PLoS Biology, volume 2, issue 6:In Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA ClonesTadashi Imanishi, Takeshi Itoh, Yutaka Suzuki, Claire O'Donovan, Satoshi Fukuchi, et al.DOI: 10.1371/journal.pbio.00201621. The abbreviation FLJ was expanded incorrectly (as full-length long Japan) in the abbreviations list and in the first paragraph of the Results/Discussion section. FLJ stands for full-length cDNA Japan.2. In Table 1, the number of cDNAs and the number of library origins for two of the cDNA sources were incorrect. The correct numbers are as follows:


The coordination around mercury is completed by two bromido ligands resulting in a distorted tetrahedral arrangement.In the title polymeric complex, [HgBr DOI: 10.1107/S1600536808030055/si2110Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Each NiII atom is chelated by two oxalate ligands and one 2,2′-bipyridine, forming a slightly distorted octahedral geometry. Oxlate acts as a bridge to link neighbouring pairs of NiII cations, forming a one-dimensional wave-like chain. The crystal showed partial inversion twinning.The title compound, [Ni(C DOI: 10.1107/S1600536808028389/cf2213Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Intermolecular N—H⋯N hydrogen bonds link neighboring molecules into extended chains parallel to [100].In the title complex, [Ni(C DOI: 10.1107/S1600536808028171/bv2105Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The four metal ions in the cluster are held together by four bridging hydroxide groups. Each NiII atom adopts a distorted octahedral geometry. The title complex, [Ni DOI: 10.1107/S1600536810003697/rz2413Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The NiII atoms are linked to each other, forming an infinite chain parallel to (In the title complex, [Ni(C DOI: 10.1107/S1600536808030377/dn2372Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The Cd atoms are connected by two dicyanamide ligands, resulting in a neutral chain propagating parallel to [010].In the title compound, [Cd(C DOI: 10.1107/S1600536809046364/pv2226Isup2.hklStructure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


At the present state of knowledge, the emergence of the Gravettian in eastern Italy is contemporaneous with several sites in Central Europe and the chronological dates support the hypothesis that the Swabian Gravettian probably dispersed from eastern Austria.In the northern Adriatic regions, which include the Venetian region and the Dalmatian coast, late Neanderthal settlements are recorded in few sites and even more ephemeral are remains of the Mid-Upper Palaeolithic occupations. A contribution to reconstruct the human presence during this time range has been produced from a recently investigated cave, Rio Secco, located in the northern Adriatic region at the foot of the Carnic Pre-Alps. Chronometric data make Rio Secco a key site in the context of recording occupation by late Neanderthals and regarding the diffusion of the Mid-Upper Palaeolithic culture in a particular district at the border of the alpine region. As for the Gravettian, its diffusion in Italy is a subject of on-going research and the aim of this paper is to provide new information on the timing of this process in Italy. In the southern end of the Peninsula the first occupation dates to around 28,000  Numerous sites throughout the Italian Peninsula and the western Balkans document key events between the late Middle Palaeolithic and the Mid-Upper Palaeolithic. Focusing on the northern Adriatic Sea rim which includes the Venetian region and the Dalmatian coast, the millennia preceding the demise of Neanderthals are recorded in very few sites which displayed data of variable relevance Even scarcer in this area is the archaeological evidence of the Mid-Upper Palaeolithic, a period better known along the Tyrrhenian Sea and the southern Adriatic coasts, where evidence of intense Gravettian occupation can be found One of the most debated issue is whether the Gravettian developed from a local Aurignacian 14C BP in Paglicci Cave in the southern end of the Peninsula 14C BP , at 580 m asl on the Pradis Plateau in the eastern part of the Carnic Pre-Alps. The Pradis Plateau comprise an area of 6 sq km, enclosed on three sides by mountains peaking from 1,148 m to 1,369 m and to the south by the foothills, facing the present-day Friulian Plain . Rio SecThe presence of Palaeolithic settlements at Rio Secco Cave was detected in 2002 after a test-pit The cave is filled with an ensemble of sedimentary bodies of differing volume, shape, composition and origin, grouped into four macro-stratigraphic units and separated by erosional and sedimentary discontinuities Macro-unit BR1 includes layer 4 and an anthropic horizon containing Gravettian flint artifacts, layer 6. The most relevant features are angular to subangular stones, with fragments of karst limestone pavement that originated from the collapse of the vault. Layer 6, with organic matter and micro-charcoal has been exposed at the entrance of the cave shelter, approximately 20 cm below the top of BR2: it is thin, planar, discontinuous, and contains rare bones and lithics .Macro-unit BR2 is a massive open-work stone-supported breccia made of angular boulders and randomly deposited stones. It lies in the external zone but ends 1 m behind the drip line in the SE zone of the cavity, where it seals the layer 5 top. Large patches have been reworked by marmots, as demonstrated by bones, an articulated skeleton found within the tunnels, several burrows and dens.The sedimentary body below BR2 is composed of stones and loamy fine fraction and is labeled BIO1 due to the intense bioturbation caused by the activity of marmots, responsible for mixing, displacing portions of anthropic sediment, and scattering Mousterian flint implements, bones and charcoals. At the top of this macro-unit, one finds layer 5 top, a brown level of variable thickness with archaeological content. Due to its variable thickness, layer 5 top has been locally divided in two arbitrary cuts, I and II. Below, the loamy, dark yellowish-brown layer 7 has been found only in some squares under the cave vault and not in the external zone, where it is cut by the burrows. The upper boundary with layer 5 top is marked by an increasing frequency of bones and lithics, some of which also bear signatures of accidental heating. Sandwiched between the two anthropic horizons layers 7 and 8, layer 5 is made of stones and loamy fine fraction with dispersed bones and lithic implements frequently affected from post-depositional alteration. Layer 8 continues in the inner cavity and is best described as 10 cm thick, stony, with dark brown loamy fine fraction, frequent tiny charcoals, small and burnt bones. Layer 8 lies over layer 9, possibly a fifth macro-unit, made of stones and yellowish brown sandy-loam, with no charcoal or other finds.The archaeological contents of BR1 and BIO1 include numerous lithic artifacts ascribed to the Middle Palaeolithic  and Upper Palaeolithic (layer 6 and correlated arbitrary cuts 4c and 4d) and a few bone retouchers Evidence for the use of fire has been found in layers 8 and 7 by tiny dispersed charcoals, burnt bones and heat-affected flints. In layer 6 two hearths have been brought to light, even if partially affected from post-depositional disturbances, labeled as US6_SI and US6_SII. The former is an agglomeration of charcoals mostly disaggregated around a large piece of charred wood . This heEvery stratigraphic unit contained animal bone remains. The colonization of the cave fill by marmots is clearly documented by diagnostic signatures observed in BR1 and BR2, such as dens, chambers and articulated skeletons. There are fewer faunal remains in the Gravettian layers in comparison with the Mousterian.Capra ibex and Rupicapra rupicapra) and remains of Bos/Bison (Bison priscus/Bos primigenius). Traces of human modification on the bones include cut-marks on shafts of caprids, partly combusted, and on a marmot clavicle. One partially burned epiphysis of the scapula of Castor fiber has been found associated to the hearth US6_SI.The archaeozoological analysis, still in progress, reveals among the ungulates the presence of caprids  predominate over the ungulates, which rather than caprids (chamois and ibex) or bovids, consist more of cervids such as red deer, roe deer, elk and wild boar . Bones are mostly fragmented, due to post-depositional processes as well as human and carnivore activity. Human interest in ungulates is evidenced by cut marks on red deer. Also the remains of Ursus spelaeus and Ursus sp. from layers 7 and 5 top show traces of butchering, skinning and deliberate fracturing of long bones This faunal association with cervids and, in particular, deer, elk, roe deer and wild boar is indicative of forest vegetation and marsh environment somewhere in the Pradis Plateau. The presence of bovids and caprids suggest the existence of patchy woodland compatible with the mountain context. Cave bears were well adapted to this kind of environment, and used the cavities for hibernation, as suggested from the faunal assemblage recovered during the last field-campaigns.-89, GRS13SP57-138, GRS13SP57-153, GRS13SP57-125, GRS13SP57-37, GRS13SP57-11, GRS13SP57-18, GRS13SP57-46, GRS13SP57-2, GRS13SP57-4.All necessary permits were obtained from the Archaeological Superintendence of the Friuli-Venezia Giulia for the described study, which complied with all relevant regulations. The identification numbers of the specimens analyzed are: GRS13SP57Repository information: the specimen is temporary housed at the University of Ferrara, in the Section of Prehistory and Anthropology, Corso Ercole I d’Este Ferrara, Italy, with the permission of the Archaeological Superintendence of the Friuli-Venezia Giulia.We selected 10 well preserved thick cortical bone fragments with and without cut marks from each layer. Four bones from layer 7 (three of them with cut marks), four bones from layer 5 (three with cut marks) and two charcoal samples from the hearth SI of layer 6.2 effervescence was observed, usually for about 4 hours. 0.1 M NaOH was added for 30 minutes to remove humics. The NaOH step was followed by a final 0.5 M HCl step for 15 minutes. The resulting solid was gelatinized following Longin (1971) at pH 3 in a heater block at 75°C for 20 h. The gelatin was then filtered in an Eeze-Filter™ (Elkay Laboratory Products (UK) Ltd.) to remove small (<80 µm) particles. The gelatin was then ultrafiltered with Sartorius “Vivaspin 15” 30 KDa ultrafilters Bone collagen was extracted at the Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany, using the ultrafiltration method described in Talamo and Richards The collagen extract was weighed into pre-cleaned tin capsules for quality control of the material. Stable isotopic analysis was evaluated using a ThermiFinnigan Flash EA coupled to a Delta V isotope ratio mass spectrometer.The two charcoal samples were sent directly to the Klaus-Tschira-AMS facility of the Curt-Engelhorn Centre in Mannheim, Germany, where they were pretreated with the ABA method At Rio Secco Cave the C:N ratio of all the samples are 3.2 which is fully within the acceptable range (between 2.9 and 3.6), and all of them displayed a high collagen yield, mostly ranging between 2.4 to 8.2%, substantially higher than 1% of weight for the standard limit Once these criteria were evaluated, between 3 and 5 mg of the collagen samples were sent to the Mannheim AMS laboratory (Lab code: MAMS), where they were graphitized and dated 14C, equivalent to ca. >48,000 14C years BP) estimated from pretreated 14C free bone samples, kindly provided by the ORAU and pretreated in the same way as the archaeological samples.The radiocarbon results are listed in 14C years BP. The four dates of layer 5 range from 45,740 to 43,210 14C years BP. The uppermost layer (layer 6), which was identified as a Gravettian layer, ranges from 29,390 to 28,995 14C years BP. There is no discrepancy between the results obtained on bones with cut marks and without cut marks.The uncalibrated radiocarbon dates of late Mousterian (layer 7) range from >49,000 to 44,560 14C BP but the charcoal result, pretreated with ABOX-SC, displayed an age of 42,000±900 14C BP. The main argument for this difference has to be found, as described above, in the stratigraphic entities of the layer, in fact it contains frequent tiny charcoals of undetermined conifer, small bones and burnt bones. Moreover deformations, removal and various crossings by marmots and other minor bioturbations affect this layer. In addition, a test-pit opened during the last field campaign (summer 2013) had detected no archaeological traces at 1,5 meters underneath this layer, thus excluding possible pollution from older deposits. For this reason we considered the youngest date  as an outlier.A series of radiocarbon dates were previously obtained from layers 8, 5 and 6 14C Age 37,790±360) Layer 5 has produced an age that is too young compared with our new results  in the layer below layer 7 (layer 8) 14C Age 42,000±900) in layer 8 is confirmed to be an outlier. OxCal finds a good agreement index , between the full set of finite radiocarbon dates and the stratigraphic information; the results of the outlier detection method confirm ideal posterior probability for all the samples.A start calibrated boundary for the lower part of the sequence (Layer 7) at Rio Secco Cave cannot be defined. What we can determine is that the lower level of Layer 7 is older than 49,000 The upper boundary of layer 7, calculated by OxCal, ranges from 49,120 to 47,940 cal BP (68.2%); the layer 5 top ranges from 47,940 to 45,840 cal BP (68.2%) .uAround the Alpine regions Neanderthal sites with comparable age ranges are rare The charcoal samples, from the archaeological horizon US 6_SI located between layers BR2 and BR1 range from 33,480 to 30,020 cal BP (68.2%) . These rIt should be noted that the charcoal samples dated at Mannheim yielded consistent age with the previous radiometric dates obtained at Poznan for the same horizon.Here it is useful to remember that strong progress has been achieved in the last decade on the radiocarbon method. Calibration is now possible back to 50,000 cal BP An accurate sample selection, more specialized pretreatment protocols, the control of isotopic values of bone collagen, in case the samples pretreated were bones and the requirement of several dated samples per layer are fundamental criteria that should be considered in order to establish the radiocarbon chronology of the archaeological sites.14C years BP.Normally the risk of underestimating the true age of the samples is higher when the samples are at the limit of the radiocarbon method. However the chronological reassessment of Geiβenklösterle, Abri Pataud, Fumane Cave and Mochi rockshelter sites Bearing in mind this fundamental issue, Rio Secco Cave layer 6 shows the newest radiometric assessment of the Italian late Mid-Upper Palaeolithic. Moreover the comparison with the single dates of layer 23 in Paglicci Cave, permits to ascribe Rio Secco as the oldest Early Gravettian site in Italy.At this stage of our investigation, the backed pieces and the burins introduced and reduced on site are an expression of short term occupations by hunter gatherers equipped with previously retouched tools made of high quality flints collected outside the Carnic Pre-Alps The appearance of the early Gravettian in Europe predates the last phases of the Aurignacian In central Europe between northern Austria, Moravia and southern Poland one finds a second early Gravettian techno-complex, named the Pavlovian, Furthermore, in the Italian Peninsula local developments of the Gravettian have not been recorded so far Current evidences make us inclined on the cultural diffusion hypothesis, and the Rio Secco site provides new insight on the two natural corridors used to reach the Italian Peninsula, the Adriatic southern coast from Croatia At the junction between the North Adriatic Plain and the eastern Alps, the chronometric refinement of a new site, Rio Secco Cave, contributes to enhance the investigation of the prehistoric human occupation during the mid-Late Pleistocene. Although not completely explored, Rio Secco Cave fills an important chronological gap and preserves an archive of potential interest for understanding the study of the late Neanderthals, the dispersal of Mid-Upper Palaeolithic populations and the diffusion of the Gravettian culture. Nevertheless, the new set of dates does not cover the millennia of the Middle-Upper Palaeolithic transition in the second half of MIS3, a period chronometrically secured from key-sequences in neighboring regions 14C results show that the excavated archaeological horizon Layer 6 belongs to the early Gravettian time period. At the present state of knowledge, with our new 14C dates, the emergence of the early Gravettian in eastern Italy is contemporaneous with the Swabian Gravettian and the Pavlovian.The continued implementation of the project with fieldwork and laboratory studies will provide new elements necessary to better understand the settlements in this area, previously considered so marginal in comparison with the North Adriatic Plain, extending towards the south. At the present stage of research, the Gravettian archaeological record at Rio Secco Cave is scarce compared with the Mousterian one, due to the thinning of layer 6 and its partial reworking produced by illegal excavations in the inner cavity. We cannot exclude that the rockfall that occurred after the late Middle Palaeolithic induced the Gravettian foragers to place their settlement under the present-day rockshelter just in front of the cave entrance. Nevertheless, the few flint artifacts give economic hints of potential interest. The The broad expansion of Swabian Gravettian and Pavlovian techno-complexes is explained by high mobility patterns of the hunter-gatherers with transport of exogenous raw materials up to 200 km Although the absence of diagnostic lithic tools at Rio Secco Cave layer 6 doesn’t allow a correlation of the lithic assemblages with the central European techno-complexes, the radiometric dates support the hypothesis of dispersal of the Swabian Gravettian probably from the eastern Austria . In the Table S1Radiocarbon dates on key Gravettian sites (DOCX)Click here for additional data file.


Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production. Influenza A virus contains eight single-stranded RNA segments. The negative-sense viral RNA (vRNA) segments act as templates for messenger RNA (mRNA) synthesis in transcription, and for complementary RNA (cRNA) synthesis which is used for replication of vRNA. Both transcription and replication are performed by viral RNA-dependent RNA polymerase (RdRp) inside the nucleus of infected cells Since 1997, sporadic human infections with highly pathogenic H5N1 viruses have been reported. Although an efficient and sustained transmission of highly pathogenic H5N1 viruses in humans has yet occurred It is clear that preferential binding of haemagglutinin (HA) to terminal α-2,3 and α-2,6-linked sialic acid receptors on host cell surface is not the sole barrier of cross-species infection in-vitro model to examine the polymerase activity of viral RNP complexes. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS)  at 33°C or 37°C in a 5% CO2 incubator.Human embryonic kidney 293T cells  were used as an cDNA clones originated from four influenza virus strains were used to generate different RNP complexes. These viruses include: A/Thailand/1(KAN-1)/2004 (H5N1), representing highly pathogenic influenza A H5N1 viruses; A/HongKong/CUHK-72079/2009 (H3N2), representing seasonal H3N2 viruses; and A/Auckland/1/2009, representing the 2009 pandemic virus (H1N1pdm09). The A/WSN/33 (WSN) H1N1 virus was used as a reference strain in this study.HindIII and NotI restriction sites, whereas the BamHI and NotI restriction sites and the KpnI and NotI restriction sites were used for PA and NP, respectively. Point mutations were introduced into the PB2 plasmid of H5N1 to generate three different mutants with the following nucleotide substitutions: (i) 473, A→G causing E158G, (ii) 811, A→G causing T271A, (iii) 1879, G→A causing E627K. The identities of these clones were confirmed by sequencing.The protein expression plasmids for three polymerase subunits  and nucleoprotein (NP) of H1N1pdm09 and H3N2 had been described previously Various combinations of PA, PB1, PB2 and NP-expression plasmids derived from different subtypes of influenza virus were used to generate hybrid viral RNP complexes. 293T cells in 48-well plates were co-transfected with 0.4 µg of each plasmid and pPolI-NA by using Lipofectamine 2000  for 48 hrs at 33°C or 37°C.Renilla luciferase gene.Luciferase reporter assay was performed as described before Total RNA was extracted from transfected cells using TRIzol plus RNA purification kit (Invitrogen) and followed by DNaseI treatment . A previously described approach was used to achieve specific quantification for each RNA species Applied Biosystems, Foster City, CA). Five microlitres of the corresponding cDNA sample were added to the 25-µL reaction. Primers were 5'-CCG GCA AAG TGA TGT GTG TGT G-3' (corresponding to nucleotide 806 to 827 of NA cRNA) and 5'-CCG AAA ACC CCA CTG CAG ATG-3' (complementary to nucleotide 900 to 920 of NA cRNA). Reactions were first incubated at 50°C for 2 min, followed by 95°C for 10 min, and subjected to 40 cycles of 95°C for 15 s and 60°C for 1 min. The concentration of viral RNA were normalized with the corresponding input 5 S rRNA by primer 5′- TAC GGC CA TAC CAC CCT GAA C -3′ and primer 5′- CGG TAT TCC CAG GCG GTC T -3′.Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify mRNA, vRNA and cRNA levels using Power SYBR® Green PCR Master Mix ; H3N2 showed no significant difference between 33°C and 37°C ; whereas H5N1 showed a significantly higher activity at 37°C .To characterize the effect of temperature on avian and human influenza viruses, we compared the polymerase activities of parental H5N1, H1N1pdm09 and H3N2 RNP complexes in human cells under incubation at 33°C and 37°C, mimicking physiological temperatures of human upper and lower respiratory tracts, respectively. H1N1pdm09 showed a significantly higher activity at 33°C than 37°C  (The polymerase activities of hybrid RNP complexes are shown in P<0.001) . In lineP<0.001) .P = 0.002) compared to 37°C  (P<0.001) than at 33°C   . In contP<0.001) .P<0.001; H1N1pdm09∶0.3-fold at 33°C, P<0.001; 0.1-fold at 37°C, P<0.001)  .P = 0.002), but not at 37°C , but a lower activity was observed at 33°C   .On the other hand, when the H3N2 NP or H1N1pdm09 NP was replaced by H5N1 NP, a slight decrease in activity was observed at 37°C .Taken together, substitution of the H5N1 RNP complex with PB2 derived from either H3N2 or H1N1pdm09 resulted in a substantial increase in polymerase activity, and the effect was more pronounced at 33°C. Substitution of the H5N1 RNP complex with PA derived from H1N1pdm09 also achieved a pronounced increase in activity at 33°C. In contrast, substitution of the H5N1 RNP complex with PB1 derived either from H3N2 or H1N1pdm09 decreased the polymerase activity of H5N1.P<0.001), respectively (The effects of H5N1 PB2 mutations on polymerase activity are shown in ectively . These mectively . Among tP = 0.01) and 3.4-fold (P = 0.03), respectively (P<0.001), 2.8-fold (P = 0.04) and 3.2-fold (P = 0.03), respectively. Among all the substitutions, E158G showed a stronger transcription activity compared to others at 33°C, whereas no differences among the three mutations were observed at 37°C.We further investigated whether the higher activity associated with those mutations was linked to an increase in transcription (mRNA) and/or replication (cRNA and vRNA synthesis) by using a real-time PCR-based quantitative assay ectively . On the The cross-species infection of influenza virus, probably of avian origin, has caused a serious pandemic in 1918 We found that the polymerase activity of H5N1 could be increased to a very large extent by substituting the RNP complex with a PB2 derived from either H3N2 or H1N1pdm09, and the effect was more pronounced at 33°C. This is an important concern as such reassortants may be able to adapt to human upper airway with increased human to human transmission efficiency. By contrast, mammalian PB1 abolished polymerase activity of H5N1-originated polymerase in human cells. These results clearly demonstrated that optimal polymerase activity, due to a better compatibility of avian PB1 and mammalian PB2, is probably required for efficient viral replication in human cells In addition to PB2, mutations in PA subunit have been reported to affect viral RNA replication The function of avian polymerase in human cells may be improved by substitution of H5N1 PB2 subunit with “human” residues. Previous studies have shown that 627K in PB2 enhanced polymerase activity The differential quantitative analysis of different viral RNA species revealed that mutations E158G, T271A and E627K on PB2 enhanced both the transcription and replication activity of viral polymerase. However, notable differences in the profile of transcriptive (mRNA) and replicative (vRNA and cRNA) intermediates were observed between 33°C and 37°C. Similar to earlier study, the K627E mutation significantly reduced vRNA and cRNA promoter binding activities of PB2 in avian H5N1 virus The mechanism behind how mutations and subunits of different species origin influence the transcriptional and replication activity of RNP complex remains elusive. One possibility is differential thermal stability of RNP complex with positive and negative strand template RNA at restrictive and permissive temperatures, as suggested in seasonal H1N1 virus In conclusion, substitution of H5N1 RNP complexes with subunits, especially PB2, derived from H3N2 or H1N1pdm09 viruses could remarkably increase its replication and transcription activity in human cells. This indicates that some residues in human PB2 subunit might be involved in human adaptation. By using a highly sensitive quantitative RT-PCR, consistent with the result of polymerase activity, an obvious enhancement in replication and transcription activity of RNPs was observed by introduction of lysine at residue 627 in PB2 subunit. Although less strongly in polymerase activity, the temperature dependency of E158G mutation appeared to alter the accumulation of viral RNA levels, suggesting a temperature-dependent mechanism in regulating transcription and replication exists.


Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood.Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated.Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes. Orthomyxoviridae, and classified into subtypes by antigenic differences of the two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA) Influenza A virus is a member of the family The segmented genome structure of influenza virus facilitates genetic reassortment with other influenza strains which is co-infected in the same cells and has played a pivotal role in the emergence of pandemics. The pandemic strains of the past century have incorporated genes expressing the superficial HA and NA glycoproteins giving rise to new subtypes with novel surface antigens. Mutations of avian HA for acquiring human receptor α2, 6 sialic acid binding specificity is a prerequisite for human adaptation. In addition to acquiring novel genes for superficial glycoproteins, at least one internal gene of the RNA polymerase from avian strains has been concurrently incorporated into human strains. The1957 and 1968 influenza pandemics coincided with the introduction of an avian PB1 gene The influenza virus RNA polymerase is a heterotrimeric complex composed of three subunits, PB1, PB2 and PA, which assembles with nucleoprotein (NP) and viral RNA, forming ribonucleoprotein complex (RNP) In this study, we attempted to characterize and dissect the role of the polymerase genes in giving rise to a functional genetic reassortant. We generated RNP containing hybrid polymerase between human-isolated avian H5N1 and human H1N1 or H3N2. Our results revealed that the PB2 subunit of H5N1 has a strong inhibitory effect on the RNP activity when introduced into the polymerase of other influenza strains. Importantly, H5N1 PB2 could form functional 3P complex properly, but significantly reduced the accumulation of RNP, specifically through the properties of four amino acids in PB2 at position 108, 508, 524 and 627.cDNA clones isolated from the following influenza strains were used: A/HongKong/156/1997 (H5N1) (abbreviated as HK or H), A/Vietnam/1194/2004 (H5N1) (abbreviated as VN or V), A/WSN/1933 (H1N1) (abbreviated as WSN or W), a newly pandemic A/Kurume/K0910/2009 (H1N1) (abbreviated as SW or S), A/NT/60/1968 (H3N2) (abbreviated as NT or N) PB1, PB2, PA and NP-expressing plasmids of influenza viruses HK, VN, WSN, SW and NT have previously been described 293T human embryonic kidney cells For a preparation of the polymerase, 293T cells were transfected with expression vectors containing PB1, PB2 and TAP-tagged PA subunit of each strain. For a preparation of the RNP, NP and vNA expression vectors were also transfected simultaneously. Cells were harvested 2 days posttransfection and the polymerase or RNP was purified by the tandem affinity purification (TAP) method described previously In order to investigate the role of the RNA polymerase on the restriction of genetic reassortment, we reconstituted reassortant RNP by introducing the polymerase subunit of H5N1 into the polymerase of H1N1 or H3N2. We used two human-isolated H5N1 strains of A/HongKong/156/1997 [HK] and a closely related A/Vietnam/1194/04 [VN], two human H1N1 strains of A/WSN/33 [WSN] and the new pandemic influenza A/Kurume/K0910/09 [SW], and H3N2 strain of A/NT/60/68 [NT], because these strains were extensively analyzed previously In the case of the HK strain of H5N1 , lane 5,Despite originating from the same H5N1 subtype, the amino acid sequence of PB2 between HK and VN differs at 26 positions out of 759 amino acids . Since HSince four amino acids in HK PB2 were found to be critical for rescuing activity of the hybrid RNP, it is conceivable that substitution of amino acids at the same position in VN PB2 by the HK PB2 sequence might decrease the RNP activity. Single mutant R508Q or double mutant R508Q/T524M showed a slight decrease in activity compared with VN PB2 , lane 5.To address the question why HK PB2 severely inhibited the activity of RNP reconstituted from hybrid polymerase, we initially examined whether HK PB2 affects the correct assembly of the trimeric complex of PB1, PB2, and PA. To allow the purification of trimeric complex, a C-terminally TAP-tagged PA was co-expressed with PB1 and PB2 in 293T cells, and the trimeric complex was affinity purified by a TAP method and quantitatively adjusted see . In the An alternative explanation for the defect of RNP activity is a loss of the polymerase activity. The binding of RNA polymerase to the promoter is an essential step to initiate RNA synthesis. Therefore, the promoter binding activity of the polymerase in vitro was assayed by UV cross-linking, then subsequent initiation of RNA synthesis was analyzed by dinucleotide replication assay see . SurprisAnother possible explanation for the loss of RNP activity is a defect in the assembly of RNP. To test this possibility, we purified and evaluated the amount of RNP accumulated in vivo. To allow RNP purification, a C-terminally TAP-tagged PA was coexpressed and the reconstituted RNP was affinity purified by a TAP method see . The amo8) genotypes theoretically. However, systematic studies by using reverse genetics have shown that the number of replicative reassortant viruses is apparently limited The genetic reassortment between two different influenza A viruses can generate 256  significantly decreased the accumulation of RNP reconstituted in human 293T cells and resulted in the concomitant decrease in the RNP activity The E→K mutation at HK PB2 627 showed a significant reduction in vRNA promoter and cRNA promoter bindings in cross-linking experiments . PB2 hasA purified H5N1polymerase shows significantly higher polymerase activity in vitro when compared to human strain A/WSN/33 (H1N1) Introduction of HK PA into SW and NT polymerases increased the synthesis of mRNA, cRNA and vRNA  has a strong inhibitory effect on the RNP activity when introduced into the polymerase of other influenza strains. In addition, four residues at positions 108, 508, 524 and 627 of the PB2 subunit appear to be important determinants that are involved in the accumulation of functional RNP and in modulating the polymerase activity. These results may suggest a possible mechanism by which the generation of replicative reassortant virus of influenza is highly restricted.


The soluble CD14 subtype, Presepsin, appears to be an accurate sepsis diagnostic marker, but data from intensive care units (ICUs) are scarce. This study was conducted to evaluate the diagnostic and prognostic value of Presepsin in ICU patients with severe sepsis (SS), septic shock (SSh) and severe community-acquired pneumonia (sCAP).Presepsin and procalcitonin (PCT) levels were determined for patients at admission to ICU. Four groups have been differentiated: (1) absence or (2) presence of systemic inflammatory response syndrome, (3) SS or (4) SSh; and 2 groups, among the patients admitted for acute respiratory failure: absence or presence of sCAP. Biomarkers were tested for diagnosis of SS, SSh and sCAP and for prediction of ICU mortality.One hundred and forty-four patients were included: 44 SS and 56 SSh. Plasma levels of Presepsin and PCT were significantly higher in septic than in non-septic patients and in SSh as compared to others. The sepsis diagnostic accuracy of Presepsin was not superior to that of PCT (AUC: 0.75 vs 0.80). In the 72/144 patients admitted for acute respiratory failure, the capability of Presepsin to diagnose sCAP was significantly better than PCT. Presepsin levels were also predictive of ICU mortality in sepsis and in sCAP patients.Plasma levels of Presepsin were useful for the diagnosis of SS, SSh and sCAP and may predict ICU mortality in these patients. Despite advances in therapy, sepsis is the leading cause of death in critical care settings . To imprMore recently, the soluble CD14 subtype, Presepsin, appears to be an accurate sepsis diagnostic marker and rises up a great clinical interest. Levels of Presepsin were found significantly higher in septic than in non-septic patients or those with SIRS . MoreoveTherefore, this study aimed to evaluate the diagnostic and prognostic utility of Presepsin measurements using the new fast method in severe sepsis and septic shock intensive care unit (ICU) patients. We also aimed to evaluate the diagnostic and prognostic utility of Presepsin measurements for severe community-acquired pneumonia (sCAP) in the subgroup of patients admitted to the ICU with acute respiratory failure.This observational prospective study was performed at 2 ICUs of Lapeyronie and Gui de Chauliac University hospitals of Montpellier, France. These two ICUs admit preferentially patients with suspected infectious diseases. It was carried out according to the principles of the Declaration of Helsinki and was approved by the Ethic Committee of Montpellier (Comité de protection des Personnes: CPP du CHU de Montpellier). Written informed consent was obtained from all participating patients or their closest relatives or legal representatives.All consecutive patients admitted to ICUs from January to May 2014 were included. Exclusion criteria were pregnancy, age < 18 years, previous congestive heart failure (class NYHA ≥ III), right ventricular failure, chronic renal failure stage III KDOQI or more, hepatic failure and acute pulmonary embolism.Baseline clinical variables including age, gender, cause of sepsis, and comorbidities were collected. The severity of disease was assessed by SAPS II  and SOFADiagnosis of systemic inflammatory response syndrome (SIRS) and of sepsis severity was based on established criteria of the American College of Chest Physicians/Society of Critical Care Medicine . MicrobiCommunity-acquired pneumonia (CAP) was defined as the presence of a new infiltrate on a chest radiograph and at least one of the following signs: cough, sputum production, dyspnea, core body temperature > 38.0 °C, auscultatory findings of abnormal breath sounds and rales . Diagnos®  recently evaluated [® immunoanalyzer  following the manufacturers’ instructions. Determination of hsCRP was run on the Cobas8000/e502® analyzer  using immunoturbidimetric method.Venous samples were taken from all patients at admission and immediately performed for Presepsin, PCT and hsCRP measurements. Presepsin concentration was measured by a chemiluminescent enzyme immunoassay (CLEIA) on a compact automatized immunoanalyzer PATHFASTvaluated . The refvaluated . PCT wasTwo study physicians (KK and VG) independently reviewed all available clinical, biological and radiological patients’ data and classified all patients into four disease groups: absence (non-SIRS) or presence of SIRS, severe sepsis (SS) or septic shock (SSh). The two study physicians followed recommended definitions and algorithms (20). Briefly, patients with SIRS and positive cultures were considered as septic. When cultures were non-contributive, clinical and biological picture , successful treatment by antibiotics and rule out of other diagnosis were main elements of sepsis diagnosis. Among the subgroup of patients who were admitted for acute respiratory failure, they reviewed also their data and classified them into two disease groups: absence or presence of sCAP (even in the absence of identified causative agent). When the study physicians cannot statute on the presence or not of sepsis, the patient was not included in the study. The study physicians and those on charge of patients were blinded to the results of Presepsin and PCT.t test, or two-tailed Mann–Whitney–Wilcoxon’s test when appropriate. Results were adjusted for multiple comparisons using Bonferroni’s method. Levels of significance for all tests were set at p < 0.05. Sensitivity, specificity and positive predictive value (PPV) and negative predictive value (NPV) of Presepsin and PCT for the diagnosis of sepsis and pneumonia were calculated using final diagnosis categorization based on clinical data, clinical scores and routinely used biomarkers levels. A receiver operating characteristic (ROC) analysis was performed for each of the biomarkers, and their diagnostic performance for sepsis and for other pathological condition was compared. The optimal threshold value was set for each ROC curve through the Youden Index (corresponding to the maximum of the sum “sensibility + specificity”). Mortality was displayed as Kaplan–Meier (log-rank test) plots according to the quartiles of Presepsin levels.The statistical analyses were performed using the STAT-VIEW II . We first performed a descriptive analysis by computing the frequencies and the percents for categorical data, means, standard deviations, quartiles and extreme values for continuous data. We also checked for the normality of the continuous data distribution using the Shapiro–Wilks tests. We compared septic to non-septic patients and patients with and without sCAP for Presepsin, CRP and PCT measurements. The univariate analysis was performed using two-tailed Student’s During the study period, a total of 222 critically ill patients were admitted in ICUs. After the exclusion of 78 patients, 144 were included: 88 males and 56 females. One hundred patients conformed to the criteria of bacterial sepsis: 44 with SS and 56 with SSh. Among the 44 non-septic patients, 19 were assigned for non-SIRS and 25 for SIRS. The screening process is shown in Fig. Patient’s baseline characteristics are summarized in Table p = 0.574). In contrast, they were significantly higher in SSh versus SS and SIRS groups  and non-infectious respiratory failure (AUC = 0.85) was higher than that of PCT (0.79), SAPS II (0.72), SOFA (0.78) scores, and similar to that of the combination of Presepsin and PCT 0.84) Fig. b. Using  Fig. b. p = 0.04)  died during ICU stay. Deceased septic patients showed significantly higher Presepsin, PCT levels and severity scores at ICU admission Table a. HoweveAt ICU admission, plasma levels of Presepsin were found to be significantly higher in critically ill patients with sepsis in comparison with those without sepsis. Presepsin plasma levels of SIRS and SS patients were not significantly different, but patients with SSh had significant higher levels as compared to others. The sepsis diagnostic accuracy of Presepsin was not superior to that of PCT. With the combination of Presepsin and PCT, specificity and predictive positive value for sepsis were enhanced. We also demonstrated the usefulness of Presepsin for the diagnosis of sCAP in settings of ARF with an even better accuracy than PCT. Also, plasma Presepsin levels best predict ICU mortality in septic patients and those with sCAP at cutoff values of 1925 and 714 pg/mL, respectively.It is now well demonstrated that sepsis, especially SS and SSh, should be diagnosed early and treated within 1 h after diagnosis . ConsequMore than half (58 %) of our septic patients have a sepsis from pulmonary origin. Diagnosis and severity of CAP are difficult and largely depend on the clinician’s experience since they are based on clinical and radiological arguments –33. CircWe must acknowledge some limitations to our study. First, our study was a bi-center study, and the results may not be directly applicable to all ICUs. Second, our population included a relative limited number of patients. Third, only plasma Presepsin levels at ICU admission were determined and dynamic and follow-up changes of this biomarker were not investigated . Fourth,Our results demonstrated the usefulness of Presepsin levels in the diagnosis and prognosis of septic shock patients admitted to ICUs, but its diagnostic ability remains moderate as recently demonstrated . Its spe


The aim of this study was to evaluate the diagnostic and prognostic value of presepsin in patients with severe sepsis and septic shock during the first week of ICU treatment.In total, 116 patients with suspected severe sepsis or septic shock were included during the first 24 hours of ICU treatment. Blood samples for biomarker measurements of presepsin, procalcitonin (PCT), interleukin 6 (IL-6), C reactive protein (CRP) and white blood cells (WBC) were drawn at days 1, 3 and 8. All patients were followed up for six months. Biomarkers were tested for diagnosis of sepsis, severe sepsis, septic shock and for prognosis of 30-days and 6-months all-cause mortality at days 1, 3 and 8. Diagnostic and prognostic utilities were tested by determining diagnostic cutoff levels, goodness criteria, C-statistics and multivariable Cox regression models.P <0.03). Presepsin levels revealed valuable diagnostic capacity to diagnose severe sepsis and septic shock at days 1, 3 and 8 (range of diagnostic area under the curves (AUC) 0.72 to 0.84, P = 0.0001) compared to IL-6, PCT, CRP and WBC. Goodness criteria for diagnosis of sepsis severity were analyzed . Presepsin levels revealed significant prognostic value for 30 days and 6 months all-cause mortality . Patients with presepsin levels of the 4th quartile were 5 to 7 times more likely to die after six months than patients with lower levels. The prognostic value for all-cause mortality of presepsin was comparable to that of IL-6 and better than that of PCT, CRP or WBC.Presepsin increased significantly from the lowest to most severe sepsis groups at days 1, 3 and 8 (test for linear trend In patients with suspected severe sepsis and septic shock, precipices reveals valuable diagnostic capacity to differentiate sepsis severity compared to PCT, IL-6, CRP, WBC. Additionally, presepsin and IL-6 reveal prognostic value with respect to 30 days and 6 months all-cause mortality throughout the first week of ICU treatment.NCT01535534. Registered 14 February 2012.ClinicalTrials.gov  Severe sepsis and septic shock represent major challenges of modern intensive care medicine, and still recently published international guidelines demand ongoing research about the pathophysiology, diagnostics and treatment . WorldwiSoluble cluster of differentiation 14 subtype (sCD14-ST) - so-called presepsin - is cleaved from the monocyte/macrophage-specific CD14 receptor complex after binding with lipopolysaccharides (LPS) and LPS binding protein (LPB) during systemic infections -10. PresThe Mannheim Sepsis Study  was conducted as a mono-centric prospective controlled study at the University Medical Centre Mannheim (UMM), Germany. Patient enrolment started in October 2011. The study was carried out according to the principles of the declaration of Helsinki and was approved by the medical ethics commission II of the Faculty of Medicine Mannheim, University of Heidelberg, Germany. Informed consent was obtained from all participating patients or their legal representatives.2) to the fraction of inspired oxygen (FiO2) PaO2/FiO2 < 250, renal organ failure with urine output <0.5 ml/kg/h, hematological organ failure with platelet count <100,000/mm3 or unexplained metabolic acidosis with pH <7.3 and lactate levels >1.5 times the upper limit of normal. Sepsis-induced organ failures in these patients were strongly connected to infection and were present for less than 24 h. Patients developing cardiovascular organ failure with need for vasopressors longer than 1 h were categorized as suffering from septic shock. Disease severity on the ICU was documented by the acute physiology and chronic health evaluation II (APACHE II) and the sequential organ failure assessment (SOFA) score [The study was designed to reflect a representative cohort of patients with a minimum age of 18 years, who had proven criteria of severe sepsis or septic shock, found at a typical internal ICU. Main exclusion criteria were any traumatic or postoperative cause of sepsis development . Diagnosis of systemic inflammatory response syndrome (SIRS) and of sepsis severity was based on established criteria ,16: whenA) score ,18.All patient data, such as creatinine levels, hemoglobin, hematocrit, WBC count, platelet count, CRP, bilirubin, sodium, potassium, urea, IL-6, PCT, body temperature, respiratory rate, heart rate, blood pressure, partial pressure of O2 and CO2, bicarbonate, base excess, lactate, pH value, Glasgow coma scale (GCS) were documented. Additionally, prior medical history, age, sex, body weight and the germ spectrum were documented.After the end of each hospital treatment, two study physicians independently reviewed all available clinical data of the study patients and classified all patients into four disease groups: SIRS, sepsis, severe sepsis or septic shock. The study physicians were blinded to the results of tested biomarker measurements, such as presepsin, PCT and IL-6.Blood samples for presepsin measurements were taken within 24 h after clinical onset of severe sepsis or septic shock on the ICU (day 1) as well as on day 3 and 8 of ICU treatment. All patients were followed up until 30 days and 6 months after study inclusion by direct telephone visits with the patients or their general practitioners. The main prognostic outcome was all-cause mortality after 30 days and 6 months: 60 people without any clinically proven systemic infection served as a control group.g at 4°C for 15 minutes. Serum/plasma was separated, frozen and stored at −80°C.Blood samples were obtained by venipuncture into serum and ethylenediaminetetraacetic acid (EDTA) monovettes® . Within 30 minutes all blood samples were centrifuged at 1,000 × Presepsin measurements were performed with the PATHFAST® immunoassay analytical system  using plasma from EDTA monovettes® ,19. IL-6t-test was applied. Otherwise, the Mann–Whitney U-test was used as a nonparametric test. Deviations from a Gaussian distribution were tested by the Kolmogorov-Smirnov test. Spearman’s rank correlation for nonparametric data was used to test the association of presepsin blood levels with medical parameters. Qualitative parameters were analyzed by use of a 2 × 2 contingency table and Chi2 test or Fisher’s exact test as appropriate. Quantitative data are presented as mean ± standard error of mean (SEM) or as median and interquartile ranges (25th to 75th percentiles), depending on the distribution of the data. For qualitative parameters absolute and relative frequencies are presented. A test for linear trend was applied to compare the biomarker levels in the different groups of sepsis severity. Post-hoc statistical power analyses were performed. All analyses were exploratory and utilized a P-value of 0.05 (two-tailed) for significance.For normally distributed data, the Student C-statistics: receiver-operating characteristic (ROC) curve analyses were performed with calculation of area under the curve (AUC) for diagnosis of sepsis, severe sepsis and septic shock during the first week of ICU treatment at days 1, 3 and 8. A minimal AUC was set at 0.75 to define valuable discriminative diagnostic capacity of any biomarker. Accordingly, diagnostic goodness criteria , and relative risk) of the biomarkers were calculated. Accuracy was defined as the sum of true positives plus true negatives divided by all measured patients. Diagnostic AUCs were compared by the method of Hanley et al. [For y et al. .C-statistics: ROC analysis with calculation of the AUC was performed for prognosis of all-cause mortality in all patients after 30 days and 6 months for all biomarkers , APACHE II and SOFA score. Prognostic AUCs were compared by the method of Hanley et al. [For y et al. . Log-traThe calculations were performed with InStat and StatMate (GraphPad Software), SPSS software (SPSS Software GmbH), and SAS version, release 9.2 .Baseline characteristics are given in Table Mean APACHE II score at day 1 was highest in patients with septic shock (mean ± SEM = 27 ± 1). The most common primary site of infection was the lung in at least 50% of patients in each group, followed by abdominal and urinary tract infections (up to 16% of patients).r = 0.28, P = 0.002) as well as with the days of renal replacement therapy (RRT) during ICU treatment . Additionally, presepsin correlated with WBC, CRP, PCT, IL-6 and bilirubin. Interestingly, presepsin was also significantly correlated with the number of days of intensive care treatment, mechanical ventilation and catecholamine therapy (P <0.05). Presepsin levels were not correlated with patients’ age and gender in this cohort (P >0.05) (data not shown).Presepsin levels were significantly correlated with clinical and laboratory parameters at day 1. As shown in Table P ≤0.03), which was not observed for PCT or IL-6 (P >0.05).Figure Presepsin levels (pg/ml) were as follows ): day 1: SIRS 393 (249 to 745), sepsis 362 (249 to 745), severe sepsis 947 , septic shock 2,330 ; day 3: SIRS 448 (350 to 844), sepsis 651 , severe sepsis 1,479 , septic shock 2,060 ; day 8: SIRS 604 (223 to 965), sepsis 1,528 , severe sepsis 1,556 , septic shock 3,041 ; and controls 216 (146 to 350).P >0.05) (Table P = 0.05) and comparable to that of IL-6 (AUC = 0.81) . Interestingly, presepsin (AUC = 0.80) levels still revealed valuable diagnostic capacity to diagnose at least severe sepsis when compared to IL-6 (AUC = 0.71) and PCT (AUC = 0.66) at day 3. However, presepsin was not able to differentiate septic shock at day 3 , whereas the AUC of IL-6 was 0.76 at day 3. At day 8 of ICU treatment, the diagnostic value of presepsin was evident for all different groups of sepsis severity , whereas IL-6, PCT, WBC and CRP did not exceed an AUC ≥0.75  was comparable to that of IL-6 (AUC = 0.86) and PCT AUC = 0.83) at day 1 of ICU treatment  with a minimum sensitivity of 89% to diagnose either patients with at least sepsis, severe sepsis or septic shock at all points in time Table . At day P = 0.008).All-cause mortality rates were 50% after 30 days (58/116) and 62% after 6 months (72/116). Six months of follow up were completed in all patients. Presepsin levels were significantly higher in patients who died by 30 days or 6 months compared to those who survived (P >0.05) (Table P >0.05).The prognostic AUCs of presepsin were statistically significant at all time points and for all-cause mortality at 30 days and at 6 months Table . AUCs ofP <0.001) increased in non-survivors  compared to survivors  consistently at days 1, 3 and 8 of ICU treatment  both in survivors and non-survivors  in patients who died from septic shock within 6 months  compared to those patients surviving septic shock , when measured at day 1 of ICU treatment (data not shown).Presepsin and IL-6 levels were significantly (P <0.03) . However, presepsin was not statistically significant in adjusted Cox models at day 3 of ICU treatment. In contrast, IL-6 had significant prognostic value at all time points of ICU treatment and for both 30-day and 6-month all-cause mortality  (data not shown).Despite replacing the APACHE II with the SOFA score within Cox regression models to predict 30-day and 6-month all-cause mortality, presepsin retained its significant prognostic value at days 1 and 8 of ICU treatment  for diagnosis of septic shock on day 1 and 3 and for sepsis at day 3. PCT, CRP and WBC mostly failed to have any discriminative capacity for the different groups of sepsis severity at the different time points (AUCs <0.75).et al. previously demonstrated that presepsin levels had the best diagnostic capacity for diagnosis of sepsis (AUC = 0.82), severe sepsis (AUC = 0.84) and septic shock (AUC = 0.79) compared to PCT  in 859 patients presenting at the emergency department in Bejing, China [et al. [In accordance with the presented results, Liu g, China , while c [et al.  evaluateIn the present study we tried to avoid this inconsistency by choosing optimal and uniform cutoff levels  at a maximum achievable sensitivity on days 1, 3 and 8 . This approach was based on clinical considerations to capture as many patients as possible who were truly diseased. Interestingly, within the present analysis presepsin levels had weak diagnostic value for the differentiation of septic shock at day 3 of ICU treatment. This might be explained by a longer half-life of presepsin keeping higher concentrations at least until day 3 of intensive care treatment, which might also being influenced by the presence of acute kidney injury (AKI) in patients with severe sepsis/septic shock . At day et al. [In contrast, Endo et al. ,21 evaluRecent trials mostly evaluated single measurements of presepsin in patients presenting to the emergency department, trying to establish presepsin as an early one-shot guiding biomarker for emergency care ,14. HoweAccordingly, further influencing factors on presepsin despite age and renal function are rarely known . NotablyC-statistics and within multivariable Cox regression models being adjusted to age, sex, intensive care days and APACHE II/SOFA score.Second, it was demonstrated that measurements of presepsin levels revealed valuable prognostic capacity to predict short- and long-term  all-cause mortality compared to PCT, CRP and WBC consistently throughout days 1, 3 and 8 of ICU treatment. Patients with presepsin levels of the fourth quartile were up to five to seven times more likely to die after 6 months than patients with lower levels. APACHE II and SOFA scores solely revealed acceptable prognostic values for all-cause mortality. IL-6 was the only inflammatory biomarker with comparable prognostic value at all time points as demonstrated both in et al. [et al. demonstrated constantly increased presepsin levels in decedents and revealed significant prognostic value for both 28-day and 90-day all-cause mortality, whereas PCT failed to provide any prognostic information [The prognostic value of presepsin in severe sepsis/septic shock has not yet been evaluated in detail. Presepsin has been described as a powerful prognostic biomarker compared to PCT and APACHE II scores for short-term 28-day all-cause mortality  ,13. Ullaet al.  demonstret al. . In a reormation . It was Therefore, the present study delivered new evidence about both presepsin and IL-6 as powerful prognostic biomarkers of short- and long-term prognosis in patients with severe sepsis and septic shock -27. SpecP-values and relatively marginal differences in these biomarker levels with regard to diagnostic and prognostic differentiation in the present study cohort of 116 patients suggest that there might not be a real clinically relevant difference. Within our study, we did not find any significant associations of presepsin levels with age and sex of the patients, which were previously described [The present study was performed on the internal ICU at the University Medical Centre Mannheim, Germany. Primarily, this study included patients with severe sepsis or septic shock 24 h after admission to the medical ICU or 24 h after disease onset during ICU treatment. If statistically significant diagnostic and prognostic values for presepsin and IL-6 were calculated, sufficient statistical power of at least 80% could have been presumed. If statistically non-significant differences were calculated, a larger number of study patients of at least 300 to 2,000 patients in each group would have been required to guarantee sufficient statistical power, for example, for the inflammatory biomarkers PCT, CRP and WBC with respect to diagnosis or prognosis. However, high escribed . AnalyseTaken together, it has been demonstrated that measurements of presepsin levels had independent diagnostic and prognostic value in patients with severe sepsis and septic shock during the first week of intensive care treatment. Presepsin levels had valuable diagnostic value for the diagnosis of sepsis, severe sepsis and septic shock at days 1, 3 and 8 of ICU treatment compared to PCT, IL-6, CRP and WBC. Additionally, presepsin levels had valuable prognostic capacity to predict short- and long-term all-cause mortality when compared to PCT, IL-6, CRP, WBC and APACHE II score.Presepsin reveals valuable diagnostic capacity for stages of sepsis severity compared to PCT, IL-6, CRP, and WBC in patients being treated on an internal ICU.Diagnostic cutoffs of presepsin were set at ≥530 pg/ml for ≥ sepsis, at ≥600 pg/ml for ≥ severe sepsis and ≥700 pg/ml for septic shock.Presepsin levels had valuable prognostic capacity to predict short- and long-term all-cause mortality at 30 days and 6 months compared to PCT, CRP, WBC, SOFA and APACHE II scores.IL-6 had comparable prognostic value to presepsin levels.Diagnostic and prognostic capacity of presepsin was consistently demonstrated throughout days 1, 3 and 8 of ICU treatment.


Sepsis, a leading cause of death in critical care patients, is the result of complex interactions between the infecting microorganisms and the host responses that influence clinical outcomes . ReliablCritical patients with suspected sepsis admitted to the Unit of Intensive Care of the University Hospital of Catanzaro  were recruited into this study; healthy volunteers were also included as controls. Plasma samples in EDTA from each patient were collected at multiple time points; samples were tested for CRP, PCT and presepsin. Blood cultures were also evaluated and processed by a BacT/Alert 3D system ; CRP was measured by immunonephelometry  and PCT was assayed by an enzyme-linked fluorescent assay ; presepsin levels were measured by rapid automated PATHFAST immunoanalyzer , based on chemiluminescent enzyme immunoassay. A statistical analysis was carried out by Mann-Whitney test.Presepsin and PCT levels were significantly higher in culture-positive subjects versus negative controls; such difference was found even at the admission time. The presepsin values in worsening/dead patients exhibited a significantly higher level at admission time. On the contrary, in the same group of patients, PCT exhibited a decrease of its level. In poor prognosis patients CRP showed a quite irregular kinetic, although in such a group the admission value was higher than the same marker in live subjects.In this preliminary study, presepsin and PCT levels exhibited substantial higher values in culture-positive patients. The kinetic curves of presepsin, obtained from both survival and worsening/dead subjects, revealed the optimal performance of this biomarker, particularly in severely ill patients, as also shown in other studies. During sepsis, increase of presepsin levels may be a more reliable marker, indicating an unfavorable outcome ,4. Furth


Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker.Presepsin was determined at presentation (T0), after 8, 24 and 72 hours in 123 individuals admitted with signs of SIRS and/ or infection. Primary endpoint was death within 30 days. Presepsin was determined using the POC assay PATHFAST Presepsin .P < 0.0001). Baseline presepsin differed highly significant between patients with SIRS, sepsis, severe sepsis and septic shock. Twenty-four patients died during 30 days. The 30-day mortality was 19.5% in total, ranging from 10 to 32% between the first and the fourth quartile of presepsin concentration. Nonsurvivors showed high presepsin values with increasing tendency during the course of the disease while in surviving patients this tendency was decreasing. See Table Mean presepsin concentrations of the patient group at presentation and of the control group were 1,945 and 130 pg/ml, respectively (Presepsin demonstrated a strong relationship with disease severity and outcome. Presepsin provided reliable discrimination between SIRS and sepsis as well as prognosis and early prediction of 30day mortality already at admission. Presepsin showed close association with the course of the disease.


This study was performed to assess the value of procalcitonin (PCT) for the differential diagnosis between infectious and non-infectious systemic inflammatory response syndrome (SIRS) after cardiac surgery.n = 47) or non-infectious (n = 95), were included. The patients with infectious SIRS included 11 with sepsis, 12 with severe sepsis without shock, and 24 with septic shock.Patients diagnosed with SIRS after cardiac surgery between April 1, 2011 and March 31, 2013 were retrospectively studied. A total of 142 patients with SIRS, infectious , and the white blood cell (WBC) count were significantly higher in the infectious SIRS group than in the non-infectious SIRS group. PCT had the highest sensitivity and specificity for differential diagnosis, with a cut-off value for infectious SIRS of 0.47 ng/mL. PCT was more reliable than CRP in diagnosing severe sepsis without shock, but it was not useful for diagnosing septic shock. The PCT cut-off value for diagnosing severe sepsis without shock was 2.28 ng/mL.PCT was a useful marker for the diagnosis of infectious SIRS after cardiac surgery. The optimal PCT cut-off value for diagnosing infectious SIRS was 0.47 ng/mL. According to an epidemiological survey, the incidence of sepsis in the USA rose at an average annual rate of 8.7% from 1979 to 2000 . Early dThis study was a retrospective investigation of 142 SIRS patients who were admitted to the ICU after cardiac surgery at the Tokyo Medical and Dental University Hospital between April 1, 2011 and March 31, 2013 when a total of 376 patients after cardiac surgery were screened. The study was approved by the ethical review board of Tokyo Medical and Dental University Faculty of Medicine. The SIRS patients were divided into an infectious SIRS group and a non-infectious SIRS group according to the results of microbiological testing. The infectious SIRS group was further divided into three groups, namely, sepsis, severe sepsis without shock, and septic shock, according to the diagnostic criteria of the Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock: 2012 (SSCG2012) [3/μL = 0; <100 × 103/μL = 1; <50 × 103/μL = 2), elevated fibrin-related marker , prolonged prothrombin time , and fibrinogen level  derived from the International Society on Thrombosis and Haemostasis [The pre-operation data, operation-related data, and post-operation data of both groups were analyzed and compared. The pre-operation data included the patient age, gender, body mass index (BMI), and concomitant diseases. The operation-related data included the cardiopulmonary bypass (CPB) duration, aortic cross clamping, operation time, and blood loss. The post-operation data were as follows: mechanical ventilation (MV) duration, disseminated intravascular coagulation (DIC) incidence, serum levels of PCT, CRP, and WBC, Acute Physiology and Chronic Health Evaluation (APACHE) II score, Sequential Organ Failure Assessment (SOFA) score, ICU stay, hospital stay, postoperative blood purification treatment ratio such as continuous renal replacement therapy (CRRT) and polymyxin B-immobilized fiber column-direct hemoperfusion (PMX-DHP), and postoperative extracorporeal membrane oxygenation (ECMO) therapy. DIC was identified using a score based on platelet count . The serum CRP level was determined by a latex coagulation detection method with a nephelometer. The PCT, CRP, and WBC levels measured on the first day after the diagnosis of SIRS in the ICU were used in this analysis.t test and comparisons of more than two groups were analyzed by the one-way analysis of variance test. If the variance was not homogeneous, the data were shown in median and interquartile ranges. Comparisons between two groups were done by the Mann-Whitney test and comparisons of more than two groups were done by the Kruskal-Wallis test. The categorical data comparisons between groups were analyzed by the Pearson Chi-square test or Fisher’s exact test. Statistical significance was assumed at p values of less than 0.05 on both sides. The abilities of PCT, WBC, and CRP to diagnose infection were evaluated by comparing the infectious and non-infectious groups by receiver operating characteristic (ROC) curve analyses. The abilities of PCT and CRP to diagnose severe sepsis without shock or septic shock were evaluated by ROC curve analyses comparing the sepsis group with the severe sepsis without shock/septic shock groups and comparing the severe sepsis without shock group with the septic shock group. The cut-off values for diagnosing infection, severe sepsis without shock, and septic shock were determined by the ROC curves. The diagnosis sensitivity, specificity, and positive and negative predictive values were calculated and compared.Statistical analyses were performed using a Statistical Package for Social Sciences  for Windows. For the numerical data, the homogeneity test of variance was done first. If the variance was homogeneous, the data were shown as mean ± SD. The numerical data comparisons between two groups were analyzed by the two-sided Student’s In total, 142 patients were treated for SIRS after cardiac surgery. Among them, 47 were diagnosed with infectious SIRS and 95 were diagnosed with non-infectious SIRS. Table The infectious pathogens were Gram-positive bacteria in 19 patients, Gram-negative bacteria in 14 patients, and fungus in 16 patients. The positive culture specimens were sputum in 14, urine in 15, blood in 24, and drainage in 1.ROC) were 0.966, 0.875, and 0.799 for PCT, CRP, and WBC, respectively. According to the ROC curves, the cut-off values of PCT, CRP, and WBC were 0.47 ng/mL, 11.95 mg/dL, and 10.85 × 103/μL, respectively. The sensitivity and specificity of PCT for predicting infection were 91.5% and 93.7%, respectively.PCT, CRP, and WBC levels were significantly higher in the infectious SIRS group than in the non-infectious SIRS group Table . Preopern = 11), severe sepsis without shock (n = 12), and septic shock (n = 24). The patient characteristics of the three subgroups are shown in Table According to the diagnostic criteria, 47 patients from the infectious group were further divided into sepsis  or interleukin-6 (IL-6) induces high expression of the calcitonin-I gene, which activates the continuous release of PCT .p < 0.001) higher than those in the non-infectious SIRS group. Therefore, PCT becomes a tool to distinguish infectious and non-infectious SIRS, although some factors such as CKD and multiple organ dysfunction syndrome may affect the PCT level.The surgical injury to the body and extracorporeal circulation during cardiac surgery can activate the complement system, which in turn induces the massive release of inflammatory cytokines and the subsequent onset of SIRS . Once deClec’h et al. reported a wide variability of PCT cut-off values in an analysis of 143 patients with different diseases . By theiThe utility of PCT for evaluating infection severity has also been controversial. One study suggested that the PCT was a valuable parameter for determining infection, but was no better than CRP in evaluating the infection severity . In contThe MV duration, postoperative blood purification therapy, postoperative ECMO therapy, DIC incidence, ICU stay, and hospital mortality were all significantly higher in the infectious SIRS group than in the non-infectious SIRS group. These findings indicate that infection will increase the disease severity and necessitate more invasive treatments such as MV, CRRT, and ECMO. This finding was consistent with the results of Rahmanian et al. . The sepThere are three limitations to this study worthy of note: The study was retrospective; the samples were too small, especially for the infectious group; and no long-term follow-up was performed.Infection after cardiac surgery significantly increased the disease severity and necessitated more invasive treatments such as mechanical ventilation, CRRT, and ECMO. PCT was a useful marker for the diagnosis of infectious SIRS after cardiac surgery. PCT had a better diagnostic value than CRP or WBC. The optimal PCT cut-off value for detecting infection was 0.47 ng/mL. The serum level of PCT rose significantly according to the degree of infection. Prospective, large-scale, controlled, and randomized studies are awaited.


Many of the processes behind the decline of farmland birds can be related to modifications in landscape structure (composition and configuration), which can partly be expressed quantitatively with measurable or computable indices, i.e. landscape metrics. This paper aims to identify statistical relationships between the occurrence of birds and the landscape structure. We present a method that combines two comprehensive procedures: the “landscape-centred approach” and “guild classification”. Our study is based on more than 20,000 individual bird observations based on a 4-year bird monitoring approach in a typical agricultural area in the north-eastern German lowlands. Five characteristic bird guilds, each with three characteristic species, are defined for the typical habitat types of that area: farmland, grassland, hedgerow, forest and settlement. The suitability of each sample plot for each guild is indicated by the level of persistence (LOP) of occurrence of three respective species. Thus, the sample plots can be classified as “preferred” or “less preferred” depending on the lower and upper quartiles of the LOP values. The landscape structure is characterized by 16 different landscape metrics expressing various aspects of landscape composition and configuration. For each guild, the three landscape metrics with the strongest rank correlation with the LOP values and that are not mutually dependent were identified. For four of the bird guilds, the classification success was better than 80%, compared with only 66% for the grassland bird guild. A subset of six landscape metrics proved to be the most meaningful and sufficiently classified the sample areas with respect to bird guild suitability. In addition, derived logistic functions allowed the production of guild-specific habitat suitability maps for the whole landscape. The analytical results show that the proposed approach is appropriate to assess the habitat suitability of agricultural landscapes for characteristic bird guilds.The online version of this article (doi:10.1007/s10661-017-5837-2) contains supplementary material, which is available to authorized users. The decline of biological diversity in European landscapes is well documented by many regional and Europe-wide studies Defra . Of all Suitable landscape data can be retrieved from digital maps, which are becoming abundant and available for many regions, based on aerial photos, satellite images, ground mapping, etc. Modern methods, including GIS and spatial statistics, allow for area-wide analyses of landscape metrics and, if time series are available, the characterization of trends. Because many of these data already exist, the calculation of landscape metrics is often well established  has the potential to improve area-wide assessments of landscapes as habitat for birds. Thus, to contribute to the development of an appropriate methodology, we draw on an extensive set of bird occurrence data of an agricultural landscape , and we test a variety of landscape metrics as potential indicators of defined groups of bird species.Generalizations of case studies are needed to address the habitat suitability for birds on a landscape scale. What is the essence of locally detected relationships between bird occurrence and landscape characteristics that may be transferable to other regions? Answers to this question may help support the efficiency of empirical work (e.g. bird monitoring schemes), the design of meaningful biodiversity supporting schemes (e.g. changes in habitat configurations) and the comparison and assessment of landscape and land use development scenarios. While it is obvious that appropriate conservation means depend on the specific regional or local situation , with a decreasing trend. Parts of the investigation area belong to a landscape conservation area. The significance of the region for birds is high, as BirdLife International identifies the region as one of the Important Bird and Biodiversity Areas (IBAs) in Europe  or absence (0) of the three characteristic species, neglecting the actual number of individuals. The occurrences were summarized as follows: in each of the surveys, a maximum of three was possible on each sample plot when all species of the guild could be observed, and 0 when no species was observed. These values are taken to construct a so-called level of persistence (LOP) for every guild at each sample plot, which is the sum of all presence/absence values over the whole survey area and duration, with a theoretical minimum of 0 and a theoretical maximum of 57 . It is true that the LOP approach introduced here has no direct ecological meaning. Due to its construction, it may be considered a further development of the common presence-absence approach. The addition of the frequency of bird occurrences at particular points may furthermore indicate the habitat quality of the sample areas in an aggregated quantitative way. A high LOP value means that species of a guild are frequently observed, so the area is considered to be a rather preferred habitat of that guild. The LOPs were derived for each of the five guilds, and statistical analysis was conducted for each guild for all sample plots. In total, 120 of the 125 potential sample plots were visited at all occasions and used for the analysis. Five sample plots were excluded due to missing data.Data analyses were conducted using SPSS Statistics 22 software from IBM and ArcMap 10.2.2 software from ESRI. Landscape metrics were calculated based on the spatial data of the “Comprehensive Biotope and Land use Map” of the Federal State of Brandenburg MLUL . These cHabitat data were pre-processed, i.e. the polygon and line layer were joined to cover the area as accurately and in as much detail as possible. For this purpose, line data were buffered with a buffer distance of 5 m to ensure that line elements were not discarded during the conversion from polygon to raster format. The processed spatial information had a standard deviation of 0.99% with respect to the original polygon data. The standard deviation of the habitat coverage and original and combined data sample plots was less than 1% and was 0.92% smaller than the whole sample area. The sample plots were tested for representativeness using the biotope inventory of the whole area. The total classification comparison revealed a significant correlation of 0.993 between the sample plots and the total Quillow investigation area , which was confirmed by Spearman’s rank correlation coefficient at the 5% error level. Therefore, the sample plot area was considered to represent the entire Quillow observation area reasonably well  registered during 8,432 listed individual sightings; the 15 selected species represent 42% of the observed individuals of the overall bird community. The composition of the different guilds is shown in Fig. To obtain a visual impression of the spatial distribution of the selected bird guilds in the investigation area, the occurrence information for the guilds in the sample plots was displayed on spatial maps of related landscape metrics. Figure The main objective of the statistical analysis of the bird occurrence data was to determine whether it was possible to predict the guild occurrence within the sample areas based on the values of the specific landscape metrics around the sample areas. The occurrence of a guild in a sample plot was expressed by the occurrence of its 3 characteristic species and their LOP values. The derived LOP values serve as a target variable for subsequent analytical statistics. Due to the statistical properties of the LOP values and of the considered landscape metrics, only analytical methods without special requirements concerning the statistical distributions of the included features were applied.The pairwise relationships between landscape metrics and the LOPs for the particular guilds were analysed with Spearman rank correlation coefficients. The classification of “preferred” and “less preferred” habitats was then accomplished with a binary logistic regression using input data derived from the LOPs. The binary logistic regression was selected as a principal analytical method because the analytical focus was on the qualitative distinction between rather unsuitable habitats (less preferred) and rather suitable habitats (preferred) based on landscape metrics. The binary logistic regression model is able to predict a binary response dependent on one or more independent inputs and has no particular requirements concerning the scale level of these inputs. Practically any appropriate quantitative or qualitative variable including landscape metrics may serve as input. The binary logistics regression allows a direct and transparent interpretation of input effects, and it is furthermore possible to evaluate the reliability of the classification and to rank the importance of inputs. Mathematically, the binary logistic regression function takes values between 0 and 1, and the binary response is assigned to the function values using a cut point between 0 and 1. In most practical situations, 0.5 serves as the cut point. Mathematical details and the implementation in SPSS are described by Field .Here, the binary response (preferred vs. less preferred habitats) is derived from the LOPs as a dependent variable, and a set of landscape metrics is used as independent variables. Among the landscape metrics, those three metrics were selected as inputs that showed the greatest Spearman correlation coefficient with the particular LOP. In case of obviously functionally dependent landscape metrics , only one of them was used as input in order to keep the inputs as mutually independent as possible. The number of input variables was limited to three to enable a transparent interpretation of the mutual interconnections.R2 values were calculated.The members of the two alternative classes (preferred vs. less preferred habitats) were taken from the cases that fell below the 25% quartile of the LOPs and above the 75% quartile of the LOPs of every particular guild and observation point. The “lower” class indicates rather unsuitable habitats (less preferred), and the “upper” class indicates rather suitable habitats (preferred). Each class contains approximately 30 cases. Within SPSS, the binary logistic regression was executed with the variable selection method “Enter”, i.e. all independent input variables are entered in a single step Field . To evalp value of the Wald chi-square statistics. The p values indicate the strength of evidence of the relationship between target and input , the shape index distribution (SHAPE_MN) and the edge density (ED). All three act in the opposite direction of the LOP and are statistically significant at the 1% error level, that is, the higher the value of the metrics, the lower the occurrence of farmland birds. Greater shape mean, with a 5% error.The correlation analysis results of the other four guilds can be interpreted in an analogous manner. With the exception of the grassland bird guild, all selected landscape metrics were significantly correlated with the particular guild LOP. In the case of the grassland birds, the only significant landscape metric was the R2 value is reported. Additionally, the output of the logistic regression indicates what type of misclassification occurs, i.e. whether there is a systematic preference among misclassifications for preferred or less preferred areas.Table R2 value in a comparative test of the classifications. It was not possible to derive the overall statistical significance of the classification, but it was possible to use the Nagelkerke R2 value to interpret the reliability of the classification. In this respect, it was obviously easier to classify the preferred and less preferred habitats for species of the forest and settlement guilds than for species of the grassland and hedgerow guilds. The rate of correct classifications and the considerably lower Nagelkerke R2 value for the grassland guild indicate that the classification of grassland habitats seems to be the most difficult.For four of the five considered guilds, it was possible to distinguish between less preferred and preferred habitats with success rates better than 80%. Only in the case of the grassland guild was the rate of correct classifications (66%) apparently poorer. Because an identical number of cases was used for all five guilds, it was possible to use the Nagelkerke p value of the Wald statistic. The latter two provide information concerning the relative importance of individual input variables (i.e. habitat suitability expressed by landscape metrics) and their statistical significance. The input variable acts independently of the input variables. In the estimated logistic regression function for the farmland guild, all three unstandardized regression coefficients were statistically significant at the 5% error level. The standardized regression coefficient for Simpson’s diversity index (SIDI) indicates that this landscape metric had the greatest relative importance. In the case of identical relative changes among the three inputs, changes in the SIDI would have the greatest effect.Table p value of the Wald chi-square statistic . The relative importance of the input metrics can be derived from the standardized regression coefficient .Tables Of the 16 different landscape metrics initially chosen, only 6 were retained in the final statistical models describing the preferred vs. less preferred habitats for the five selected bird guilds. The habitats for farmland and forest guilds as well as for hedgerow and settlement guilds were described with the same set of indices but with varying directions and combinations. Table The relationship between landscape structure and biota has been extensively described mostly for species richness issues, including birds  and the highest uncertainties of all guilds. This might be due to the lowest numbers of sightings of these bird species in our survey. The preferred areas for the grassland species were identified by the contiguity index distribution, patch density and shape index distribution set and are described as small, isolated grassland plots (low contiguity and low patch density) without highly complex shapes (low shape index). The occurrence of small closed drainage basins with grassland vegetation, which are extensively used or not managed at all, is a specific feature of many North-Middle European landscapes. The preference of the grassland birds for these features, as found in our study, might be strongly affected by the extensive management of such plots. The meadow pipit is known to inhabit open grassy areas with dense, low vegetation cover, such as extensive meadows, and to avoid very short grass in intensive meadows or grazed pastures  between the different habitat types (high Simpson’s diversity), in contrast to the forest habitats due to their small sizes (largest patches). Batáry et al.  found clForest birds preferred areas with high shape index values, high Simpson’s diversity index values and low edge density values in our analysis. They can be differentiated from the hedgerow guild as preferring different habitat types, including forests (higher Simpson’s diversity index), that are of irregular shape (high shape index) and as avoiding transient zones (edges) between different habitat types. Within the agricultural matrix, forests are mostly singular patches and seldom share borders with other forest patches. In contrast, in forest-dominated landscapes, the forest patch area could be the most significant variable explaining the patch occupancy of residents and summering forest birds, as reported by Suk et al. . BarbaroBird species that prefer settlements and urban environments can be described with the same indices as the hedge breeding birds but with different factor levels. Settlement birds prefer the transient area between settlements and arable land (high edge density), the patchy environment around the settlements (high largest patch index LPI) and the high diversity between habitat types (high Simpson’s diversity index). Birds in urban environments can be classified into groups of distinct habitat requirements  would lose comparability with respect to the full models.Many papers emphasize that the spatial scale affects the performance of the landscape metrics for terrestrial birds  landscapes with different natural pre-conditioning (setting) and to (ii) different bird fauna compositions (inventories), which we suggest to investigate further.ESM 1(PDF 7018 kb)


Furthermore it was demonstrated that although APTES was fully removed from the silicon surface following four hours incubation in water, the gold nanoparticle-amino surface complex was stable under the same conditions. Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to study these affects.This study evaluates the effectiveness of vapour-phase deposition for creating sub-monolayer coverage of aminopropyl triethoxysilane (APTES) on silicon in order to exert control over subsequent gold nanoparticle deposition. Surface coverage was evaluated indirectly by observing the extent to which gold nanoparticles (AuNPs) deposited onto the modified silicon surface. By varying the distance of the silicon wafer from the APTES source and concentration of APTES in the evaporating media, control over subsequent gold nanoparticle deposition was achievable to an extent. Fine control over AuNP deposition (AuNPs/μm Fabrication and manipulation of nano-sized features is a fast-growing science playing an important role in the development of electronics, materials and biotechnology . Whilst The aim of this work was to evaluate the effectiveness of a vapour phase deposition protocol for preparing sub-monolayers of aminopropyl triethoxysilane (APTES) on silicon surfaces to control gold nanosphere surface densities. The effect of changing vapour phase deposition parameters on resulting APTES density and the propensity of the modified surface to adsorb gold nanoparticles (AuNPs), have been systematically evaluated. Additionally, the effect of varying the ionic concentration of the incubating AuNP solution on nanoparticle deposition and the protection of underlying chemical functionality conferred by the nanoparticles was investigated. The methods described are simple and practical, allowing reproducible deposition of various AuNP densities and may find application in the patterning of a range of substrates that are amenable to silanisation, including mica, quartz and glass.et al. [AuNPs stabilised with anionic citrate ions bind electrostatically to cationic APTES molecules and have widely been used to confirm the presence of APTES on derivatized silicon wafer ,10,11. Tw/w in paraffin oil (PO)) as the evaporative solution and with silicon surfaces centered at 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 cm from the edge of the Eppendorf lid. AFM images of the surfaces following immersion in AuNP solution  are shown in et al. [2. In this current study the maximum observed number density was 242 ± 17 AuNPs/μm2. This discrepancy in AuNP number density is more than likely attributable to a difference in the particle size and ionic concentrations of the AuNP solutions used in the studies. Surfaces positioned up to 3.0 cm . A clear gradient in coverage (represented as nitrogen content) was observed as the APTES concentration increased from 2% to 20%. Further increasing the concentration had no effect on nitrogen concentration suggesting that monolayer coverage was achieved at 20% APTES concentration. There was no evidence for multilayer coverage since there was no significant increase in surface nitrogen at APTES concentrations >20% . Following immersion in 10 nm AuNP solution, no AuNP deposition was observed on the surfaces silanised with 2% APTES but almost complete coverage was seen at 4% and 8% (2); the clear gradient observed in the XPS data  for 10, 30, 120 and 240 min. AuNP surface density reached a maximum (~200 AuNPs/μm2) between 10 and 30 min (−3 M)   inset. T .The stability of nanocolloidal solutions is based upon repulsion between individual nanoparticles. In aqueous solution, negatively-charged citrate-stabilized AuNPs are surrounded by an electrical double layer of positive ions. Increasing the thickness of the double layer directly increases the electrostatic repulsion and interparticle distance of colloids in solution (Deyagin-Landau and Verwy-Overbeek (DLVO) theory) ,15,16. Ai.e., below the immobilized pKa of APTES (immobilized pKa 7.6 [450) values [The AuNP solutions used in this study were stabilised in a citrate buffer (0.04% trisodium citrate) that was sequentially diluted to give rise to solutions of varying ionic concentration. All solutions, regardless of ionic concentration, were shown to have a pH of between 6.4 and 6.7,  pKa 7.6 ). Ionisa) values  and mult−3 M). It was observed that on increased exposure to water, a decrease in AuNP surface densities resulted . All experiments were carried out at room temperature and pressure. Surfaces were degreased by ultrasonication in ethanol (15 min) followed by dimethylformamide (15 min), then dried under a stream of nitrogen. Surfaces were submerged in Piranha solution  and maintained at 80–90 °C . Surfaces were washed thoroughly in diH2O and dried under a stream of nitrogen. Unless otherwise stated, the activated surfaces were placed 2 cm from the edge of an upturned Eppendorf tube lid and enclosed within a Petri dish ) were cut into ~0.5 cmtri dish . A mixtu2O to remove physisorbed AuNPs. The ionic concentration of the solution, with respect to trisodium citrate, is given for each solution.Unless otherwise stated, surfaces were incubated in AuNP solution ) for 14–24 h and then rinsed thoroughly in dHA Veeco Scanning Probe Microscope  was used in tapping mode for AFsM analysis of the surfaces using RTESP 1–10 Ohm-cm phosphorous (n) doped silicon AFM tips. All consumables were purchased from Veeco Instruments Inc. AFM images were processed using “WSXM” image analysis . To detei.e., 90° grazing angle). Surfaces were mounted using double-sided adhesive tape. The data was analyzed using Casa XPS [X-ray photoelectron spectroscopy (XPS) was carried out using a Kratos Axis Ultra DLD spectrometer . A monochromatic AlKα X-ray source (75–150 W) with an analyzer pass-energy of 160 eV (survey scans) or 40 eV (detailed scans) was used. Photoelectrons were detected in a direction normal to the surface  distribution, onto silanised surfaces was shown to be readily achievable by varying the ionic concentration of the AuNP solutions. The protective nature of AuNPs on the underlying APTES groups will allow for chemical nanopatterning, through the selective modification of the amine groups not involved in the binding interaction with the nanoparticles, to generate a bi-functional surface.