively similar to those of Axisporcinus . However, Axisaxis differs from Axisporcinus in having less developed anterior cingulids on the lower molars and the presence of back fossae on the m3. Recent Axisaxis represents an intermediate size between Axisporcinus and two cervid species (Panoliaeldii and Rusaunicolor)  Tab. . Axisaxons Figs  and 18.Axisaxis from Khok Sung are smaller than those of Axisshansius from Anhui and Yunnan (China) and of Axisjavanicus from Ngandong and Buitenzorg in Java and Carnul Cave in India, but are larger than those of Axislydekkeri from Trinil H. K. (Java)  Figs  and 18. porcinus  are muchCambodia . The exiTaxon classificationAnimaliaArtiodactylaCervidaePageBreakdistal part), DMR-KS-05-03-00-119 , DMR-KS-05-03-19-2 , and DMR-KS-05-08-16-1 ; three left metatarsi—DMR-KS-05-03-25-8, DMR-KS-05-03-28-17, and DMR-KS-05-03-15-15.A cranium with a right partial antler, DMR-KS-05-04-20-4; a right P2, DMR-KS-05-03-15-11; two left M1—DMR-KS-05-03-00-24 and DMR-KS-05-03-00-25; six M2—DMR-KS-05-03-00-23 (right), DMR-KS-05-03-30-5 (right), DMR-KS-05-04-3-4 (right), DMR-KS-05-03-30-6 (left posterior lobe), DMR-KS-05-03-27-7 (left), and DMR-KS-05-04-3-5 (left); five M3—DMR-KS-05-03-27-6 (right), DMR-KS-05-04-9-1 (right), DMR-KS-05-04-8-3 (right), DMR-KS-05-03-00-22 (left), and DMR-KS-05-04-9-2 (left); two left mandibles—DMR-KS-05-03-27-2 (with p2–m3) and DMR-KS-05-04-9-5 (with p2–m2); a right i1, DMR-KS-05-03-29-2; a right scapula, DMR-KS-05-06-24-4; a left humerus, DMR-KS-05-04-11-35; a right fragmentary humerus, DMR-KS-05-03-18-1 ; three radii—DMR-KS-05-03-31-10 (right), DMR-KS-05-04-11-3 (right), and DMR-KS-05-03-19-16 (left); a right metacarpus, DMR-KS-05-03-24-2; two right femora—DMR-KS-05-03-27-11 and DMR-KS-05-03-17-36; five fragmentary femora—DMR-KS-05-04-05-38 , DMR-KS-05-03-28-20  . The foramina ovale of DMR-KS-05-04-20-4 are more circular and open more anteriorly than those of Axis. The right partial antler contains a half of the slender main beam, but lacks a brow tine entirely . The antler surface is smooth and the burr is poorly developed in relation to the ontogenetic stages. The preserved shed antler shows a typical character of Panoliaeldii, whose main beams strongly project and curve laterally  Fig.   Fig. , differeely Fig. . The divlly Fig. .P2 exhibits a prominent medial crista which divides the fossette into two islands Fig. . The sepMandibles and lower dentition: Two mandibles   Fig. . Lower mds) Fig. . The m3 ds) Fig. . The shaPostcranial remains: postcranial bones include a scapula  suggested that placement of the Eld’s deer in the genus Panolia is an acceptable alternative based on mtDNA analysis . However, we suggest that some isolated teeth of cervids from Thum Wiman Nakin  due to their larger sizes. Our identification thus confirms the existence of Panoliaeldii in Thailand during the late Middle Pleistocene.The shed antler of the Eld’s deer, ion Tab.  and scation Tab.  and 22, an Nakin  reveal aTaxon classificationAnimaliaArtiodactylaCervidaePageBreaka left M3, DMR-KS-05-03-31-1; two right mandibles—DMR-KS-05-03-31-2 (with m2) and DMR-KS-05-03-13 (with p4–m3); two left mandibles—DMR-KS-05-03-00-101 (with p3–m3) and DMR-KS-05-03-27-4 (with m3); a right m1, DMR-KS-05-03-00-5; a left fragmentary humerus, DMR-KS-05-03-15-43 ; three right fragmentary radii—DMR-KS-05-03-25-9 , DMR-KS-05-03-19-14 , and DMR-KS-05-03-26-19 ; a left metacarpus, DMR-KS-05-03-17-26; six fragmentary femora—DMR-KS-05-03-19-7 , DMR-KS-05-03-12-2 , DMR-KS-05-04-11-2 , DMR-KS-05-03-26-5 , DMR-KS-05-04-30-9 , and DMR-KS-05-04-19-10 ; a right tibia, DMR-KS-05-03-28-16; a right metatarsus, DMR-KS-05-03-19-11Three right antlers—DMR-KS-05-03-20-11 (nearly complete specimen), DMR-KS-05-03-26-2 (fragment), and DMR-KS-05-03-28-23 (fragment); a right M1, DMR-KS-05-03-22-10; two left M2—DMR-KS-05-04-9-3 and DMR-KS-05-04-3-3; PageBreakPageBreakAntlers: DMR-KS-05-03-20-11 is a nearly complete antler, slightly broken at the middle part of the main beam . The divergent angle between the main beam and brow tine ranges from 50° to 90°. The shed antlers of Rusaunicolor are different from those of Axisaxis in having slightly rougher surfaces, more divergent insertion relative to the frontal orientation, a shorter main beam, and a smaller angle between the main beam and the brow tine, and in lacking small-ornamented tines or knobs on the brow tine , Rusamarianna (Philippine deer), Rusatimorensis (rusa), and Rusaalfredi (Prince Alfred’s deer).According to Leslie (2011), we regard here Rusaunicolor are characterized by its typical three tines and forked beams at the tip, similar in shape to those of Axisporcinus but much more robust. The sambar deer shares a similar dental morphology with the Eld’s deer. But it differs from Panoliaeldii as well as Axisaxis in being larger-sized and in having more developed anterior cingulids on lower molars. The sambar deer is much larger than Axisaxis  probably do not belong to Rusaunicolor according to their smaller sizes. The taxonomic revision of fossil cervids from Thum Wiman Nakin would lead to the recognition of either higher or lower diversity of cervids in Southeast Asia during the Middle Pleistocene.Antlers of xis Figs  and 22. lor Tab. . As demolor Tab.  and 22, kai Phet , Phnom Lkai Phet , and Ma kai Phet  , DMR-KS-05-03-23-2; a right M3, DMR-KS-05-03-29-6; a right mandible with m1–m3, DMR-KS-05-03-9-1; two left mandibles—DMR-KS-05-04-9-1  and DMR-KS-05-04-29-1 (with m3); a left i2, DMR-KS-05-03-15-12; a right i3, DMR-KS-05-03-23-4; a right p2, DMR-KS-05-04-01-6; a right m1, DMR-KS-05-03-15-10; a right m2, DMR-KS-05-03-29-7; two m3—DMR-KS-05-04-28-4 (right broken posterior lobe) and DMR-KS-05-03-24-5 (left); a left humerus, DMR-KS-05-03-20-2(1).Upper dentition: DP3 (DMR-KS-05-03-29-8) is molariform and elongated, characterized by well-developed anterior and posterior cingula, buccal styles, and medial fossettes, a slightly-developed entostyle, and a reduction of the anterior lobe width and height compared to the posterior lobe  and i3 (DMR-KS-05-03-23-4) are spatulate and small, compared to other species of  m3 Fig. . The ant m3 Fig. . The pos m3 Fig. . The pos m3 Fig. . The entPostcranial remains: a humerus, DMR-KS-05-03-20-2(1), preserves the shaft and distal part . We attribute this humerus to Bossauveli according to the proportional correlation with the extant specimens  collected from Khok Sung, we identify these fossils on the basis of dental features.Southeast Asian large bovids are accurately identified by differences in cranial features , although they show sexual and ontogenetic variation in morphology. Lacking the cranial remains, it is difficult to make a distinction within the species of MNHN-ZMO-1940-51 and MNHN-ZMO-10801), the cheek teeth of koupreys are similar to those of other species of Bos, characterized by having hypsodont crowns, well-developed styles and stylids, a horse shoe-shaped infundibulum (anterior and posterior fossettes), and bifurcated or trifurcated entostyles depending on the wear stage. Among Southeast Asian PageBreakPageBreakPageBreaklarge bovids, it differs from Bosjavanicus (banteng) and Bosgaurus (gaur) in having a more developed postprotocristid on the p3 and p4, a metacentric chromosome-shaped molar in relation to the middle wear stage, a single large medial fossette on the upper molars, a flat lingual surface of the entostyle on the moderately to heavily worn molars. The M1 and M3 of Bossauveli are almost more square and rectangular in outline, respectively, compared to those of other Bos species. Bossauveli is usually smaller than Bosgaurus and Bubalusarnee , but is often comparable in size to Bosjavanicus   . Howevers (zebu) . These aTaxon classificationAnimaliaArtiodactylaBovidaeA left horn core, DMR-KS-05-03-26-22; a right DP2, DMR-KS-05-03-20-4; two right P2—DMR-KS-05-03-19-27 and DMR-KS-05-04-03-3; a right DP3, DMR-KS-05-03-20-3; a right DP4, DMR-KS-05-03-17-3; a right M1, DMR-KS-05-03-00-20; a right M3, DMR-KS-05-03-17-1; a right mandible with m1–m3, DMR-KS-05-03-00-1; a left mandible with p2–m3, DMR-KS-05-04-3-1; a left i1, DMR-KS-05-03-00-27; two left m2—DMR-KS-05-03-19-26 and DMR-KS-05-03-16-1; two humeri—DMR-KS-05-05-1-1 (right) and DMR-KS-05-03-00-62 (left); a right metacarpus, DMR-KS-05-03-26-27; two left femora—DMR-KS-05-03-9-2 and DMR-KS-05-04-30-1 .Horn core: a single horn core (DMR-KS-05-03-26-22) is small, curved upward  is small and elongated, characterized by three main cones  and a well-developed metastyle  to  forward .Bosgaurus are also observed. The cheek teeth of Bosgaurus are distinguished from Bossauveli and Bosjavanicus by having two separate fossettes on the P2, more developed metaconids and more anteroposteriorly constricted PageBreakpostprotocristids on the p3 and p4, and more robust cheek teeth , similar to those of Bosjavanicus. But the entostyle is not bifurcated, when the molar is extremely worn, as seen on the specimen DMR-KS-05-03-00-20 , in Khok Sung.Mandibles and isolated teeth of eth Figs . The ent-20 Fig. . This chion Figs . We elucPageBreakTaxon classificationAnimaliaArtiodactylaBovidaeA nearly complete cranium associated with a right mandible, DMR-KS-05-03-20-1; a cranium with a right tooth row (P3–M3), DMR-KS-05-03-21-1; a partial cranium with two tooth rows (P3–M1), DMR-KS-05-03-16-3; a partial cranium with a right tooth row (P3–M3), DMR-KS-05-03-11-1; three horn cores—DMR-KS-05-03-16-2 (right), DMR-KS-05-03-31-6 (right), and DMR-KS-05-03-19-28 (left); a left P2, DMR-KS-05-03-18-14; a left DP3, DMR-KS-05-03-00-103; two right P3—DMR-KS-05-03-22-14 and DMR-KS-05-04-05-3; a right DP4, DMR-KS-05-04-29-8 (broken anterior lobe); two P4—DMR-KS-05-03-18-13 (right) and DMR-KS-05-03-18-9 (left); four M1—DMR-KS-05-03-31-5 (right), DMR-KS-05-03-18-12 (right), DMR-KS-05-03-18-6 (left), and DMR-KS-05-03-22-13 (left); five M2—DMR-KS-05-03-00-2 (right), DMR-KS-05-03-25-21 (right), DMR-KS-05-03-18-5 (right), DMR-KS-05-03-16-2(1) (left), and DMR-KS-05-03-18-7 (left); four M3—DMR-KS-05-03-00-7 (right), DMR-KS-05-03-22-12 (left), DMR-KS-05-03-14-1 (left), and DMR-KS-05-03-18-10 (left); a right mandible with p2–m1, DMR-KS-05-03-20-2; three left mandibles—DMR-KS-05-03-10-3 (with p2–m3), DMR-KS-05-03-20-10 (with p2–m1), and DMR-KS-05-03-20-20 (with m1 and m2); a right i1, DMR-KS-05-03-18-8; a right i2, DMR-KS-05-03-22-15; a left i3, DMR-KS-05-03-00-106; a right i4, DMR-KS-05-03-16-3; a right p3, DMR-KS-05-03-14-4; a left dp4, DMR-KS-05-03-00-4; a right p4, DMR-KS-05-03-19-6; four m1—DMR-KS-05-03-25-3 (right), DMR-KS-05-03-18-18 (right), DMR-KS-05-03-00-105 (left), and DMR-KS-05-03-00-3 (left); two m2—DMR-KS-05-03-27-12 (right) and DMR-KS-05-03-25-2 (left); two m3—DMR-KS-05-03-18-11 and DMR-KS-05-04-29-2 (left posterior lobe); eleven thoracic vertebrae—DMR-KS-05-04-1-11 (T3), DMR-KS-05-04-1-26 (T4), DMR-KS-05-04-1-13 (T5), DMR-KS-05-04-1-14 (T6), DMR-KS-05-04-1-15 (T7), DMR-KS-05-04-1-16 (T8), DMR-KS-05-04-1-12 (T9), DMR-KS-05-04-1-17 (T10), DMR-KS-05-04-1-18 (T11), DMR-KS-05-04-1-19 (T12), and DMR-KS-05-04-1-20 (T13); four lumbar vertebrae—DMR-KS-05-04-1-24 (L1), DMR-KS-05-04-1-23 (L2), DMR-KS-05-04-1-22 (L3), and DMR-KS-05-04-1-21 (L4); two humeri—DMR-KS-05-03-31-1 (right) and DMR-KS-05-03-31-8 (left); two scapulae—DMR-KS-05-03-26-2 (right) and DMR-KS-05-02-20-4 (left); three ulnae and radii—DMR-KS-05-03-00-61 (right), DMR-KS-05-03-31-2 (right) and DMR-KS-05-03-31-9 (left); a right metacarpus, DMR-KS-05-03-26-3(1); a pelvis, DMR-KS-05-04-1-25; two femora—DMR-KS-05-04-1-1 (right) and DMR-KS-05-04-1-2 (left); a right fragmentary femur, DMR-KS-05-03-20-8 ; three tibiae—DMR-KS-05-4-1-11 (right), DMR-KS-05-04-1-3 (left), and DMR-KS-05-03-20-9 (left); two fourth tarsal bones—DMR-KS-05-04-1-7 (right) and DMR-KS-05-04-1-5 (left); three metatarsi—DMR-KS-05-04-1-8 (right), DMR-KS-05-04-1-6 (left), and DMR-KS-05-03-28-30 (left); a left astragalus, DMR-KS-05-04-1-4; a left phalanx I, DMR-KS-05-04-1-9; a left phalanx II, DMR-KS-05-04-1-10.PageBreakCrania and upper dentition: DMR-KS-05-03-20-1 is undeformed and nearly complete . The horn cores are subtriangular in cross-section base, becoming subrounded toward the apex  Fig. . Anotherlus Fig. .Three isolated horn cores  but are more robust than most species of Bos   is well-developed, often separating into two or three islands with wear  Tab. . Howeverear Fig. . The infeli Fig. . In occleli Fig. . The smaeli Fig. .Mandibles and lower dentition: five mandibles: DMR-KS-05-03-20-1  were found separately. The articulated skeletons show a typical character of Bubalusarnee whose postcranial bones are more massive and thicker than those of Bos  and third molar features are more informative for the species identification than others . However, tooth dimensions are informative to make an ongoing distinction among the Khok Sung large bovids. The largest bovid in this locality is Bubalusarnee, followed by Bosgaurus and Bossauveli, respectively, similar to the size tendency of their recent population A left M2, DMR-KS-05-03-18-16; three m3—DMR-KS-05-04-05-4 (right), DMR-KS-05-03-27-5 (left), and DMR-KS-05-03-28-10 (left posterior fragment).PageBreakonly a posterior lobe  because they are comparable in size and morphology to the extant specimens , but is smaller than Capricornismilneedwardsi. In addition, it differs from Capricorniscrispus in having more developed metastylid and entostylid and a presence of PageBreakback fossettes on the slightly worn m3 and from Capricornismilneedwardsi in having less developed metastylid and posthypoconulidcristid on the m3.We assign these isolated teeth from Khok Sung to ens Fig. . Among cCapricornissumatraensis from Khok Sung is smaller than that from the Late Pleistocene of Lang Trang in Vietnam   . HoweverPageBreakTaxon classificationAnimaliaSquamataBoidaeFour trunk vertebrae—DMR-KS-05-03-00-21, DMR-KS-05-03-00-16 (two attached vertebrae), and DMR-KS-05-04-28-12.Vertebrae are almost complete and represent a large-sized snake  and fossil  python vertebrae. According to the fact that the species-level distinction based on the vertebral morphology is poorly known, we therefore assign these vertebrae to Python sp.These four vertebrae are attributed to the family PageBreakTaxon classificationAnimaliaSquamataVaranidaeTwo trunk vertebrae—DMR-KS-05-03-08-36 and DMR-KS-05-03-29-36.The vertebra DMR-KS-05-03-08-36 is more complete than the specimen DMR-KS-05-03-29-36 , an oval-shaped cotyle, a short neural spine, and an absence of the zygosphenes and zygantra (Varanus sp. is reported from the Middle Pleistocene of Phnom Loang (Varanuscf.komodoensis (larger) and Varanussalvator, are described from the Middle Pleistocene of Trinil H. K.  and fossil  specimens. Identifying these vertebrae more precisely to the species-level, more detailed morphological comparisons need to be made in the future.We assign these two vertebrae to the the family  condyle . The Khozygantra . Varanusom Loang . Two varil H. K. . The KhoTestudines, an extinct gharial , and a spotted hyaena (Crocutacrocutaultima), have been previously described from Khok Sung by Testudines is the most diverse group of non-mammalian taxa in the locality (22% of the fauna). In addition, other vertebrates such as birds and fish are tentatively observed. A single fragmentary cervical vertebra of the bird order Galliformes is also present  and reptilian (Batagurcf.trivittata) species in the Khok Sung fauna are no longer present in the region but occur far away from Thailand or even from Southeast Asia. Moreover, two taxa, Stegodoncf.orientalis and Gavialiscf.bengawanicus were present PageBreakin the locality but became globally extinct later. The Khok Sung vertebrate fauna totally contains 19 of 27 identified taxa that are currently present in Thailand , the absence of medium- and small-sized mammal remains is likely due to taphonomic conditions and/or fossil collecting methods. Similarly to most of the Middle and Late and Tab. .Stegodonorientalis and its co-occuring species, Rhinocerossondaicus, Rhinocerosunicornis, Susbarbatus, Axisaxis, Panoliaeldii, Rusaunicolor, Bossauveli, Bosgaurus, Bubalusarnee, and Capricornissumatraensis.Past records and recent distribution patterns of large mammalian species present in Khok Sung are revealed in this work. Paleontological sites in Southeast Asia as well as South China are examined for the Early, Middle, and Late Pleistocene, compared with the modern distribution patterns. We only focus on mammalian taxa assigned to the species-level, including Stegodon  and Keo Leng (northern Vietnam) caves  and Vie., Daxin ). Anothecalities . During nce Fig. . The twonce Fig. , 2010 innce Fig. , 2015 innce Fig. , 2014. Sm) caves . Perhapsm) caves . The numcalities . Althougene Fig. , the PlePalaeoloxodon is reported from several Middle Pleistocene localities in mainland Southeast Asia . Palaeo Vietnam , 2006, sHolocene . The cauElephasmaximus is known from the late Middle Pleistocene of Thum Wiman Nakin (northeastern Thailand)   , 2001, aVietnam) . The IndVietnam) . Those aVietnam) . HoweverVietnam) . It is pRhinocerossondaicus is reported from the upper part of the Irrawaddy Formation, near Pauk Township in central Myanmar   sensu . Accordi species , so therPageBreaknone are reported from Trinil H. K.  is known from the caves of Thum Wiman Nakin and Thum Prakai Phet  in northern Thailand  have never been previously recorded from Thailand but were present in mainland Southeast Asia, at least in Khok Sung, during the late Middle Pleistocene   Fig. . Its habka) Fig. . The Pleistocene . The locPanoliaeldii are collected from the caves of Thum Wiman Nakin and Kao Pah Nam , northern Thailand , 2010. TPanoliaeldii is restricted to the Indochinese province  , Bosjavanicus (banteng), Bosgaurus (gaur), and Bubalusarnee . Bantengs, gaurs, and koupreys presumably shared a common ancestor at 2.6 Ma (Plio-Pleistocene) and their lineages split in a short period of time  based on the molecular estimations of divergence times  yielded remains of these bovid species (cf.) , 2010. OThe historical distribution of koupreys during the last century is restricted to Cambodia, southern Laos, southeastern Thailand, and western Vietnam . They bePageBreakscapes seem to have caused the reduction of large bovid population in several areas during the past decade. The koupreys is more widely distributed during the Pleistocene than today . The advocated cause for the local extinction of serows is possibly related to the unfavorable climatic conditions. The drier and cooler climate that occurred after 81 ka in Java  of the Indochinese subregion  and on the island of Sumatra  Fig. 4747. Capri in Java  probablyPageBreakof the family level referred to “indet.” and the species level designated “sp.” are herein excluded from our comparisons. The Khok Sung large mammalian assemblage yields most extant and some extinct taxa, which are characteristic of the Ailuropoda-Stegodon assemblage. Compared to other Thai Pleistocene faunas, the Khok Sung mammalian assemblage shares 10 ten species with Thum Wiman Nakin , Ursusthibetanus (Asiatic black bear), Pongopygmaeus (orang-utan), Muntiacusmuntjak (Southern red muntjac), and Tapirusindicus  are absent in Khok Sung. The paleoenvironments of Khok Sung corresponded to a floodplain near the river channel (Elephasmaximus and Capricornissumatraensis) are found in the locality, these fossils  were transported from the surrounding upland forests by the river.The Khok Sung assemblage shares at least one similar archaic mammal taxon such as  channel . The absThe degree of the faunal similarity also depends on the number of identified taxa for each site. We further analyse the relationships between the geographic regions and faunas in Southeast Asia, using the Simpson coefficient of faunal similarity Tab.  performePageBreakPageBreakWiman Nakin according to the Simpson’s index  Fig. . Within dex Tab. . This isOverall, this analysis suggests initially that the differences in species composition and distribution do not follow a trend of the latitudinal gradient north to south, but show spatial and time variability of large mammalian fauna in Southeast Asia. The main problems of mammalian fauna comparisons in Southeast Asia are likely due to the poorly-known species diversity and/or the imprecisely chronological determination in several localities.PageBreakKhok Sung is possibly different, slightly older or younger, from those three localities according to some of the compositional dissimilarity. Two early Late Pleistocene sites: Nam Lot  present in Khok Sung are possibly geographical remnants of the former Siva-Malayan fauna that survived until the late Middle Pleistocene as they occurred earlier in Java. Otherwise, these vertebrates possibly appeared either firstly or repeatedly  in Thailand during the late PageBreakMiddle Pleistocene. Several cyclic occurrences of high amplitude glacial periods , Macacanemestrina (pig-tailed macaque), Pantheratigris (tiger), Dicerorhinussumatrensis (Sumatran rhinoceros), Helarctosmalayanus (sun bear), Capricornissumatraensis, Bubalusarnee, Susscrofa, and Susbarbatus. The Khok Sung mammalian assemblage consists of at least 4 of forest dwelling mammals: Capricornissumatraensis, Bubalusarnee, Susbarbatus, and Elephas sp.  may indicate the faunal exchange from Indonesia to the mainland Southeast Asia  subsequently in Java  that evidently continued to exist up until today in Thailand.The Khok Sung mammalian assemblage supports that Thailand was a biogeographic gateway of the Sino-Malayan migration event as the mainland forested faunal association replaced the earlier Siva-Malayan fauna ( in Java . The gla


Influence maximization is the task of finding a set of seed nodes in a social network such that the influence spread of these seed nodes based on certain influence diffusion model is maximized. Topic-aware influence diffusion models have been recently proposed to address the issue that influence between a pair of users are often topic-dependent and information, ideas, innovations etc. being propagated in networks are typically mixtures of topics.In this paper, we focus on the topic-aware influence maximization task. In particular, we study preprocessing methods to avoid redoing influence maximization for each mixture from scratch.We explore two preprocessing algorithms with theoretical justifications.Our empirical results on data obtained in a couple of existing studies demonstrate that one of our algorithms stands out as a strong candidate providing microsecond online response time and competitive influence spread, with reasonable preprocessing effort.The online version of this article (doi:10.1186/s40649-016-0033-z) contains supplementary material, which is available to authorized users. Influence maximization is the task of finding a set of seed nodes in a social network such that the influence propagated from the seed nodes can reach the largest number of people in the network. More technically, a social network is modeled as a graph with nodes representing individuals and directed edges representing influence relationships. The network is associated with a stochastic diffusion model  through influence diffusion starting from the seed nodes, is maximized . Then for any influence probability function p, we select edge  as a live edge if blocked edge. All live edges form a random live-edge graphS in random graph p and S in S in We use the following coupling method. For any edge We remark that using a similar idea as above we could show that influence spread in the linear threshold (LT) model  is also p, and a budget k, influence maximization is the task of selecting at most k seed nodes such that the influence spread is maximized, i.e., finding set p, the marginal influence (MI) of a node v under seed set S is defined as k seeds in k iterations, and in the j-th iteration it selects a node S guarantees that the greedy algorithm selects a seed set with approximation ratio Given a graph mentclass2pt{minim #P-hard . The mond base topics, and we use set notation topic mixture. Given a directed social graph p for any diffusion item p on edges.Topic-aware independent cascade (TIC) model  is an exG, base topics [d], influence probability function i, a budget k and an item topic-aware influence maximization is the task of finding optimal seeds Given a social graph There are relatively few studies on topic-aware influence analysis. For our study, we are able to obtain datasets from two prior studies, one is on social movie rating network Flixster  and the We obtain two real-world datasets, Flixster and Arnetminer, which include influence analysis results from their respective raw data, from the authors of the prior studies , 16.u and v are connected by a directed edge  if they are friends both rating the same movie and v rates the movie shortly later after u does so. The network contains 29,357 nodes, 425,228 directed edges and 10 topics , BTS[Greedy] and MIS[PMIA], BTS[PMIA], respectively.We conduct all the experiments on a computer with 2.4 GHz Intel(R) Xeon(R) E5530 CPU, 2 processors (16 cores), 48G memory, and an operating system of Windows Server 2008 R2 Enterprise (64 bits). The code is written in C++ and compiled by Visual Studio 2010.We test these algorithms on the Flixster and Arnetminer datasets as we described in "In terms of topic mixtures, in practice and also supported by our data, an item is usually a mixture of a small number of topics, thus our tests focus on testing topic mixtures from two topics. First, we test random samples to cover most common mixtures as follows. For these three datasets, we use 50 topic mixtures as testing samples.In the preprocessing stage, we use two algorithms, Greedy and PMIA, to pre-compute seed sets for MIS and BTS, except that for the DBLP dataset, which is too large to run the greedy algorithm, we only run PMIA. Algorithms MIS and BTS need to pre-select landmarks We choose S, its influence spread plus the sum of top k marginal influence spread of k other nodes is an upper bound on the optimal k seed influence spread. We use the minimum of the upper bounds among the cases of S being one of the greedy seed sets selected.In addition, we apply offline bound and online bound to estimate influence spread of optimal solutions. Offline bound is the influence spread of any greedy seeds multiplied by factor rem 4 in : for anyAdditional file As is shown in Table For the Arnetminer dataset (Additional file The detailed analyses are listed as follows: First, topic-oblivious algorithms does not perform well in topic-aware environment. Based on Observation ds Table b, which me Table a.What we can conclude from tests on Arnetminer is that, for networks where topics are well separated among nodes and edges such as in academic networks, utilizing preprocessing can greatly save the online processing time. In particular, MIS algorithm is well suited for this environment achieving microsecond response time with very small degradation in seed quality.For Flixster dataset (Additional file entclass1pt{minimaIn DBLP (Additional file To summarize, the greedy algorithm has the best influence spread performance, but is slow and not suitable for large-scale networks or fast response time requirements. PMIA as a fast heuristic can achieve reasonable performance in both influence spread and online processing time, but its response time varies significantly depending on graph size and influence probability parameters, and could take minutes or longer to complete. Our proposed MIS emerges as a strong candidate for fast real-time processing of topic-aware influence maximization task: it achieves microsecond response time, which does not depend on graph size or influence probability parameters, while its influence spread matches or is very close to the best greedy algorithm and outperforms other simple heuristics. Furthermore, in large graphs where greedy is too slow to finish, PMIA is a viable choice for preprocessing, and our MIS using PMIA as the preprocessing algorithm achieves almost the same influence spread as MIS using the greedy algorithm for preprocessing.Domingos and Richardson , 4 are tA large body of work follows the framework of . One linA recent independent work by Aslay et al.  is the cOne possible follow-up work is to combine the advantages of our approach and the approach in  to furth


Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of The online version of this article (doi:10.1007/s11240-017-1168-9) contains supplementary material, which is available to authorized users. However, of these, ferulic acid has been found to be the most prevalent in most grass cell walls -d-xylose], FAXX {4-0-[3-0-(5-0-feruloyl-α-l-arabinofuranosyl)-β-d-xylanopyronosyl]- d-xylose} and the corresponding p-coumaroyl esters (PAX and PAXX)  genes, which increased activities of superoxide dismutase, peroxidase and catalase, and the proline content of plants resulting in enhanced salt tolerance and superior plant growth   from A. niger  barley aleurain signal sequence  or a linker (PVAAA) followed by the KDEL reading frame for the ER vector .For the C-terminals two different sequences were utilized, the native or Table . In ordeor Table , to geneAspergillus excretion signal for apoplast targeting (pTR2).For stable transformations vectors were finally used with FAE driven either by the rice actin promoter,  or the soybean 2019E 23 kD heat shock promoter (pTT3) with either an intact barley aleurain vacuolar targeting sequence, (pTT3), or with modified versions for apoplast targeting (NPIR to NPGR) and with modification to incorporate the KDEL sequence for ER retention (pTU5) or containing the native hph) resistant gene driven by a CaMV35S promoter (pROB5) in pUC18  under the control of a CaMV35S promoter. Further details of vector construction can be found in Dunn-Coleman et al. , cultivar 20BN3 were initiated from shoot-tip-derived embryogenic callus grown on medium 133 MS pH 5.6, with 3% sucrose and 3 mg l−1 2–4-D solidified with 0.8% agar. Eight to ten weeks old embryogenic suspension cultures were bombarded either with a single co-integration plasmid vector containing FAE and hyg resistance genes, or with a co-transformation vector containing FAE and with plasmid pROB5 conferring hygromycin resistance, using a Particle Inflow Gun  over a 10–12 weeks selection period at 25 °C under continuous white fluorescent light (60 μE m2 s−1).Cell suspensions of tall fescue . Transformed cells were grown in medium containing 150 units of hygromycin per litre at 25 °C and shaken at 150 rpm on an orbital shaker and were subcultured every 7 days.2 and 1U Taq DNA polymerase (Perkin-Elmer). PCR reactions were carried out in a Perkin-Elmer 480 under the following conditions: 1 cycle of 94 °C for 1 min and 30 s, 30 cycles of 30 s denaturation at 94 °C, 30 s primer annealing at 65 °C, 1 min extension at 72 °C. PCR amplification products were analysed by electrophoresis on 1.0% agarose/TAE gels in Tris-borate-EDTA buffer  cultures was used. Callus tissue was placed on a 5 × 2 cm strip of sterile 180 water proof latex paper (Rhynowet) and ground between the paper using 120 µl of buffer S  and then transferred to 0.5 ml Eppendorf tubes. 80 µl phenol/chloroform was added, vortexed, centrifuged and the aqueous top layer transferred to Eppendorfs containing 70 µl of isopropanol. Pellets were then resuspended in 10 µl TE buffer. PCR analysis was carried out using primers specific for the FAE gene. PCR reaction mixtures of 25 µl volume contained: 0.5 µl of final DNA extract, 48 µM dATP, dTTP, dCTP, dGTP, 0.2 µM primers (FAEN5- CTAAA GCTTA CCATGG CGGCC GCCTC CACGC AGGGC ATCTC CGA and FAE3-TCTAA GCTTG CGGCC GCGCA CGGCC AGGTC CATGC GTCGT CATCCC), 1× Amplitaq commercial buffer (as supplied), 3mM MgClHindIII (which excises the FAE gene) or with BglII (which restricts at one site in the plasmid DNA) restriction enzymes (Boehringer-Mannhein). Ten micrograms of digested DNA were separated by electrophoresis through 1.0% agarose gels in Tris-borate-EDTA buffer . DNA was fixed to the membrane by UV cross-linking, using a CL-1000 ultraviolet crosslinker (UVP). A digoxigenin-labelled FAE probe was prepared by PCR of 100 pg plasmid DNA using primers and conditions described above, except that the final nucleotide concentration was: 48 µM dATP, dCTP, dGTP; 36 µM dTTP, 12 µM DIG-dUTP (Boehringer–Mannhein). The product of the reaction was extracted from a 0.8% low-melting point agarose/TAE gel.Genomic DNA was isolated from PCR positive cell cultures following the procedure of Dellaporta et al.  and digeMembranes were pre-hybridised in 6.5 ml EasyHyb solution (Boehinger–Mannehein) at 42 °C for 3–5 h, in a hybridisation oven (Stuart Scientific). The DIG labelled FAE probe, corresponding to the entire 1.2Kb was boiled for 10 min and 10 µl was added to 6.5 ml of fresh, pre-warmed EasyHyb solution and hybridisation was carried out overnight. After hybridisation, membranes were washed in trays: twice in 2× SSC/0.1% SDS for 10 min each at room temperature, then twice in 0.2× SSC/0.1% SDS for 30 min each at 68 °C. Blocking and detection of the hybridised probe DNA was carried out as described by the manufacturer (Boehringer–Mannheim) except for blocking which was for 90 min. Exposure times were 1–2 h.Cell growth was determined from increases in fresh and dry weight by sampling 10 ml of suspension from triplicate flasks at each time point and filtering onto pre-wetted Whatman No. 1 filter paper under vacuum. Dry matter determinations were obtained after drying cells at 50 °C to constant weight.Ferulic acid esterase activity was determined in soluble extracts from 0.5 g of fresh or frozen cell cultures. Cells were ground in a precooled mortar and pestle with 0.1 M sodium acetate, pH 5.0 extraction buffer containing 0.5% Triton. Extracts were incubated with 24mM ethyl ferulate (ethyl 4-hydroxy-3-methoxycinnamate) at 28 °C for 24 h. Following incubation, and centrifugation, soluble extracts were loaded onto an activated reverse phase C18 µNova Sep-Pak column (Waters), and eluted with 4 ml 100% methanol (MeOH). FAE activity was calculated from the amount of ferulic acid released determined by HPLC. One unit of FAE activity equals 1 µg ferulic acid released from ethyl ferulate at 28 °C in 24 h.Heat-shocked cells were assayed in the same way as above, but prior to assay the cells were routinely heat shocked at 38 ± 1 °C for 2 h in a shaken incubator and then allowed to recover for 24 h at 25 ± 1 °C on an orbital shaker. At the end of the 24-hour period cells were assayed for FAE activity by HPLC. FAE activity was also determined by measuring the release of monomeric and dimeric ferulic acids from self-digested cell culture samples.2. After centrifugation, acidification and precipitation of solubilised carbohydrates with MeOH at 4 °C, the extracted phenolics in the aqueous phase were loaded onto an activated reverse phase C18 µNova Sep-Pak column and eluted with 100% MeOH, and analysed by HPLC.Following exhaustive extraction of soluble phenolics with aqueous methanol, ester bound phenolics were extracted from the cell walls of freeze dried powdered cell cultures (50 mg), with 1 M NaOH (5 ml) followed by incubation at 25 °C for 24 h, in the dark under NHPLC was carried out on a µNova Pak C18 8 × 10 Radial Compression Module (Waters) with MeOH: 5% acetic acid either with a 35–65% linear MeOH gradient in 15 min (for FAE assay) or with a 30–70% linear MeOH gradient in 25 min  at 2 ml/min. Hydroxycinnamic acids were monitored and quantified at 340 nm, with a Waters 996 photo-diode array detector with UV /visible spectra collected at 240–400 nm, and analysed with Millennium software (Waters Inc) against authentic monomer standards, or using response factors for the various dehydrodiferulate dimers reported by Waldron et al. .18 µNova Sep-Pak column (Waters Inc), eluted with 100% MeOH and analysed by HPLC as described above.An autodigestion procedure was used to investigate the effect of the expression of FAEA on the release of cell wall HCAs on cell death. Callus cultures (0.2–0.5 g fresh wt) were ground in 0.1 M Na acetate extraction buffer pH 5.0, in the presence and absence of excess β-1-4-endoxylanase (1000 U/sample) (GC140 Genencor Inc), without additional substrate, and incubated at 28 °C for 24 to 72 h with shaking at 120 rpm. Following centrifugation, soluble extracts were loaded onto a reverse phase CDigestibility parameters were also determined from in vitro gas production measurements. Freeze dried powdered cell suspension cells (1.0 g samples) were fermented in triplicate in 165-ml capacity serum bottles in 90 ml of bicarbonate buffered medium and were inoculated with rumen micro-organisms in 10 ml of clarified rumen fluid, at 30 °C for 48 h according to the pressure transducer technique of Theodorou et al. . This tefaeA driven by a rice actin promoter, with a number of C and N terminal ends, , and with modifications to the core ORF, (to enhance expression in plants with the codon choice based on published barley preferences) were made and tested in transient expression for FAE activity following micro-projectile bombardment of grass cell cultures. A smaller number of functional constructs were chosen for stable transformations and used to construct vectors containing constitutive and inducible heat-shock promoters.A number of expression vectors in pUC plasmids with Festuca cell cultures with FAXX or ethyl-ferulate as substrates. No FAE activity was detected with the constructs lacking an N terminal signal sequence, irrespective of C terminal modifications and consistently lower FAE activity was detected with the native FAE N terminal signal than with the barley aleurain signal (data not shown). Mutation of the NPIR vacuolar localisation motif of the barley aleurain transit peptide to NPGR was found to be as effective as removal of this motif but both resulted in a decrease in transient FAE activity compared with the native NPIR . Removing the putative glycosylation site or truncating the ORF at the 32 amino acid site also severely reduced FEA expression from the aleurain signal. In some cases the activities of FAE were lower with the native Aspergillus C terminal end (CTW) than when a linker sequence was added (CTW-PVAAA) suggesting that the native Aspergillus C terminal end has unusual and probably deleterious effects in grasses (data not shown).Reproducible transient FAE activity was detected in soluble cell extracts of The co-integration vectors containing the bacterial gene conferring hygromycin resistance were made following a number of experiments to compare the levels of FAE expression obtained from constructs in which the orientation of FAE and the resistance gene were varied. A tandem arrangement of both genes, with FAE followed by the resistance gene with the same reading frame polarity, was found to be at least as effective as other arrangements in transient expression assays  and was therefore used for the final series of constructs.sgfp) were constructed and the cellular distribution of gfp fluorescence determined in transient expression studies in embryogenic cell cultures of Festuca. Figure In order to obtain information on the effectiveness of targeting sequences in delivering FAE to the different compartments, parallel vectors containing a green fluorescent protein , and further Southern blot analysis hybridised with FAE probes, showed at least one intact copy of FAE in line TT3 and TU5 and multiple intact copies in line TR2 together with a number of higher molecular weight bands probably corresponding to multiple tandem rearrangements of the FAE gene  and TT3(Vac) lines and by 30% in the TU5(ER) line Fig. a, and feThe very low levels of ester bound ferulates found in the TT3 line are at first slightly surprising, given the low levels of cellular FAE activity, sequestration of FAE in the vacuole, and the finding of little excretion of FAE into the medium. It should be noted however that the heat-shock induced FAE activities shown in Fig. Self-digestion of the hemicellulose component of the cell wall after disruption of callus cultures of vacuolar, apoplast or ER targeted FAE-expressing cells resulted in the release of small but significant quantities of esterified ferulic acid at higher levels compared to non-transformed control cultures Fig. . FerulicTraditionally tissue digestibility is determined as in-vitro-dry matter digestibility (IVDMD) and is an estimate of end point digestion , and were further enhanced by the addition of 1000U β-1-4 endoxylanase . Furthermore initial rates of digestion in the presence of added xylanase were substantially higher than control cells in the presence of added FAE and xylanase  Fig. a, indicaSimilar but less pronounced results were found for maximum rates of gas evolution Fig. b. The tiFestuca arundinacea plants expressing FAE  and both modifications were incorporated into subsequent FAE vectors for plant transformation . This may indicate that poor translation of Below is the link to the electronic supplementary material.Supplementary material 1 (DOC 5712 KB)


Diarylethene-derived molecules alter their electronic structure upon transformation between the open and closed forms of the diarylethene core, when exposed to ultraviolet (UV) or visible light. This transformation results in a significant variation of electrical conductance and vibrational properties of corresponding molecular junctions. We report here a combined experimental and theoretical analysis of charge transport through diarylethene-derived single-molecule devices, which are created using the mechanically controlled break-junction technique. Inelastic electron tunneling (IET) spectroscopy measurements performed at 4.2 K are compared with first-principles calculations in the two distinct forms of diarylethenes connected to gold electrodes. The combined approach clearly demonstrates that the IET spectra of single-molecule junctions show specific vibrational features that can be used to identify different isomeric molecular states by transport experiments. Molecular junctions hold promise for the realization of novel miniaturized electronic circuits –6 as welIn this study, we investigate both elastic and inelastic charge transport through single-molecule junctions of a bis(furanylmethanthiol)ethene with a fluorinated cyclopentene bridging unit . Experi3, denoted as Ac) groups are cleaved off by adding a droplet of ammonium hydroxide (NH4OH), resulting in a thiol (SH) group, before assembly on the metal surfaces of the sample. The MCBJ sample preparation /2, since the vibronic excitations are expected to show antisymmetric features in the IET spectra with respect to bias reversal [Subsequently, we measured IET spectra for the two different forms of C5F-ThM molecular junctions and compared them with computed IET spectra, as shown in /(dI/dV) . IET meareversal . This rereversal . The proTo elucidate the origin of the different IET spectra in the two forms, we have computed the inelastic transport of the optimized geometries shown in The comparison of the theoretically computed IET spectra with the measured ones is not easy due to the rather broad peak features found in the experiments. We want to stress again the challenge to record these spectra for such low-conducting molecules, requiring an averaging procedure over several spectra, as explained above. Both experimental and theoretical results show, however, that IET spectra can distinguish between the different states of the functional molecular switching core through the different vibrational properties of either the open or closed form.We have studied the elastic and inelastic charge transport behavior of difurylethene-derived single-molecule junctions at low temperatures both experimentally and theoretically. We have found that the HOMO is much closer to the Fermi energy than the LUMO, but the energy-dependent transmission still suggests that the single-level toy model is not straightforwardly applicable. Based on the position of the HOMO level and its broadening in the transport calculations, we have nevertheless argued that experimental trends of an effective level that is better aligned with the metal’s Fermi energy and better coupled to the electronic states of the electrodes, when going from the open to the closed molecular state, are consistent with theoretical expectations. These trends naturally result in a higher electrical conductance of the closed form, as observed both experimentally and theoretically. In this context, we have also seen that the HOMO–LUMO gap in the closed form is smaller than in the open one. Quantitatively, theory and experiment differ by one order of magnitude in the conductance of the open form but two orders in the closed one, which requires further investigations. Points to examine are the accuracy of theoretically described level alignments for molecules with extended π-electron systems and ideally a representative ensemble of junction geometries as well as the possibility of the existence of several single-molecule junction geometries with differing conductance. Finally, we have performed an IET spectroscopy study and have shown that different IET spectra are obtained experimentally and theoretically for open and closed forms. IET spectroscopy can hence be used to distinguish between different molecular “on” and “off” states by means of their vibrational features. These findings advance the development of functional molecular electronics and prove at the same time that IET spectroscopy is a valuable technique to probe the isomeric states of individual molecules.


Hypotensive neonates who have been treated with dopamine have poorer neurodevelopmental outcome than those who have not been treated with dopamine. We speculate that dopamine stimulates adrenoceptors on cerebral arteries causing cerebral vasoconstriction. This vasoconstriction might lead to a rightward shift of the cerebral autoregulatory curve; consequently, infants treated with dopamine would have a higher risk of low cerebral blood flow at a blood pressure that is otherwise considered “safe”.In anaesthetized piglets, perfusion of the brain, monitored with laser-doppler flowmetry, and cerebral venous saturation was measured at different levels of hypotension. Each piglet was studied in two phases: a phase with stepwise decreases in MAP and a phase with stepwise increases in MAP. We randomized the order of the two phases, whether dopamine was given in the first or second phase, and the infusion rate of dopamine . In/deflation of a balloon catheter, placed in vena cava, induced different levels of hypotension. At each level of hypotension, fluctuations in MAP were induced by in/deflations of a balloon catheter in descending aorta.2 and arterial saturation were stable. MAP levels ranged between 14 and 82 mmHg. Cerebral autoregulation (CA) capacity was calculated as the ratio between %-change in cerebrovascular resistance and %-change in MAP induced by the in/deflation of the arterial balloon. A breakpoint in CA capacity was identified at a MAP of 38±18 mmHg without dopamine and at 44±18, 31±14, and 24±14 mmHg with dopamine infusion rates of 10, 25, and 40 μg/kg/min (p = 0.057). Neither the index of steady-state cerebral perfusion nor cerebral venous saturation were affected by dopamine infusion.During measurements, PaCODopamine infusion tended to improve CA capacity at low blood pressures while an index of steady-state cerebral blood flow and cerebral venous saturation were unaffected by dopamine infusion. Thus, dopamine does not appear to impair CA in newborn piglets. Hypotension in newborn infants is associated with higher incidence of mortality and cerebral injury–5. CerebHypotensive neonates treated with dopamine have poorer long-term neurodevelopmental outcome than those who have not received dopamine,12–14. TCerebral autoregulation (CA) is a protective mechanism that maintains a fairly stable cerebral blood flow despite fluctuations in MAP. Cerebral arteries reach their maximal compensatory vasodilation when MAP is at the lower limit of CA. Reduction in MAP below the lower limit of CA will reduce cerebral blood flow and ultimately expose the brain to ischaemia. We have previously described an association between impaired CA and dopamine infusion in very preterm infants, unfortuThe observed association between poor neurodevelopmental outcome and dopamine therapy in hypotensive infants might be explained in different ways: dopamine usage might rather be an indicator of disease and vulnerability, or maybe dopamine has a direct deleterious effect on the developing brain as dopamine has been shown to cross the blood-brain-barrier in preterm infants. AnotherThe Danish Animal Experiments Inspectorate approved the experimental protocol: 2014-15-0201-00123.The sample size calculation was based on a previous study where dopamine infusion increased cerebral perfusion in piglets (gain-laser doppler flowmetry 3±1.4%/mmHg). If we a2 between 4–6 kPa and arterial saturation above 95%. Arterial saturation and heart rate were monitored by pulse oximetry from the forepaw . Piglets were kept on a heating pad to maintain rectal temperature 38.5–39.5°C.Anaesthesia was induced by 3% isoflurane and supplemented by 5 mg/kg propofol and 20 μg/kg fentanyl and maintained on propofol 15–20 mg/kg/h and fentanyl 7 μg/kg/h. Fentanyl was discontinued after the preparation of the piglet and at least one hour before start of the protocol. A bolus of propofol or fentanyl was given if the piglet showed signs of superficial anaesthesia or pain. After tracheostomy, the piglet was connected to a pressure-controlled mechanical ventilator. Settings were adjusted to maintain PaCOThe femoral arteries were cannulated with (i) an arterial line for MAP monitoring and blood gas sampling (placed in the thoracic part of aorta), and (ii) a 4 FR embolectomy-catheter with a balloon near its tip  with the purpose of increasing MAP in the upper part of the body by inflating of the balloon. The arterial balloon catheter was positioned by inflating the balloon: first inflation of the balloon above the arterial line, resulting in a flat arterial blood pressure waveform. Then the arterial balloon catheter was drawn back until inflation of the balloon resulted in increased MAP along with an intact blood pressure waveform and left there. The femoral veins were cannulated with (i) a 4 FR double lumen central venous line for infusion of glucose and dopamine, and (ii) another 4 FR embolectomy-catheter with the balloon in the inferior vena cava just before the inlet into right atrium (16cm) with the purpose of decreasing cardiac preload when inflated and hereby to induce arterial hypotension. Control of the placement was done by checking that MAP was easily manipulated. In some cases, position was confirmed by x-ray.We monitored micro-vascular perfusion by laser-doppler flowmetry (LDF) . For placement of the LDF probe in the right parietal region, a biopsy punch was used to perform a craniotomy and dura was leaved intact. The LDF probe was fixated in a specially designed metal washer glued to the skull of the piglet. In this way, we secured that measurements were from the same point on the cerebral cortex. LDF, measured as perfusion units (PU), are non-absolute values that depend on the number of moving red blood cells as well as the velocity of these cells. Therefore, relative changes in PU were used as an index of steady-state cerebral blood flow. Superior sagittal sinus was cannulised and cerebral venous blood was sampled in order to determine cerebral venous saturation. All piglets recovered for one hour after preparation.The experimental protocol is illustrated in At each level of hypotension, blood samples were drawn from the arterial line and from superior sagittal sinus. Then, to test the autoregulatory capacity, five fluctuations in MAP were induced by in- and deflating 0.4 ml saline into the aortic balloon catheter, causing rapid in- and decreases in the blood pressure, each cycle lasting 60 sec. Between the two phases (i.e MAP being either in- or decreased), the venous balloon was fully deflated while the piglet recovered. If dopamine was given during the first phase dopamine infusion rate was reduced by 10 μg/kg/min every 10 mins during the recovery phase. After dopamine infusion was stopped the piglet recovered for 30 mins. If dopamine was given in the second phase the piglet recovered for 30 mins after dopamine infusion was initiated. At the end of the protocol, piglets were euthanized by pentobarbitone (100 mg/kg) while they were still anaesthetized.CA capacity was estimated, as described by Tiecks et al, as a meThe relation between MAP and the outcome variables  were best described by a regression line with a break, i.e. two intersecting regression lines with a breakpoint: a positive slope below the breakpoint, and a horizontal line above the breakpoint. This method has previously been used to describe the relationship between MAP and cerebral blood flow,23. We uThe following equation was used to describe relationship between one outcome variable and MAP: ‘Outcome variable’ = (MAP < breakpoint) * ((MAP—breakpoint) * slope) + plateau. The first bracket in the equation gives a dichotomous outcome– 0 when false and 1 when true. Thus, if MAP is above the determined breakpoint, the value of the first bracket is zero and the ‘outcome variable’ = plateau. On the other hand, if MAP is below the breakpoint, the value of the bracket is one and it is possible to calculate the slope of the curve below the breakpoint. We assumed ‘perfect’ autoregulation above the breakpoint, the ‘plateau’. Since each piglet only contributed five to seven data points with and without dopamine, respectively, non-linear regression fitting three parameters  will not converge. Therefore, we determined the common slope and plateau for all the piglets in one analysis. Thereafter we performed non-linear regression analysis for each piglet to determine individual breakpoints with the slope and plateau as fixed parameters as determined in the common analysis. These individual breakpoints were related to dopamine infusion rate by linear regression. The index of steady-state cerebral blood flow measured by LDF was logarithmic transformed to obtain variance homogeneity. We used students t-test to compare means in two groups, and paired t-test when changes in a parameter within the same piglet were tested. Values are given as mean ± standard deviation or median (range).2 were stable during the experiment. None of the piglets needed resuscitation during the experiment or were euthanized prior to termination of the experiment.Eighteen piglets were included in the study (median age 60 hours (range 4–66 hours), weight 1.9 ± 0.2 kg). MAP before start of protocol was 59 ± 10 mmHg. Arterial saturation and arterial pCONone of the outcome variables: Cerebral venous saturation, the index of steady-state cerebral blood flow, or CA capacity were affected by whether the piglets were randomized to have dopamine in the first or last phase or whether MAP went high-to-low or low-to-high.In the low-dosage dopamine group (10μg/kg/min) MAP was unaffected by dopamine infusion , whereas MAP increased when dopamine infusion rates were 25 μg/kg/min  and 40 μg/kg/min .-1*min-1 (p = 0.558) and is illustrated in During the induced hypotension, MAP ranged between 14 and 82mmHg. When including all data in one non-linear regression analysis, we found a slope of 0.78%/mmHg of the cerebral venous saturation curve below the breakpoint. The plateau was placed at a cerebral venous saturation of 45%. Based on the individual determination of the breakpoints, the mean breakpoints were at 47±2 mmHg, 43±6 mmHg, 50±4 mmHg, and 53±6mmHg, without dopamine, with 10 μg/kg/min, 25 μg/kg/min, and 40 μg/kg/min, respectively. Regression coefficient of the relation between individual breakpoints of cerebral venous saturation and dopamine infusion was 0.09 mmHg/μg*kg-1*min-1, p = 0.863).The relation between the index of steady-state cerebral blood flow, as indicated by LDF, and MAP had a positive slope of 1.06%/mmHg below a breakpoint and the plateau was placed at 96% of the baseline LDF value. When the piglets did not receive dopamine the breakpoint was at 36±18 mmHg. With increasing dopamine infusion rates the breakpoints were at 30±18 mmHg, 35±10 mmHg and 38±18 mmHg. There was no trend between the individual breakpoints of an index of steady-state cerebral blood flow and dopamine infusion rate   and the plateau was placed at a CA capacity of 26%. The breakpoint was 38±18 mmHg when the piglets did not receive dopamine. Piglets receiving dopamine infusion rates of 10 μg/kg/min, 25 μg/kg/min and 40 μg/kg/min had breakpoints at 44±18 mmHg, 31±14 mmHg and 24±14 mmHg, respectively . The rel = 0.057 .Our hypothesis was that dopamine affected CA negatively by inducing cerebral vasoconstriction. We used three different measures to examine our hypothesis. Cerebral venous saturation is a global estimate of the venous saturation from both hemispheres, whereas the two other measures: CA capacity, as an estimate of cerebral vasoreactivity, and the index of steady-state cerebral blood flow are based on perfusion in a very small area of cortex as interrogated by the laser Doppler light probe. Surprisingly, CA capacity rather tended to improve with increasing infusion rates of dopamine while there was no suggestion of an effect of dopamine treatment on the index of steady-state cerebral blood flow or cerebral venous saturation at low levels of MAP.CA maintains cerebral blood flow by cerebral vasodilation when MAP is low. Below the lower limit of CA, cerebral blood flow falls in proportion with the fall in MAP. As cerebral metabolic rate of oxygen is the product of cerebral blood flow and cerebral oxygen extraction and if cerebral metabolic rate of oxygen is constant, then changes in cerebral oxygen extraction reflect changes in cerebral blood flow . We chosSuperior sagittal sinus blood comes from both hemispheres but is dominated by blood from cortex with higher blood flow. White matter is more vulnerable to ischemia during hypotension , thus weThe MAP breakpoints, i.e. where the cerebral venous saturation began to drop in response to a decrease in MAP, were unaffected by dopamine infusion . This suWe estimated an index of steady-state cerebral blood flow at each level of hypotension in parietal cortex by LDF. Dopamine infusion did not affect the location of breakpoints . If dopa−8 M to 2 x 10−5 M[−7–10−6 M. Therefore, dopamine in the infusion rates that we used could, in theory, induce vasodilation. However, these theoretical considerations are not supported by the two other outcomes in this study. Another explanation could be that dopamine improved vasoreactivity and therefore reduced the degree of cerebral vasoparalysis at low MAP. In line with this explanation, dopamine therapy has been found to prevent loss of CA during hypotension in simulated traumatic brain injury; and restore the ability of pial arteries to vasodilate in response to hypotension[CA capacity quantifies the cerebral arteries’ ability to adjust cerebrovascular resistance in response to induced fluctuations in MAP, i.e. the vasoreactivity. We were surprised that CA capacity rather tended to improve at low MAP with higher infusion rates of dopamine Figs  and 3. A x 10−5 M. If we a x 10−5 M, our infpotension. Also, ipotension. We consThe advantage of our study is that we compared cerebrovenous oxygenation, an index of steady-state cerebral blood flow, and CA capacity at comparable levels of MAP with and without dopamine infusion. This is important because an increase in MAP, caused by dopamine infusion, in itself might affect these variables.Our study also has limitations. First, we used healthy newborns piglets to mimic hypotensive newborn infants. We do not know if the presence and development of dopaminergic and adrenergic receptors are identical in piglets and infants. The piglets were healthy, in contrast, most infants who are treated with dopamine are distressed and might have increased basal sympathetic tone. Second, we used MAP instead of cerebral perfusion pressure in our description of CA. Minor changes from normal intracranial pressure to lower intracranial pressure might be expected when we induced hypotension. However, we would not expect intracranial pressure to vary significantly in our experimental design and Brady et al showed that in gradually hypotensive newborn piglets an estimate of CA using MAP was highly comparable to an estimate of CA using cerebral perfusion pressure. Third, When we calculated the breakpoints in the non-linear regression analysis we assumed that the slope below the breakpoint was the same without and in all three dosages of dopamine. It is possible, that the slope is affected by dopamine dosage; but the study was not powered to study this.Another concern might be if our anaesthetic procedure affects our measurements. Piglets were initially sedated by isoflurane. It is well described that isoflurane impairs CA and therTo ensure equal distribution of design-induced bias, data from each piglet was obtained with and without dopamine, and with MAP both being lowered and raised. Furthermore, to exclude a time-dependent bias, the order of dopamine/not dopamine and increasing/decreasing MAP was randomized between piglets. Ideally, each piglet should have been studied without dopamine and with all three infusion rates of dopamine to eliminate the effect of variability among piglets. We did not attempt this study design because we expected, based on previous experience with experiments on newborn piglets, deterioration of the piglet model over four periods with hypotension and a long experimental time. We considered that measurements during two periods with severe hypotension were a more robust design. Actually, severe hypotension might induce cerebral ischemia and this could affect autoregulation afterwards. Despite severe hypotension lasting approximately 10 min, we did not observe any cardiac arrest. Neither did we see any influence on our measurements . In this model the short duration of hypotension and decreased blood flow did not lead to impairment of CA. In an experimental setting with hypotensive adult cats, ischemic brain lesions were only seen when MAP was lowered to 50% of the lower limit of CA for at least 15 min.Dopamine tended to improve cerebral autoregulation capacity at low arterial blood pressure; however, a beneficial effect of dopamine was not confirmed by improved cerebral blood flow or cerebrovenous oxygen saturation. We conclude that dopamine therapy does not appear to affect cerebral autoregulation negatively in hypotensive newborn piglets.S1 File(SAV)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Wolfiporia cocos is an edible and medicinal fungus that grows in association with pine trees, and its dried sclerotium, known as Fuling in China, has been used as a traditional medicine in East Asian countries for centuries. Nearly 10% of the traditional Chinese medicinal preparations contain W. cocos. Currently, the commercial production of Fuling is limited because of the lack of pine-based substrate and paucity of knowledge about the sclerotial development of the fungus. Since protein kinase (PKs) play significant roles in the regulation of growth, development, reproduction, and environmental responses in filamentous fungi, the kinome of W. cocos was analyzed by identifying the PKs genes, studying transcript profiles and assigning PKs to orthologous groups. Of the 10 putative PKs, 11 encode atypical PKs, and 13, 10, 2, 22, and 11 could encoded PKs from the AGC, CAMK, CK, CMGC, STE, and TLK Groups, respectively. The level of transcripts from PK genes associated with sclerotia formation in the mycelium and sclerotium stages were analyzed by qRT-PCR. Based on the functions of the orthologs in Sclerotinia sclerotiorum (a sclerotia-formation fungus) and Saccharomyces cerevisiae, the potential roles of these W. cocos PKs were assigned. To the best of our knowledge, our study is the first identification and functional discussion of the kinome in the edible and medicinal fungus W. cocos. Our study systematically suggests potential roles of W. cocos PKs and provide comprehensive and novel insights into W. cocos sclerotial development and other economically important traits. Additionally, based on our result, genetic engineering can be employed for over expression or interference of some significant PKs genes to promote sclerotial growth and the accumulation of active compounds. Wolfiporia cocos (Schwein.) Ryvarden et Gilb.  is an edible fungus, and its sclerotia, known as Fuling in China, have been reported to possess important medicinal value  gene SMK1 resulted in impaired sclerotial formation  levels  catalyze reversible phosphorylation of serine, threonine or tyrosine residues to control the activity of functional proteins, and they play significant roles in regulating growth, reproduction, developmental processes, and environmental stress responses . For exaormation . Sclerot) levels , indicatelopment  and deleormation .W. cocos genome based on homologous sequences searching by using BLASTx program against the Saccharomyces cerevisiae and S. sclerotiorum databases. Based on known and presumed functions of the orthologs of these PK genes found in other fungi, the putative roles of these W. cocos PKs in colonization, mycelial growth, development and response to environmental stress were assigned. The data from our study contribute to a better understanding of the potential roles of PKs in various processes of W. cocos and will help to illuminate the mechanisms of sclerotial formation.In the present study, 103 putative PK genes were putatively identified in the W. cocos  collected from Yingshan county, Hubei province, China (W. cocos mycelium was grown on a cellophane membrane place on the surface of potato dextrose agar (PDA) medium at 28°C for 7 days. Both the mycelium and sclerotium were frozen in liquid nitrogen and stored at -80°C for total RNA extraction.Sclerotium and mycelium of e, China  were kin1. Genome data was retrieved from the JGI database2.RNA-seq data from two samples of mycelium and sclerotium was from a previous study  and is aS. cerevisiae database 3 and S. sclerotiorum database4 (≥e-5) as described  was used to calculate RNA levels as previously described . To idenh DEGSeq .TM, Carlsbad, CA, USA) and treated with RNase-free DNase I  to remove residual DNA according to manufacturer’s protocols. The High Capacity cDNA Reverse Transcription Kit  was used to generate the first strand cDNA according to the manufacturer’s instructions. Gene expression was analyzed by qRT-PCR using a Bio-Rad CFX96 Real Time System  and SYBR Premix Ex Taq II , according to the manufacturer’s instructions. The PCR conditions were as follows: denaturation at 95°C for 3 min; 40 cycles of 95°C for 15 s, 55°C for 15 s and 72°C for 30 s; final step of 72°C for 10 min. The primers for qRT-PCR are listed in Table 1. The mRNA levels of the W. cocos alpha-tubulin gene  to one or more of the 92 S. sclerotiorum and/or 115 S. cerevisiae PKs (Supplementary Table S1) (Supplementary Table S1). Compared with S. cerevisiae, W. cocos is predicted to have fewer PKs genes. It lacks orthologs of 30 S. sclerotiorum and/or S. cerevisiae PKs, including DBF2/4, ELM1, ALK1/2, PTK1/2, NPR1, HSL1, ISR1, YGR052W, YLR253W, YMR291W, HAL5, KKQ8, NNK1, PRR1/2, YPL150W, KCC4, MEK1, DUN1, KSP1, CAK1, SRB10, SSK2/SSK22, MPS1, PSK1/2, BUD32, TOS3, SAK1, SCY1, IKS1/2, and RAD53 (Supplementary Table S1). Moreover, W. cocos has two orthologs of CBK1 (Wolco1| 74908 and Wolco1| 70675), COQ8 (Wolco1| 138133 and Wolco1| 144299), PKP1 (Wolco1| 110818 and Wolco1| 92641), TEL1 (Wolco1| 108405 and Wolco1| 91919), VHS1 (Wolco1| 106038 and Wolco1| 155089), CDC28 (Wolco1| 91184 and Wolco1| 93694), SAT4 (Wolco1| 159823 and Wolco1| 134885), EVN7 (Wolco1| 137941 and Wolco1| 132412), ATG1 (Wolco1| 20796 and Wolco1| 109315), SPS1 (Wolco1| 95124 and Wolco1| 111584) and three orthologs of YAK1 , SKY1 , Pan3p  (Supplementary Table S1). W. cocos also contains 16 putative PKs that have no distinct orthologs in S. cerevisiae and/or S. sclerotiorum.Our search of the able S1) . On the W. cocos AGC Group has 13 members. The PKs from the AGC Group are important in the regulation of signaling pathways in response to cell wall or membrane stress and limited nutrients. In S. cerevisiae, in response to limited nutrients and other stresses, the activity of YPK1/2, SCH9, RIM15, DBF2/20, TPK1/2  and PKC1 are directly or indirectly regulated by the atypical PKs TOR1 and TOR2  activity, thereby regulating downstream signaling pathways  and S. sclerotiorum SS1G_13577, increased at the sclerotia stage. However, the mRNA level of the other PKA, Wolco1| 115209, which is homologous to S. cerevisiae TPK1 (NP_012371.2) and S. sclerotiorum SS1G_03171, did not change (Supplementary Table S1), indicating that Wolco1| 107737, but not Wolco1| 115209, may play a major role in the sclerotial development of W. cocos. The aurora kinase IPL1 (NP_015115.1) of S. cerevisiae and Fg06959 of F. graminearum are essential; their deletion is lethal , indicating that W. cocos IPL1 may not be involved in sclerotial formation.Like many other filamentous fungi, enotypes . In S. cpathways . In S. shenotype , indicats lethal . HoweverWolfiporia cocos Wolco1| 71589 and Wolco1| 24527 encode PKs orthologous to S. cerevisiae SCH9 and RIM15, respectively. SCH9 is structurally related to PKA and works with PKA to negatively control RIM15 activity, thereby regulating the response to nutrient starvation or stress in yeast  is involved in both deoxynivalenol (DON) production and growth, and Fgrim15 (Fg01312) plays important roles in DON production and conidiation (W. cocos SCH9 (Wolco1| 71589) demonstrated higher mRNA levels in the sclerotial than the mycelial stage. In contrast, mRNA levels for W. cocos RIM15 (Wolco1| 24527) decreased at the sclerotial stage (Supplementary Table S1).in yeast . In F. gidiation . In the S. cerevisiae, YPK1/YPK2 are involved in cell-wall integrity  results in a reduced growth rate and increased sensitivity to osmotic and oxidative stress  mutants decreased by more than 90% and mutant strain formed compact colonies (Table 2). In W. cocos, there are two PKs; one of them (Wolco1| 70675) is highly similar to the yeast CBK1 , indicating their potential roles in growth and cell-wall integrity (Table 2).Several AGC Group PKs, including YPK1/YPK2, PKC1 and SCH9, are phosphorylated and activated by PHK1/PHK2 . In S. cntegrity , and YPKntegrity . In F. ge stress . PKC1  kinases . Wolco1| 160811 encodes sucrose non-fermenting (SNF1) kinase, and its homolog in S. sclerotiorum and S. cerevisiae are involved in carbon catabolite repression  is important for DNA damage repair but not necessary for pathogenesis deletion . MAPK cascades and MAPK signaling pathways are known to be involved in many major cell processes in fungi (W. cocos. Wolco1| 95503 orthologous to the HOG1-style MAPK in S. sclerotiorum (SS1G_07590) and S. cerevisiae (NP_013214.1) with high similarity. In B. cinerea, the closely related phytopathogen of S. sclerotiorum, the HOG1 ortholog is involved in osmotic stress, oxidative stress, conidia formation and sclerotial development  and S. cerevisiae SLT2-style MAPK (NP_011895.1). Deletion of SMK3 in S. sclerotiorum inhibits the production of sclerotia  that regulate cell division in eukaryotes . Wolco1|nfection . W. cocooformans . In yeasoformans . In Aspeogenesis .Wolfiporia cocos has three orthologs of YAK1 . The YAK1 members control cell proliferation, differentiation and homeostasis . SKY1 is a serine-arginine PK that is involved in metabolic signaling, cell-cycle regulation and chromatin reorganization . Wolco1| 166770, Wolco1| 43954 and Wolco1| 150169 are the orthologs of yeast STE20, STE11, and STE7, respectively. In the MAPK cascade, MAPKKs (STE7) phosphorylate MAPKs, MAPKKKs (STE11) phosphorylate MAPKKs (STE7) and STE20 is the upstream kinase . Wolco1| 22440 is orthologous to S. cerevisiae HRR25. HRR25 is a multifunctional kinase that is involved in autophagy and endocytosis . Wolco1| 159823 and Wolco1| 134885 are orthologous to S. cerevisiae SAT4, which is involved in regulating permeases activity and salt tolerance , HOG1 (Wolco1| 95503), FUS3 (Wolco1| 107181), SLT2 (Wolco1| 92114), STE11 (Wolco1| 43954) and STE7 (Wolco1| 150169) were present in higher amounts during the sclerotial stage; CLA4 (Wolco1| 121529), STE20 (Wolco1| 166770), and MKK2 (Wolco1| 75024) mRNAs were present in lower amounts . These results are consistent with the de novo transcriptome sequencing data (Supplementary Table S1), indicating that W. cocos PKs regulate sclerotial formation in both positive and negative ways.Nine W. cocos sclerotia, it is important to find ways to improve the sclerotial yield and their content of pharmacologically active components. We hypothesize that better knowledge of the kinome of the fungus can point to ways of achieving this goal.Since shortages in pine wood resources currently limit the commercial production of S. sclerotiorum, sclerotial formation is dependent on mycelial differentiation and the response to stress, nutrient and environmental changes via PK signaling pathways . Besides SMK1, Wolco1| 92114, which is orthologous to SLT2-style MAPKs, is also upregulated in the sclerotial stage , indicating that MAPKs also potentially positively regulate sclerotial formation in W. cocos. In B. cinerea, deletion of the bmp3 gene encoding a homolog of the yeast SLT2-style MAPKs results in the loss of sclerotial formation , suggesting that MAPKs play significant roles in the sclerotial formation of multiple fungi. In addition, sclerotial development of S. sclerotiorum is also associated with the cAMP-PKA signaling pathway in a complicated way , indicating that Wolco1| 107737, but not Wolco1| 115209, potentially plays the major role in sclerotial development in W. cocos.In pathways . Silenciormation . In W. cormation . Additioormation , and theated way . W. cocohenotype . In the W. cocos-pine wood interactions remain largely unknown. This paucity of knowledge is also the case particularly for sclerotogenesis, which is regulated via PK signaling pathways. In addition, since W. cocos is only able to form sclerotia after colonization of pine wood, we hypothesize that some components from pine woods may induce mycelial differentiation and sclerotial development via PK signaling pathways. Since multiple PK genes whose homologs regulate sclerotial formation in other fungi were upregulated in mature sclerotia of W. cocos, it is likely that sclerotogenesis and sclerotial development occur in response to stress and/or nutrient and environmental change via PK signaling pathways.Although sclerotial development has recently been studied , the mecGanoderma lucidum, another traditional Chinese medicinal mushroom, over-expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene by using Agrobacterium tumefaciens-mediated transformation method led to a two fold increase in ganoderic acid content, increased accumulation of intermediates and the up-regulation of downstream genes (W. cocos has been developed (W. cocos) biosynthesis are known , indicating that these PKs potentially positively regulate sclerotial formation in W. cocos. Therefore, we have selected these six PKs as the target genes for eventual over-expression in W. cocos by genetic engineering to improve the sclerotial yield.Because PK pathways integrate multiple external and internal signals to co-regulate the key processes of the fungal life cycle, such as growth, infection, nutrient or stress responses, metabolism and sclerotial development , over-exam genes , indicateveloped , and there known . Based oW. cocos PK genes in growth, sclerotial developmental, morphological changes and environmental stress responses. Characterization of these PK genes will help illuminate the underlying mechanisms of sclerotogenesis and improve the sclerotial yield.In summary, we have identified and discussed the potential roles of W. cocos sclerotial development and other important physiological processes. And it also provides a valuable data for illuminating the mechanisms of W. cocos sclerotial development and promoting commercial cultivation of W. cocos sclerotia.This study firstly contributes to an understanding of putative functions of PKs in Conceived and designed the experiments: WZ and WW. Performed the experiments: WW, SS, and WZ. Analyzed the transcription data: WW, WZ, and YX. Contributed reagents/materials/analysis tools: WW, SS, WZ, YX, and FP. Wrote the paper: WZ and WW. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.