A substantial proportion of both the mouse and human genomes comprise of endogenous retroelements (REs), which include endogenous retroviruses. Over evolutionary time, REs accumulate inactivating mutations or deletions and thus lose the ability to replicate. Additionally, REs can be transcriptionally repressed by dedicated mechanisms of the host. Nevertheless, many of them still possess and express intact open reading frames, and their transcriptional activity has been associated with many physiological and pathological processes of the host. However, this association remains tenuous due to incomplete understanding of the mechanism by which RE transcription is regulated. Here, we use a bioinformatics tool to examine RE transcriptional activity, measured by microarrays, in murine and human immune cells responding to microbial stimulation.in vitro caused extensive changes in the transcription not only of the host genes involved in the immune response, but also of numerous REs. Modulated REs were frequently found near or embedded within similarly-modulated host genes. Focusing on probes reporting single-integration, intergenic REs, revealed extensive transcriptional responsiveness of these elements to microbial signals. Microbial stimulation modulated RE expression in a cell-intrinsic manner. In line with these results, the transcriptional activity of numerous REs followed characteristics in different tissues according to exposure to environmental microbes and was further heavily altered during viral infection or imbalances with intestinal microbiota, both in mice and humans.Immune cell activation by microbial signals Together, these results highlight the utility of improved methodologies in assessing RE transcription profiles in both archived and new microarray data sets. More importantly, application of this methodology suggests that immune activation, as a result of infection with pathogens or dysbiosis with commensal microbes, causes global modulation of RE transcription. RE responsiveness to external stimuli should, therefore, be considered in any association between RE transcription and disease.The online version of this article (doi:10.1186/1742-4690-11-59) contains supplementary material, which is available to authorized users. While the existence of repetitive genetic elements has been recognized since the 1950s, the scale of their contribution to overall genome size was only fully realized through the sequencing of the human and mouse genomes . In totaOriginally identified as leukemogenic agents in mice, both exogenous and endogenous retroviruses have been extensively studied for potential contributions to cancer and disease in many species . Many ERDependent on their relative distance and orientation, REs have been suggested to act as transcriptional promoters and enhancers, canonical and alternative transcription initiation and termination points, splice donor and acceptor sites  and polyFv1 and Fv4[Through co-option by the host, REs, and ERVs in particular, can have more direct effects. The fusogenic and immunomodulatory roles of certain ERV envelope sequences have been acquired as ‘syncytins’ separately in a variety of placental mammals . Knock-o and Fv4–16.Despite the lack of infectious ERVs in the human genome, ERV-encoded envelope glycoprotein antigens have been suggested as putative autoantigens in human autoimmune conditions and viral-like particles have been observed in a variety of human diseases , 18. ComUsing mice with distinct immunodeficiencies, we have previously reported the spontaneous emergence and establishment of replication-competent murine leukemia viruses (MLVs) through recombination between replication-defective ERVs . The appin situ synthesized microarrays [Studies of RE transcription have to date relied primarily on PCR-based methods , 24, whiroarrays , 27 haveroarrays  and cytoroarrays .et al.[While it has been known for some time that microarray platforms from various commercial manufacturers contain probe sequences corresponding to repetitive genetic elements, the major focus in the literature has been on the removal of such probes from analysis pipelines , 31. Recet al. detailedet al. and RepBet al. librarieet al. (data noUsing the Affymetrix Mouse Genome 430v2 (MG430v2) platform, where a probeset was noted as containing RE-reporting probes, a median of 3 probes from the group were identified Figure A. Only 1Tnnt2  within heart tissue [Ldb3 (LIM domain binding 3) within skeletal muscle [Ighv14-2 (immunoglobulin heavy variable 14–2) within the spleen [To assess the potential impact of such co-regulation, three independent experiments using MG430v2, originally designed to determine tissue-specific expression patterns, were analyzed for significantly regulated RE-reporting probes. While obvious clustering of tissues was observed (data not shown), the most highly expressed RE-reporting probes were members of probesets reporting the expression of known tissue specific genes, including t tissue , Ldb3  being observed as the result of the enhanced filtering of RE-reporting probes  genetic background . To inveMicrobial products are recognized by pattern recognition receptors, such as TLRs, and previous work has shown the widespread and diverse impacts of various TLR agonists on ERV expression in both murine and human cells . SubsequMyd88−/− mice housed in both specific pathogen-free (SPF) and germ-free (GF) conditions , MT2B (ERVL), and MMTV expression was also noticeably increased in SPF mice, albeit in tissue-specific manners  reduced MLV expression across all tissues sampled in the absence of Myd88 , the expression of which is lipopolysaccharide (LPS)-inducible [Emv2[Emv2 expression [Emv2 seen upon BrdU treatment  of HIV-infected or uninfected individuals  viral-like particles, a treatment that allows DC infection, exhibited an altered RE expression profile in comparison with all other treatment groups  script was produced to run and query BLASTn of the downloaded probes against the relevant genome using the ‘-task blastn-short’ parameter. The number of times an individual probe could be localized to the genome with ≥95% identity was recorded, along with the location of the highest scoring hit. A further Python script was used to parse these data to identify probes falling entirely within regions masked by RepeatMasker and to identify those in the correct orientation to report sense expression of the particular element. For technologies hybridizing antisense cRNA (e.g. Affymetrix 3′ microarrays), probes are sense to the retroelement, whereas for technologies hybridizing sense cDNA (e.g. Affymetrix Gene microarrays), probes are required to be antisense to the retroelement. The nearest genes chromosomally 5′ and 3′, as well as their locations, were recorded from the gene annotation files and, together, this information was compiled to form an annotation file for probes identified as reporting retroelement expression. Where probes were originally identified as reporting expression from multiple genomic loci, annotation information requiring a specific genomic context was omitted. This probe list was filtered using an additional script for probes derived from probesets where >75% of probes report retroelement expression, and where the probe was identified as >1 kb from the nearest protein coding gene. Annotation files are supplied as Additional files A Python (http://www.ebi.ac.uk/arrayexpress). Pseudo-images of the array chips were visually inspected for spatial artifacts and arrays that passed this inspection were analyzed at the probe level with a custom R script utilizing routines provided within ‘oligo’. Perfect-match (PM) probe expression data for the entire dataset were RMA background corrected and quantile normalized before log2 transformation and export. Downstream analysis, probe annotation, batch-effect correction (where appropriate), and heatmap production was thereafter performed with Qlucore Omics Explorer . To reduce the size of heatmaps and to decrease artificial clustering resulting from multiple probes from the same probeset, probes identified as significant were collapsed into their respective probesets using facilities build into Qlucore Omics Explorer.Raw CEL files corresponding to accessions E-GEOD-97 , E-GEOD-Other figure production and statistical analysis was performed with SigmaPlot v12 .w-estimator for probeset expression followed the algorithms defined by Affymetrix [N, of probe expression values, x, where x, and S denotes the median absolute deviation of x, the w-estimator is calculated as c = 5 and ϵ = 0.0001.Calculation of the one-step Tukey’s biweight fymetrix . For a nMyd88tm1Aki (Myd88−/−) and TLR4-deficient B6.129P2-Tlr4tm1Aki (Tlr4−/−) mice have been described [Myd88tm1AkiTicam1tm1Aki (Myd88−/−Ticam1−/−) mice, additionally deficient for toll-like receptor adaptor molecule 1 (TICAM-1) [Inbred B6 and 129 wild-type strains, as well as B6-backcrossed MyD88-deficient B6.129P2-escribed , 67. MicTICAM-1) , were al2. Adherent DCs could typically be obtained after this time at a purity of 50-70%. TLR agonists were introduced for 48 hours at 1 μg/ml for LPS , 10 μg/ml for poly(I:C) (Sigma-Aldrich) and 0.25 μg/ml for Pam3CSK4 (Axxora). BrdU (Sigma-Aldrich) was introduced at 20 μg/ml.For the production of BMDCs, bone marrow was flushed from the femurs and tibiae of culled mice and incubated in IMDM supplemented with 5% FCS  and 10% GM-CSF for 7 days at 37°C and 5% COPrior to cDNA preparation, all samples were stored in RNAlater  at −20°C. Where tissues were processed, samples were disrupted using a TissueLyser LT (Qiagen). RNA was extracted from samples using RNeasy spin columns (Qiagen) and extracted nucleic acids were subjected to DNaseI (Qiagen) treatment in solution and a further column cleanup. RNA for qRT-PCR was reverse transcribed using the Applied Biosystems  high capacity reverse transcription kit with an added RNase-inhibitor  and cDNA was cleaned using QIAquick spin columns (Qiagen). All elutions were conducted with nuclease-free water (Qiagen).Hprt using the ΔCT method using previously-described primer sets and methodology [Purified cDNA was used as template for the amplification of target gene transcripts with SYBR Green PCR master mix (Applied Biosystems) using the ABI Prism SDS 7000 and 7900HT machines (Applied Biosystems). Target gene expression was determined relative to hodology . In plotGRY and BM are post-doctoral Career Development Fellows in GK’s laboratory. GK is a program leader at MRC National Institute for Medical Research, UK, and Professor of Retrovirology at Imperial College London, UK.p < 0.001 by ANOVA comparing tissues and eliminating experiment). Data are obtained from E-GEOD-1986, −9954, and −10246, which are identified with numbers. (PDF 6 MB)Additional file 1: Figure S1: Tissue-specific RE expression patterns. Hierarchally-clustered heatmap of RE-reporting probes significantly regulated between B6 tissues for three independent experiments using the Mouse Genome 430 v2 microarray platform (Archive of mouse annotation files. Probe annotation files (csv format), as defined in the Methods, for the following Affymetrix platforms: mg_u74a, mg_u74a_v2, mg_b74b, mg_u74b_v2, mg_b74c, mg_u74c_v2, moe_430a, moe_430b, ht_mg_430a, mogene_1_0_st, mogene_2_0, mouse430_v2, mouse430a_v2. Shortened platform names correspond to identifiers used within the ‘oligo’ Bioconductor R package. Column identifiers are pid – probe id, probeset – Affymetrix probeset, plen – probe length, sid – target chromosome, sstart – start position of probe on sid, send – end position of probe on sid, nident – identity within the region sstart to send, numhits – number of hits recorded by BLASTn, repeat – RepBase-defined repeat, repclass – RepBase-defined repeat class, rstart – start position of repetitive element, rend – end position of repetitive element, 5id – symbol of nearest 5′ protein coding gene, 5start – start position of 5id, 5stop – end position of 5id, 3id – symbol of nearest 3′ protein coding gene, 3start – start position of 3id, 3stop – end position of 3id. (ZIP 846 KB)Additional file 2:Archive of human annotation files. Probe annotation files (csv format), as defined in the Methods, for the following Affymetrix platforms: hg_u95a, hg_u95a_v2, hg_u95b, hg_u95c, hg_u95e, hu6800, hg_u133a, hg_u133a_v2, hg_u133b, hg_u133_plus_v2, hg_u219, hg_focus, hugene_1_0_st, and hugene_2_0_st. Shortened platform names correspond to identifiers used within the ‘oligo’ Bioconductor R package. Column identifiers are pid – probe id, probeset – Affymetrix probeset, plen – probe length, sid – target chromosome, sstart – start position of probe on sid, send – end position of probe on sid, nident – identity within the region sstart to send, numhits – number of hits recorded by BLASTn, repeat – RepBase-defined repeat, repclass – RepBase-defined repeat class, rstart – start position of repetitive element, rend – end position of repetitive element, 5id – symbol of nearest 5′ protein coding gene, 5start – start position of 5id, 5stop – end position of 5id, 3id – symbol of nearest 3′ protein coding gene, 3start – start position of 3id, 3stop – end position of 3id. (ZIP 3 MB)Additional file 3:


Little evidence exists on the physical activity and sedentary time of Canadian toddlers; this study objectively measured such behaviors and compared participants’ activity levels to national guidelines. Levels of screen-viewing among toddlers were also explored.T-tests were used to assess whether toddlers’ activity levels differed based on cut-points applied and various demographic and screen-related variables. Regression analyses were conducted to examine associations between toddlers’ sedentary time and screen-viewing levels.Forty toddlers (mean age = 25.7 months) wore Actical accelerometers for seven consecutive days (15 s epoch). Parents/guardians completed a wear-time log and a demographic and screen-viewing questionnaire. Descriptive analyses were used to determine participants’ levels of physical activity and sedentary time, to identify whether toddlers were meeting physical activity/sedentary guidelines, and to explore demographic variables. SD = 3.79) to 49.40 (SD = 3.29) mins/hr of sedentary time, 9.79 (SD = 2.90) to 18.78 (SD = 3.22) mins/hr of light-intensity physical activity (LPA), 0.82 (SD = 0.72) to 3.95 (SD = 1.93) mins/hr of moderate- to vigorous-intensity physical activity (MVPA), and 10.60 (SD =3.29) to 22.73 (SD = 3.97) mins/hr of total physical activity (TPA), based on the Trost et al. and the Canadian Health Measures Survey (CHMS) cut-points respectively; these rates were significantly different (p <.001). On at least 1 day, 17.5 %  and 97.5 % (CHMS cut-points) of the sample met or exceeded the Canadian physical activity guidelines. No statistically significant differences in sedentary time or physical activity  based on sex were reported (p <.001); however, LPA (CHMS cut-points) did significantly differ based on childcare attendance (p <.05). Approximately 93.2 % of participants watched television, and 56.8 % utilized computers. Only 18.8 and 25.0 % of children under 2 years and 70.8 and 62.5 % of 2–3 years olds met the screen-use recommendation of the sedentary behavior guidelines on weekdays and weekend days, respectively.Toddlers engaged in 37.27 (The implications of this work suggest that a greater understanding of toddlers’ activity patterns is needed; additional mechanisms of promoting active behaviors among this group should be explored. Physical activity plays a pivotal role in the overall health and well-being of children. Among young children under the age of 5 years, regular physical activity has been linked to decreases in cardiovascular risk  as well The Canadian Society of Exercise Physiology , 16 releAccelerometers represent one popular method for objectively measuring levels of physical activity and sedentary time among young children , 24, 25,This exploratory study sought to objectively measure the physical activity levels and sedentary time of a sample of toddlers in London, Canada using two sets of cut-points in comparison to the national physical activity guidelines. Because a variety of demographic variables have been identified as influencing young children’s activity levels, the impact of sex , parentaUsing a cross-sectional study design, English-speaking toddlers (between the ages of 18–29 months) from London, Canada were invited to participate. In an effort to target a geographically-representative sample, parents/guardians of participants were recruited at a mother and child exhibition, at various playgroups offered by the Ontario Early Years Centers (spanning various socio-economic areas), and via posters placed in locations frequented by parents/guardians and young children . Where appropriate, snowball sampling was also utilized as a means of maximizing the reach of our recruitment methods.Data collection occurred between August 2013 and November 2014 . Participants were asked to wear an accelerometer for seven consecutive days  during all waking hours; parents/guardians were asked to fit their child with the device upon them waking in the morning, and to remove it prior to their bedtime. In addition, parents/guardians were asked to keep a log of the on/off times of the accelerometers. Accelerometers and logs were dropped off to participants’ parents/guardians a few days prior to the first day of data collection . Following the week of data collection, a researcher returned to the participants’ homes to collect the accelerometers and logs. Ethical approval for the study protocol and related documents was obtained from the Office of the Research Ethics Board at the University of Western Ontario. Written informed consent was provided by parents/guardians of all participating children.Toddlers’ sedentary time and physical activity levels  were measured using Actical™  accelerometers. These lightweight omnidirectional motion sensors provide detailed data on the duration and intensity of the children’s movements . A 15-seet al. , LPA [≥115 ≤697 counts⋅15 s−1⋅epoch−1], and MVPA [≥698 counts⋅15 s−1⋅epoch−1], and TPA [≥115 counts⋅15 s−1⋅epoch−1]) to determine the amount of activity accumulated at various intensity levels – this was achieved by entering the cut-points into the program and then processing the included data files to produce a number of outcome variables using these thresholds. Because the toddler population has only recently begun to receive attention regarding physical activity levels, combined with the evidence that suggests that different accelerometers and/or their respective cut points can influence the outcome data , it was deemed important to apply a second set of cut-points (used by other researchers who included toddler participants) for comparison. As such, and in line with the Canadian Health Measures Survey (CHMS), the following cut-points  were also applied to the collected accelerometer data : sedentary activity (≤24.75 counts⋅15 s−1) , and does your child spend time on a computer? [how many minutes per week(end) day?]). To determine the number of participants that met/failed to meet the screen-use portion of the sedentary behavior guidelines , an approach undertaken by other Canadian researchers was followed  = 37.81, p <.001), LPA , MVPA , and TPA  were found to significantly differ based on cut-points applied. Using an average wear-time of 10.11 h, these values translate roughly to 376.80 and 499.43 mins/day of sedentary time, 98.97 and 189.87 mins/day of LPA, 8.29–39.93 mins/hr of MVPA, and 107.17–229.80 mins/hr of TPA when Trost et al.  = −.02, p = .98), LPA , MVPA , and TPA  did not significantly differ based on sex.While males accumulated less sedentary time and more MVPA and TPA (but not LPA) than their female counterparts, independent samples t-points . Likewist-points , 37; sedt[36] = 3.07, p = .004). When comparing weekdays to weekend days, it was found that toddlers’ rates of sedentary time , LPA , MVPA , and TPA  were statistically significantly higher during the week than on the weekends using Trost et al. cut-points [t[39] = 14.80, p <.001), LPA , MVPA , and TPA  using CHMS  months) and Johansson and colleagues’ [SD = 0.1] years) research participated in slightly higher levels of MVPA; 47 and 84 mins/day (measured via ActiGraph accelerometers), respectively.The low levels of MVPA among participating toddlers were similar to Gubbels et al.’s   devi Actical  are the  Actical  cross-se Actical , 37 to tet al.’s [et al.’s [et al.’s [et al.’s [et al.’s [Comparable to Gubbel et al.’s , Fees etet al.’s , Hnatiuket al.’s , and Johet al.’s  work, buet al.’s , levels et al.’s ). This fet al. and CHMS cut-points, respectively). Given the many negative health outcomes associated with sedentary behaviors [et al. [et al. [Participants from this study were found to engage in high levels of sedentary time . Despite this, the majority of participants had adequate wear time to be included in all analyses. Future research with toddlers may also consider defining non-wear time as 20 min of consecutive zeros (rather than 60 min) as it may be more reasonable to consider this age group remaining still for 20 min (rather than 60 min). Although efforts were made to achieve a geographically-representative sample, the generalizability of these results may be limited by the small sample size used. This is the first study to apply the Trost and colleagues  cut-poinet al. [The findings from this work highlight the challenge of accurately interpreting toddlers’ levels of physical activity an sedentary time, which consequently makes comparisons to national guidelines challenging. In comparison to the CHMS cut-points , 37, it et al.  produce


AbstractDicronocephalus are a very interesting group with a unique appearance and distinct sexual dimorphism. Only one species among them, Dicronocephalusadamsi, has been known in the Korean fauna. This species is recognized as having a wide distribution from Tibet to Korean Peninsula and is currently represented by two subspecies that have separated geographical ranges. The phylogenetic relationships of Dicronocephalusadamsi were still unclear. The phylogeny of Dicronocephalus is reconstructed with a phylogenetic study of five species including four subspecies based on a molecular approach using mitochondrial COI and 16S rRNA genes. Our results are compared with the results obtained by previous authors based on morphological characters. They show that the tested taxa are divided into two major clades. Clade A consists of two species  and Clade B includes the others . This result generally supports Kurosawa’s proposal except that Dicronocephalusdabryi and Dicranocephalusuenoi are newly recognized as members of a monophyletic group. We propose that Dicronocephalusadamsidrumonti is a junior subjective synonym of Dicronocephalusadamsiadamsi. These results show that three members of the Dicranocephaluswallichii group should be treated as species rather than subspecies. However, further research including analyses of different genetic markers is needed to reconfirm our results.The seven species belonging to the genus PageBreak Dicronocephalus Hope, 1831 is a group of medium- to large-sized beetles with a unique appearance among Cetoniinae representatives. The members of the genus show distinct sexual dimorphism such as antler-like clypeal horns and prolonged tarsomeres in males ; Dicranocephalusshimomurai Kurosawa, 1986; Dicranocephalusuenoiuenoi Kurosawa, 1968; Dicronocephalusuenoikatoi Kurosawa, 1968; Dicranocephalusbieti Pouillaude, 1914; Dicronocephaluswallichiiwallichii Hope, 1831; Dicronocephaluswallichiibourgoini Pouillaude, 1914; Dicronocephaluswallichiibowringi Pascoe, 1863; Dicranocephalusyuiyui Kurosawa, 1968; and Dicranocephalusyuicheni Kurosawa, 1986 ; 2) the wallichii species-group ; and 3) the Dicranocephalusuenoi species-group . However, he did not explain the phylogenetic relationships between these species.Dicronocephalus, only Dicronocephalusadamsi is found in the Korean fauna. This species was described from Korea, but it has been known to have a wide range across Korea, China, Tibet, and Vietnam. The range of this species is divided by a wide geographical gap between Liaoning and Shanxi provinces of China  were originally described as valid species  and the lack of phylogenetic analysis of the genus. To resolve these questions, phylogenetic analysis was performed for the genus using cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) mitochondrial gene sequences as well as examination of their morphological diagnostic characters.During a review of the genus Dicronocephalus belonging to five species and seven subspecies from four countries were obtained , Jeonju, Korea.Fifty specimens of ned Fig. , Table 1 program . Based o program , diagnosPageBreakPageBreakPageBreakPageBreakPageBreakc oxidase subunit I gene (COI) and 16S ribosomal RNA gene (16S rRNA) using Accupower PCR PreMix . The universal primer set LCO1490/HCO2198  after the product yield was monitored by 0.7% agarose gel electrophoresis. DNA sequencing was performed using an automated DNA sequencer  with the same primers used for PCR. All sequences (excepting a 198 bp fragment of COI in no. 7282) are available from GenBank under accession numbers KM390855–KM390903 for COI and KM390809–KM390854 for 16S rRNA (Table Genomic DNA (gDNA) was extracted from middle legs removed from dried specimens of all species and accomplished using a QIAamp DNA Mini Kit  in accordance with the manufacturer’s instructions. Polymerase Chain Reaction (PCR) was performed in order to amplify the cytochrome /HCO2198  for ampl/HCO2198 . We applateridae , 2010. PCOI, 520 bp fragment of 16S rRNA sequences, and the concatenated COI and 16S rRNA sequences. The data sets were aligned using ClustalW in MEGA 5.2  events, had 144 (21.9%) variable sites (Vs). Of these, 140 (21.3%) were parsimoniously informative sites (PIs). The data set of 16S rRNA, with indel events at three sites, consisted of 43 (8.3%) Vs, of which 41 (7.9%) were PIs. There was about 2.6 times more variability and the level of PIs was about 2.7 times greater in COI than in that in 16S rRNA.The data set of Dicronocephalusadamsi formed one cluster. Their terminal nodes were well supported, but the values of ML and BI were very low in Dicranocephalusyuiyui (<50% in ML and 53% in BI) and Dicronocephaluswallichiibowringi (<50% in ML and 56% in BI).Phylogenetic inferences based on three analyses  reconstructed the same topologies for COI . The two subspecies of Dicronocephalusadamsi did not separate into two distinct subgroups. The genetic divergences between the two subspecies were relatively low (0–1.7%); moreover, Dicronocephalusadamsidrumonti shared haplotypes with Dicronocephalusadamsiadamsi from Korea and China. Dicranocephalusyuiyui was sister to Dicronocephalusadamsi with distinct inter-specific divergences (5.6%–7.3%).Clade A is composed of PageBreakDicronocephalusdabryi, Dicronocephalusuenoikatoi, and three subspecies of Dicranocephaluswallichii with strong bootstrap supports by ML and BI, but relatively low support (56%–62%) by MP. Among the members of Clade B, Dicronocephalusdabryi and Dicronocephalusuenoikatoi formed a monophyletic group with very strong supporting values in all analyses and with distinct inter-specific divergences (5.6%–8.9%). The intra-specific divergences of these two species  were explicitly lower than their inter-specific values. The three subspecies of Dicranocephaluswallichii were clustered as a monophyletic group and clearly subdivided. Dicronocephaluswallichiibowringi diverged early from an ancestor, and then Dicronocephaluswallichiiwallichii and Dicronocephaluswallichiibourgoini underwent subsequent separation with strong bootstrap supports by ML (83%) and BI (99%); however, despite low divergences within each subspecies ranging from 0.3%–0.8%, the genetic divergences between these subspecies were unexpectedly variable ranging from 2.7%–8.1%. Genetic divergences were larger between Dicronocephaluswallichiibowringi and both Dicronocephaluswallichiiwallichii (4.3%–5.0%) and Dicronocephaluswallichiibourgoini (4.8%–8.1%), than those between Dicronocephaluswallichiiwallichii and Dicronocephaluswallichiibourgoini (2.7%–5.7%).Clade B is composed of Dicranocephalusyuiyui and paraphyly in Dicronocephaluswallichiibowringi with respect to Dicronocephaluswallichiiwallichii.ML, BI, and MP analyses of 16S rRNA resulted in considerably similar topologies to those of COI .The intra-specific pairwise distances of Dicronocephalusadamsi was clustered as a sister to Dicranocephalusyuiyui in Clade A with strong bootstrap support (>90%), while the remaining taxa were clustered into Clade B with relatively low supporting values (>76%) in BI and MP. The monophyly of Dicronocephalusadamsi, Dicronocephalusuenoikatoi, Dicronocephaluswallichiiwallichii, and Dicronocephaluswallichiibourgoini was well supported by bootstrap analyses (>84%). In contrast, in all analyses a polytomy was found in Dicranocephalusyuiyui and ML and BI showed paraphyly of Dicronocephaluswallichiibowringi. We showed that these phenomena were caused by few parsimony-informative nucleotide variations in conserved regions. A comparison of each of those sequences, showed that Dicronocephalusyuiyui has different substitutions at 326 nucleotide position. Two samples (7290 and 7291) have “C”, while one sample (7292) has “T”. On the other hand, Dicronocephaluswallichiibowringi has a substitution occurred in 196 nucleotide position. The 7693 sample has “G”, while the other samples  and two samples (7274 and 7275) of Dicranocephaluswallichii have “A” at this site  and moderate value in ML (74%), but not in MP  by having no angular projection at the base of the anterior edge of the clypeus  tra Fig. . Howeverens Fig. . Also thhii Fig.  was sinuini Fig. , and didies Fig. . We consies Fig.  to distiDicronocephalusadamsiadamsi and Dicronocephalusadamsidrumonti. Among them, we found four characters, namely body size, general body shape, longitudinal bands on the pronotum, and the shape of the triangular umbone of the elytra, to be ambiguous. He also illustrated the metasternal process and the parameres and explained in the key to subspecies that the ridge of the metasternal process does not reach the plate, and the process is weakly raised and more rounded anteriorly in Dicronocephalusadamsidrumonti. Also, the parameres of Dicronocephalusadamsidrumonti are shorter and with more acute lateral angles than of Dicronocephalusadamsiadamsi. However, we found that these characters were variable in the specimens from the two geographically isolated populations .ons Fig. . For exanti Fig.  is similrea Fig. , and anoina Fig.  resembleina Fig. . We did PageBreakDicronocephalus includes two major lineages, one with Dicronocephalusadamsi and Dicranocephalusyuiyui and another with Dicronocephalusdabryi, Dicronocephalusuenoikatoi, Dicronocephaluswallichiibowringi, Dicronocephaluswallichiiwallichii, and Dicronocephaluswallichiibourgoini  of COI between Dicronocephaluswallichiibowringi + Dicronocephaluswallichiiwallichii and Dicronocephaluswallichiibowringi + Dicronocephaluswallichiibourgoini. Dicronocephaluswallichiiwallichii + Dicronocephaluswallichiibourgoini were slightly lower than the average inter-specific distances of Dicronocephalusadamsi + Dicranocephalusyuiyui (6.2%) and Dicronocephalusdabryi + Dicronocephalusuenoikatoi (6.9%)  the distance between Dicronocephalusadamsi and Dicranocephalusyuiyui (0.8%–1.2%)  Table . Our phy cluster  with eacss Table . Howeveraaurata . There i


Objective.To illustrate the difference in cold/hot natural properties and therapeutic effect of coptis-evodia herb couples by using cold/hot plate differentiating technology and microcalorimetry combined with material basis analysis in vivo and in vitro. It showed that animal retention ratio in hot pad significantly decreased along with the decrease in coptis proportion in coptis-evodia herb couples. In addition, Zuojin wan markedly reduced the retention ratio of gastritis mice in the hot pad, while Fanzuojin wan displayed an opposite result. Further, Mg2+-ATPase, Ca2+-ATPase, and T-AOC activity significantly weakened in coptis-treated group in the livers of the mice. In the gastric cells from the gastritis mice, Fanzuojin wan remarkably increased calorific value for growth and metabolism, while Zuojin wan significantly reduced the calorigenic effect. It suggested that the changes in the major chemical compositions  were the material base-induced transformation between “cold” and “hot” syndromes. The material basis which affected the transformation between “cold” and “hot” syndromes might be X2, X3, X4, X8, epiberberine hydrochloride, jatrorrhizine hydrochloride, coptisine sulphate, palmatine hydrochloride, and berberine hydrochloride. The CHPD combined with microcalorimetry technology is a good method to determine the differences in the “cold” and “hot” natural properties of coptis-evodia herb couples. The “cold” (Han) or “hot” (Re) property of traditional Chinese medicine is determined by its therapeutic effect on “cold” or “hot” syndrome which involves physiological, biochemical, metabolic, and pathological changes , 2. Codonopsis, and Astragalus cured the “cold” symptom. Some studies suggested that luteinizing hormone, thyroid-stimulating hormone, and adrenocorticotropic hormone levels significantly elevated in the “cold” animal model, while the “hot” drugs attenuated the increased hormone levels .–16. coptThe mice were divided into a vehicle group, a “cold” model group, a “cold” model plus Zuojin wan group (5.0 g/kg), a “cold” model plus Ganlu san group (5.0 g/kg), a “cold” model plus Zhuyu wan (5.0 g/kg) group, a “cold” model plus Fanzuojin wan (5.0 g/kg) group, a “hot” model group, a “hot” model plus Zuojin wan group (5.0 g/kg), a “hot” model plus Ganlu san group (5.0 g/kg), a “hot” model plus Zhuyu wan (5.0 g/kg) group, and a “hot” model plus Fanzuojin wan (5.0 g/kg) group.To establish gastric “cold” symptom, the animals were given cold water (4°C) for three days and once a day. After fasting for 24 h, the mice were administrated with 4°C NaOH . Then they received various treatments for 7 days and once a day. To copy gastric “hot” symptom, the animals were administrated with 10% ethanol pepper solution (20 mL/kg) for 3 days and once a day. 10% pepper-ethanol solution was prepared as follows. Pepper oil was purchased from Tashuifang Co., Ltd. 3 mL of pepper oil was then dissolved by 10% ethanol solution to reach a total volume of 30 mL. After that, the animals received various treatments for 7 days and once a day.During the experiment, the body, food intake, water intake, and oxygen consumption of the mice were recorded for 7 days successively.+-K+, Mg2+, Ca2+ATPase, T-AOC, and SOD are in accordance with the instructions of the manufacturers by using ultraviolet spectrophotometer.Prepared 10% liver homogenates and performed biochemical assay of NaThe mice model with gastric “cold” or “hot” symptom was established as described previously. Briefly, the mice were sacrificed by cervical dislocation. Then the peritoneal cavity was open and the stomach was removed and placed in cold normal saline. The stomach was cut off along with the greater curvature. Food debris and blood were washed from the stomach. After that, the cells passing a sheet of cell sieve were cultured in prepared Dulbecco's modified eagle's medium (DMEM).Pm) is calculated in accordance with the following formula: Pt = P0ek(t−t0) .Under aseptic conditions, DMEM containing gastric cells (appropriate gastric cells from 2 mice weighing 18–22 g) were added to each ampule and then Zuojin wan and its similar formulae were added, respectively. Then the ampule was sealed and placed in a microcalorimetry instrument at a constant temperature of 37°C. The thermogram was not recorded until the curve returns to baseline. Maximum power output (μm)  was used for this part experiment. The column temperature was set at 22 ± 0.5°C. The mobile phase was 0.05% phosphoric acid/water (v/v)-acetonitrile. The measurement wave was 270 nm. The injection volume was 1 μL. Number of theoretical plates was more than 3000 according to berberine hydrochloride.ACQUITY UPLC BEH C18 column  data were present in Pm was gradually increased with the increase in evodia proportion. The Pm in FZJ-treated normal mouse gastric cell was higher than that in ZJW-treated cell.The thermogenic curve was shown in Pm) data were present in Pm in “cold” symptom mouse gastric cells, whereas Zuojin wan decreased Pm in “hot” symptom mouse gastric cells.The thermogenic curve was shown in  coptis-evodia herb couples, we compared the fingerprints and confirmed 23 specific fingerprint peaks. Among them, the area of 14 peaks was about 90% of the total area. Thus, these 14 peaks were confirmed to be specific peaks (According to the information provided by the HPLC fingerprints ofic peaks .μL of coptis-evodia herb couples solutions was injected to record the chromatograms (For the area of berberine hydrochloride peak (wave 20#) was largest in the total area (10% or more) with the highest peak height and relative stability, it was selected for the control wave. Under the above chromatographic conditions, 1 atograms .After that, we used external standard method to calculate alkaloids dissolution rate per unit area of a single herb in berberine, evodia, and Zuojin wan and its similar formulae .Among four similar formulae, the resemblance of Zuojin wan, Ganlu san, Zhuyu wan, and Fanzuojin wan to control fingerprint was gradually decreased, suggesting differences in chemical compositions among the four formulae. coptis-evodia herb couples by biothermodynamics method. Biothermodynamics is a science focusing on energy transfer and thermal variations in metabolic process of life systems [To the best of our knowledge, it is first time for us to investigate the “cold” and “hot” properties of systems , 19. ItsEnergy metabolism in a living body system will change in the presence of traditional Chinese medicine (TCM). Thus, it is necessary to obtain the changes in thermodynamic parameters to reflect the differences in biological activity of various reagents measured.Microcalorimetry (MCM) is an important method for biothermodynamics. Currently, it is becoming a key approach to studying body metabolism characteristics and rules, preliminary activity screening of drugs, drug interactions, and taxonomic identification –23. In t coptis-evodia herb couples for the biothermodynamic assay. Coptis-evodia herb couples are consisted of berberine and evodia. The therapeutic effect is completely different if the proportion changes. Secondly, the active ingredients  are identified. Thirdly, the two herbs berberine and evodia have distinct “cold” and “hot” properties. Thus, this formula is typical and representative in some degree.In this study, we selected coptis-evodia herb couples. In the mice with gastric “cold” symptom, Ganlu san, Zhuyu wan, and Fanzuojin wan significantly reduced the retention ratio in hot pad, while in the gastric “hot” model Zuojin wan markedly increased the animal retention ratio in hot pad compared with the model group. Meanwhile, related biochemical parameters changed in the normal, “cold” symptom, and “hot” symptom mice. These above results reflected differences in “cold” and “hot” properties of Zuojin wan and its similar formulae.In the temperature tropism experiment, we found that animal retention ratio in hot pad decreased along with the decrease in coptis proportion inIn vitro study revealed thermogenic changes in coptis-evodia herb couples. It showed that coptis-evodia herb couples had different effects on the growth and metabolism of the gastric cells. Zuojin wan reduced the heat production in the gastric cells from the mice with gastric “hot” symptom, while Fanzuojin wan increased the heat production in the gastric “cold” symptom cells. From the principal component analysis, we could draw that the difference in the formulae was determined by thermodynamic parameters like R and Pm. coptis-evodia herb couples were X2, X3, X4, X8, epiberberine hydrochloride, jatrorrhizine hydrochloride, coptisine sulphate, palmatine hydrochloride, and berberine hydrochloride. Further, changes in dissolution rate might be involved in them. These might be the material basis for the transfer of “cold” and “hot” properties in coptis-evodia herb couples.Principal component analysis by UPLC showed that the composition differences in coptis-evodia herb couples. Further, UPLC is efficient to confirm the material basis.In summary, the CHPD combined with microcalorimetry technology is a good method to determine the differences in the “cold” and “hot” natural properties of


Forty-four young adults  each participated in four, separate, 30-minute exercise conditions on a treadmill in a random order. During each condition, the treadmill speed display was covered and grade was fixed at zero. However, participants were able to alter treadmill speed as desired. Throughout the texting and talking conditions, research personnel used a pre-determined script to simulate cell phone conversations. During the music condition, participants used their cell phone to listen to music of their choice. Finally, participants completed a control condition with no cell phone access. For each condition, average treadmill speed, heart rate and liking  were assessed. Treadmill speed (3.4 ± 1.3 miles∙hour-1), heart rate (122.3 ± 24.3 beats∙min-1) and liking (7.5 ± 1.5 cm) in the music condition were significantly (p ≤ 0.014) greater than all other conditions. Treadmill speed in the control condition (3.1 ± 1.2 miles∙hour-1) was significantly (p = 0.04) greater than both texting and talking (2.8 ± 1.1 miles∙hour-1 each). Heart rate during the control condition (115.4 ± 22.8 beats∙min-1) was significantly (p = 0.04) greater than texting (109.9 ± 16.4 beats∙min-1) but not talking (112.6 ± 16.1 beats∙min-1). Finally, liking during the talking condition (5.4 ± 2.2 cm) was greater (p = 0.05) than the control (4.3 ± 2.2 cm) but not the texting (5.1 ± 2.2 cm) conditions. In conclusion, using a cell phone for listening to music can increase the intensity (speed and heart rate) and liking of a bout of treadmill exercise. However, other common cell phone uses  can interfere with treadmill exercise and reduce intensity.This study used a within-subjects design to assess the effect of three common cellular telephone (cell phone) functions ( According to the Pew Research Center 2013), 91% of American adults and 97% of young adults (age 18–29) own cellular telephones (cell phones). Indeed, the device is ubiquitous and its popularity is increasing along with its functionality. In addition to calling, most cell phones now provide many of the same services as an internet-connected computer. Currently, most modern cell phones allow users to access the internet, engage in diverse forms of social networking including text messaging, play video games, watch streaming video, and employ a wide array of specially designed software applications (apps). Because of the device’s inherent portability, users can now engage in all of these activities nearly anywhere and anytime. To illustrate, recent research has found that the cell phone is commonly used during work, academic pursuits such as classroom learning and studying, while watching movies and sporting events, during meals, and while going to the bathroom ..2 peak mMaybe decrease. I’m not sure. I mean, sometimes I’ll be walking and texting on the treadmill, but it’s not like I’m stopping to send a text, I’m still walking. It is the same on the elliptical if I have to send a text. I’ve kind of gotten used to, like, balancing so that I can text if I need to. Or, if it’s too long, like with my mom, then I’ll call her and I’ll still be going on the treadmill, I might not be going as fast but I’m still going.It decreases physical activity because, for instance, the other day, one of my friends called me during my work out, and like, I haven’t talked to her in a while and I had to tell her a lot of stuff. So it kind of distracted me from my work out.In an effort to explain this negative relationship between cell phone use and fitness, all participants were interviewed about their daily physical activities, sedentary behaviors, and cell phone use habits. Participants’ responses revealed two potential explanations for why cell phone use was negatively associated with cardiorespiratory fitness. First, participants tended to describe cell phone use as a sedentary behavior. Furthermore, high frequency cell phone users, relative to low frequency users, reported engaging in a greater number of sedentary behaviors . Therefore, cell phone use may be a marker for a wider array of sedentary behaviors which research suggests typically occurs at the expense of physical activity –26. SecoAs these statements suggest, cell phone use during a bout of planned exercise may cause a distraction and potentially reduce exercise intensity. If so, this could limit the ability of an exercise bout to enhance fitness and provide a partial explanation for the negative relationship between cell phone use and cardiorespiratory fitness described above.texting), talking on a cell phone , using a cell phone to listen to music (music), and no cell phone use (control). It was hypothesized that using a cell phone to listen to music would result in the greatest average speed and liking of all conditions. This was hypothesized as listening to music has been previously shown to increase exercise intensity and enjoyment ..2  repeated-measures ANOVAs were utilized to examine differences in average treadmill speed, heart rate and liking. Post-hoc analyses for all significant main effects were completed using t-tests with the Benjamini and Hochberg False Discovery Rate correction . H. H49]. H


N (eigen)states to later execute nonclassical “subroutines” involving unitary phase shifts of measured states and to produce root-rate or quadratic gain in the algorithmic time (O(N1/2)) needed to find some “target” solution m. Akin to this fast technological search algorithm, single eukaryotic cells, such as differentiated neurons, perform natural quadratic speed-up in the search for appropriate store-operated Ca2+ response regulation of, among other processes, protein and lipid biosynthesis, cell energetics, stress responses, cell fate and death, synaptic plasticity, and immunoprotection. Such speed-up in cellular decision making results from spatiotemporal dynamics of networked intracellular Ca2+-induced Ca2+ release and the search  velocity of Ca2+ wave propagation. As chemical processes, such as the duration of Ca2+ mobilization, become rate-limiting over interstore distances, Ca2+ waves quadratically decrease interstore-travel time from slow saltatory to fast continuous gradients proportional to the square-root of the classical Ca2+ diffusion coefficient, D1/2, matching the computing efficiency of Grover's quantum algorithm. In this Hypothesis and Theory article, I elaborate on these traits using a fire-diffuse-fire model of store-operated cytosolic Ca2+ signaling valid for glutamatergic neurons. Salient model features corresponding to Grover's quantum algorithm are parameterized to meet requirements for the Oracle Hadamard transform and Grover's iteration. A neuronal version of Grover's quantum algorithm figures to benefit signal coincidence detection and integration, bidirectional synaptic plasticity, and other vital cell functions by rapidly selecting, ordering, and/or counting optional response regulation choices.Grover's quantum (search) algorithm exploits principles of quantum information theory and computation to surpass the strong Church–Turing limit governing classical computers. The algorithm initializes a search field into superposed  Modern analog, digital, and quantum descriptions of phylogenetically diverse cell functions /21/2, |ψ〉01 = (|01〉 + |10〉)/21/2, |ψ〉10 = (|00〉 − |11〉)/21/2, |ψ〉11 = (|01〉 − |10〉)/21/2. Quantum entanglement between the two qubits of a state permits secure cryptographic protocols, such as quantum teleportation, and compressed information encoding and storage, such as superdense coding and quantum memory.Named respectively after John Bell and Albert Einstein, Nathan Rosen, and Boris Podolsky, these four orthonormal quantum states, spanning the two-qubit state space, form an essential computational basis for many fundamentally useful applications of quantum information theory. The states or pairs are: |ψ〉Church–Turing LimitUpper computational bound of efficiency for classical computers independently determined by Alonzo Church and Alan Turing. The Church–Turing limit emerges from the Church–Turing thesis or conjecture, which equates functions computable on a Turing machine with those computable by an algorithm. The strong limit is believed to subtend allowable complexity of computations performed by quantum computers, as captured in David Deutsch's rigorous conceptualization of universal quantum computers.Eigenstates, Eigenvalues, and Eigenspacesv〉, is a nonzero state in a state space operated on by a linear function L, so that L|v〉 = ω|v〉 with complex eigenvalue ω. Eigenstates and eigenvalues are ascertained from the characteristic function, c(λ) = det|L − λI|, where det is the determinant function. The eigenspace of ω, a subset of the state space on which L acts, is the set of eigenstates with the eigenvalue ω.An eigenstate or eigenvector, |Entropic Uncertainty PrincipleH(Q) + H(R) ≥ 2 log2[1/f], where H(Q) and H(R) are the Shannon entropies of respective spectrally decomposed measurements Q and R of quantum state |ψ〉 with probability distributions p(q) and p(r) and maximum fidelity or inner product f = maxq,r|〈q|r〉| between eigenvectors |q〉 and |r〉. As with Heisenberg's version involving the standard deviation of observables, the entropic uncertainty principle places an upper bound on attainable knowledge about quantum systems.Information or entropy principle first derived by Iwo Bialynicki-Birula and Jerzy Mycielski and by David Deutsch from Werner Heisenberg's uncertainty principle. The principle is formally expressed in the strong condition as Hadamard Transformationn times to n input qubits to initialize the data register of a quantum information system into superposition, so that multiple values of an index integer x can be simultaneously analyzed by a single function f. Common notation for the Hadamard transform is Hn⊗.An important step for quantum parallel computing, the Hadamard transformation applies the Hadamard gate Hermitian OperatorV. For a Hermitian operator L, there is a unique linear operator L† acting on V, so that an adjoint or conjugate vector |v〉† exists for every |v〉 in V. That is, L = L†, |v〉 = |v〉†, and LL† = L†L when L is normal.A linear operator, also known as a self-adjoint operator, imposed on a vector space Landauer's PrinciplekBTln2 of energy, where kB is Boltzmann's constant and T is ambient temperature in degrees Kelvin, must be transferred to the environment for erasure of one bit of information. The corresponding entropy rendering of Landauer's principle may be written without variable T.Principle postulated by Rolf Landauer to define the relationship between energy and computation. Landauer improved earlier ideas of Leó Szilárd, John von Neumann, and other theorists to concretize the minimum amount of energy/information consumed during irreversible operations. Although Landauer's principle applies generally to energy/information dissipated as heat from work, it is usually placed into the context of memory erasure. For example, for a biological or technological computer with exhausted finite memory capacity, it is necessary to erase information for further computations. Landauer's principle states at least Quantum Gates and CircuitsLogic gates and circuits constructed to perform operations based on quantum mechanics and information theory. Popular unitary single-qubit gates in matrix form include, for instance, the quantum Identity Quantum Mechanical PropertiesStatistical wave-particle features of quantum mechanical systems, such as quantum superposition, entanglement, interference, and tunneling, not observed for deterministic classical Newtonian physical or Shannon informational systems. Quantum superposition describes the linear combination or addition of state or vector solutions to Schrödinger's wave equation (or other quantum state equation permutations). When superposed states are indistinguishable, they are said to be entangled. Quantum interference is the disruption of state or vector  spacetime trajectories. And quantum tunneling is the transition of one state to another without surmounting classical energy barriers required for transformation in classical physics. Such probabilistic effects are useful for development of information technologies and additional purposes.Quantum NetworksT , which dictates system behavior, is often replaced with a computational annealing parameter, such as space, time, or the “critical tunneling field strength.” Quantum networks may display the network analogs of Bose-Einstein condensation and the Pauli exclusion principle depending on statistical parameters. In addition, rate of state transitions or computational decisions in a quantum network follows nonlinear first-order Arrhenius kinetics associated with quantum tunneling, also making it a computational or network analog of the physical phenomenon. These properties of quantum networks strongly compare with networks capable of certain associative forms of learning and memory, such as Hebbian-type learning dependent upon mutually weighted nodal or synaptic strengths.Technological and biological networks whose connectivity tends to obey either Bose-Einstein or Fermi-Dirac quantum statistics rather than classical Maxwell-Boltzmann statistics. The strength of each network node is described as a separate fitness or energy level and nodal links take on the identity of particle states functioning under associative-like preferential attachment rules. In such cases, control parameter Schrödinger Wave FunctionA solution to Erwin Schrödinger's wave equation used to describe the statistical nature of eigenstates that exhibit wave-particle duality. The wave function gives the probable energy of a wave in a 4D spacetime interval. Each wave is associated with a respective wave number related to wavelength or an energy level known as a quantum. Because the wave equation is additive, the distribution of wave numbers or energy levels occurring over the spacetime interval may be combined into a mixed or superposed wave packet. Pure states are represented by a single wave number or quantum.Unitary OperatorA mathematical operation performed on a state space that satisfies the identity operator by being normal, having a spectral decomposition , and preserving the inner product of two vectors, such as a unitary phase shift of 180° from state or vector |0〉 to |1〉 or |1〉 to |0〉 residing on the unit circle or the unit 3D sphere—the Bloch sphere.Universal Turing MachineAn idealized computational machine with unbounded memory belonging to a class of devices introduced by Alan Turing, the universal Turing machine is one of the most powerful classical computers conceived and serves as model to examine issues of computational complexity. All Turing machines are prototype modern programmable computers capable of executing algorithmic routines of different complexity. They consist of four essential components: (1) a microprocessor-like finite state control that coordinates computing action, (2) a program, (3) a memory tape, and (4) a read-write tape head that points to the tape location currently accessible for read-write operations. Unlike other Turing machines, the only variable component maintained by the universal Turing machine is the initial contents of the tape. Such a configuration is deceivably powerful and enables the universal Turing machine to emulate or simulate the processing of all other Turing machines, even more powerful ones.seem to appear in every major aspect of cell structure and function, from sensory transduction to gene expression to cellular metabolism to cell motility  compression and stretch receptors], ATP-dependent , voltage-gated , ligand-gated , and peptidergic porin  mechanisms  and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid receptors (AMPARs), and metabotropic glutamate receptors (GPCRs), critical for the induction and/or maintenance of certain forms of synaptic structural and transmission plasticity  help control endosome transport, membrane remodeling, and up- and downregulation of metabolic and catabolic processes. In classic scenarios of facilitated and depressed glutamatergic synaptic function, respectively known as long-term potentiation (LTP) and depression (LTD), extracellular Ca2+ enters the post-synaptic cell through activated NMDARs during the induction phase of plasticity. Ca2+ loading in dendritic spines often evokes either LTP or LTD in a concentration-dependent manner, with higher and lower levels of Ca2+ tending to respectively produce LTP and LTD  and IV, nitric oxide synthase, protein kinase C, tyrosine kinase Src, mitogen-activated protein kinase, and other molecular complexes that lead to enduring changes through post-synaptic CREB-dependent transcription and immediate early gene activation, post-synaptic receptor synthesis, transport, and distribution, pre- and post-synapse geometry, and pre-synaptic vesicular transport and docking at transmitter release zones -mediated CICRs assist in directing response regulation under physiological constraints of neuronal synaptic transmission and plasticity.Extensive varieties of functional Cas Clark, . The maj3R isoforms, all permeable to certain mono- and divalent cations, especially Ca2+, are expressed by animals -β and -γ, distribution and activation/inhibition properties of IP3Rs, uptake and storage of Ca2+ in the ER and other reservoirs, and influences over Ca2+ diffusion  sequester free cytosolic Ca2+ in the ER lumen, where it stays free or becomes attached to buffers. Estimates of total luminal Ca2+ concentrations are as high as 1 mM. The fraction of unbound luminal Ca2+ ranges from 100 to 700 μM  and high  concentrations /21/2 and |ψj〉 = (|0〉 − |1〉)/21/2. The state space of such a bipartite system is defined by the tensor product |ψi〉 ⊗ |ψj〉 = |ψ〉10 = (|00〉 − |11〉)/21/2, yielding, in the present case, the third Bell basis state or Einstein–Podolsky–Rosen pair. Composite quantum information systems, such as the four Bell basis states or Einstein–Podolsky–Rosen pairs, play significant roles in superdense coding, information encryption, error diagnosis and correction, and other aspects of quantum computation, including execution of quantum algorithms ]. The same search problem may be accelerated to O(N1/2) with Grover's quantum algorithm and its unique Grover's operator  = 1 or θ (x) = 0 when a solution m is or is not obtained, respectively. Given these constraints, Grover's operator consists of four distinct sequential procedures . Using a classical search algorithm to discover the shortest route  Grover,  /21/2 is the single Oracle qubit which assists in flipping or phase-shifting |x〉 only when θ(x) returns 1 as a result. A second Hadamard transformation is utilized following the Oracle call to place labeled qubits into superposition. From this state, a conditional phase shift, |x〉 → − (− 1)x)θ is the average concentration of calcium in directions perpendicular to the direction x of propagation, δ(ζ) is the δ function, H(ζ) is the Heavyside step function (and not the Hadamard transform), D is the classical Ca2+ diffusion coefficient, d is the (mean) distance between Ca2+ release sites, ti is the first time the ith Ca2+ release site reaches threshold value, σ is the total number of Ca2+ ions released per storage site, and τ is the period that receptors remain open to release Ca2+ in a single event. Notably, the evolution equation and parameters for this sort of model are general enough to fit conditions stipulating IP3R clusters or individual IP3Rs as Ca2+ release sites g−1Γ, where g−1 is an inverse function 1/2f−1Γ, where f−1 is an inverse function 1/2 with α = Γ1/2. The equations for wave velocity should not be misleading, as continuous waves are often faster than saltatory ones , large d , and extremely small or large Γ . The variable N represents the total number of search elements queried by Grover's quantum algorithm. Its value is neither quantum nor classical! Therefore, D needs to be neither quantum nor classical to effect Grover's quantum algorithm in a cellular system! That is, what makes Grover's quantum algorithm quantum in nature is its action on a search field, not necessarily the properties of the search field itself. In view that classical diffusion terms satisfy the quadratic improvements needed for Grover's quantum algorithm, it is interesting that search selectivity by such an algorithm in single cells may be enhanced, instead of being damped, by diffusion barriers sometimes causing unstable Ca2+ gradients. Anisotropic patterns of Ca2+ diffusion due to free cytosolic chaperons and buffers  quantum states needed to execute Grover's quantum algorithm, since each state embodies a long-range networked pattern of catalyst-linked receptor clusters  and, consequently, probably surpasses Wigner's mass-time uncertainty for periodic mechanical processes  d2, τ, and σ for the fire-diffuse-fire model make the diffusion coefficient variable. Sharper definitions of D may be obtained from the wave velocity equations for saltatory Ca2+ waves, v = (D/d)g−1Γ, and continuous Ca2+ waves, v = (D/τ)1/2f−1Γ. It follows that the respective diffusion coefficients for saltatory and continuous Ca2+ waves are:However, fluctuations in loading of cytosolic CaDSmin > 0 and DSmax ≪ DCmin, andwith DCmin ≫ 0, DSmax and DCmax ≪ ∞. Setting Dmax = 0 ≤ Dmax ≤ DCmax = {Dnj|j = 0, …, DCmax} and considering the one-to-one discrete mapping Dmax onto N, Dmax → N, M therefore transforms into the subset DC = DSmax ≪ M ≤ N = {im|m = DSmax ≪ m ≤ N − 1} of all correct solutions involving only intracluster continuous Ca2+ wave fronts. A major result from this interpretation is that, in order to arrive at a solution m = DC, quadratic and exponential speed-ups in respective algorithmic search time and wave velocity must coexist, with a maximum algorithmic search time of O(Dmax/DC)1/2 and wave velocity of vC = v2S! In addition, Equations 6 and 7 are especially useful for purposes of implementing Grover's quantum algorithm, since they are inversely proportional to dimensionless parameter Γ rather than β. As shown in Equation 3, reliance upon Γ permits Ca2+ diffusion coefficients to be indexed to physiologically pertinent ratios involving Ca2+ concentrations liberated by IP3R-dependent store operation and free cytosolic Ca2+ concentrations sensed by multiaffinity IP3R cytosolic Ca2+ binding sites. So that, index values of register entries i corresponding to Dmax and DC may be practically redefined by substituting Equation 5 into Equations 6 and 7, then solving for Γ for separate saltatory and continuous Ca2+ wave forms:with Smin > 0 and ΓSmax ≪ ΓCmin, andwith ΓCmin ≫ 0, ΓSmax and ΓCmax ≪ ∞. Therefore, index i = {in|n = 0, …, N − 1} = Γmax = {in|n = 0, …, ΓCmax} and solution subset index iM = {im|m = ΓCmin ≤ m ≤ ΓCmax}. Remember also from earlier reviewed content that Grover's quantum algorithm employs a function θ that accepts as input an integer x = i valued over the range 0 to N − 1. The function returns results θ(x) = 1 or θ(x) = 0 when a solution m is or is not acquired, respectively. In parameterizing the fire-diffuse-fire model for Grover's quantum algorithm, elements of the index set i yielding θ(x) = 1 readily signify the solution subset iM = {im|m = ΓCmin ≤ m ≤ ΓCmax} for intracluster continuous Ca2+ waves, whereas elements of the index set i yielding θ(x) = 0 signify the incorrect-solution or failure subset iF = {if|f = 0 ≤ f < ΓCmin} for all intracluster noncontinuous Ca2+ waves, including saltatory and possibly mixed Ca2+ wave fronts.with Γ3R molecular biology and function  and (|0〉 − |1〉)/21/2 is the superposed IP3R-Oracle qubit. The IP3R Oracle phase-shifts |x〉 only when θ(x) returns 1 as a result. Function θ(x), accordingly, may be thought of as the low- and high-affinity Ca2+ binding sites located at the cytosolic end of the IP3R emulating Grover's quantum algorithm  and high  concentrations. Low Ca2+ concentrations capable of inducing sustained Ca2+ waves bind to high-affinity binding sites, changing cation-pore permeability by repositioning the transmembrane gate and activating Ca2+ conductance. In contrast, very low or very high Ca2+ levels keep the IP3R in a closed state. Ca2+ binding sites operating as θ(x) return 1 upon ion-pore opening and otherwise return 0 as a result. Coincident with θ(x) action and all-or-none IP3R stimulation, the IP3R Oracle labels |x〉 by flipping the state from |1〉 to |0〉. Isolating the Oracle's operation to a particular IP3R molecular region and function presents some conceptual difficulties. Arguments could be made for cytoplasmic and ER-lumen channel sensors which detect the presence of cations ions and IP3R interactions with nucleotides, proteins, and other substrate. Regardless, as noted above, once an IP3R becomes active, respective high quantal Ca2+ release exceeding 10 μM near the opening of the same channel begins to cause reversible autoinhibition, presumably via low-affinity Ca2+ binding sites located proximal to IP3R cytoplasmic terminus. Inactivation may last longer than the open-channel period τ. Such an effect is tantamount to the |x〉 phase shift produced by the Oracle of Grover's quantum algorithm x)θ1/2 and wave velocity of vC = v2S. If these constraints are not met, algorithm-processing capabilities will approach a classical algorithm taking O(Dmax/DS) time. After only quick inspection, the velocity equations for saltatory, vS = (D/d)g−1Γ, and continuous waves, vC = (D/τ)1/2f−1Γ, may falsely imply to readers that a quantum-search result is impossible. Indeed, the arguments of Ponce-Dawson et al. 1/2 ≈ 69 μm/s) can exceed saltatory wave  velocity by greater than a power of 2. Despite being a simple example, these values underscore the plausibility of fast continuous Ca2+ waves displaying characteristics consistent with those predicted by the Grover's quantum-algorithm model. Importantly, the version of the fire-diffuse-fire model used in the present article assumes deterministic channel refractivity and instantaneous buffering for σ without specification of Ca2+ store re-uptake or extracellular extrusion  values for global  continuous and saltatory waves yield respective local (intracluster) continuous waves of β = 19000 with v ≈ 69 μm/s and β = 1500 with v ≈ 19 μm/s. The large values for β indicate conditions supporting saltatory waves at a global scale do not necessarily ensure presence of saltatory waves for intracluster dimensions. Thus, although the algorithm searches for and finds optimal solution m, the boundary between local and global waves needs to be better resolved through numerical examination of Γmax—the critical Grover's quantum-algorithm index of effective diffusion and solution m.A fundamental prediction of the above analytical treatment unaddressed by standard classical interpretations is that, for an individual IPn et al. , for insmax unsatisfactorily weakens the explanatory power of the current model in regard to channel gating kinetics and quantum molecular action underlying formation and interrogation of superposition states, such as |ψ〉. Resolution of state boundaries for the IP3R Grover's quantum-algorithm model therefore should be perfected by redefining Γmax and, naturally, constituent Γ to include buffering, re-uptake, and gating kinetics terms, as is done with more complex wave-evolution equations , with [Ca2+]ωti, [Ca2+]ωt0 = [Ca2+]T − [Ca2+]b. The role of molecular dynamics in channel state is evident in each Φ ([Ca2+]ER − [Ca2+]C), Φ2 = FU[Ca2+]2C/[Ca2+]2C + A2, and Φ3 = KB[Ca2+]C[B]C, where CER/C is the ratio of Ca2+ in ER and cytoplasmic volumes, FMO is maximum outward IP3R Ca2+ flux, FL is IP3R Ca2+ leak flux, FU is maximum ER Ca2+ uptake, pO is the probability of IP3R-channel opening, A is the ER-uptake-activation constant, KB is the Ca2+-buffer-binding constant, [Ca2+]ER and [Ca2+]CF are free ER and cytoplasmic Ca2+ concentrations, and [B]C is the free Ca2+ buffer concentration. While the complexity of these equations may be increased, addition of the open-channel-probability term,Variables ΦKCaI is the dissociation constant for IP3R channel inhibition, KIP31 and KIP32 are IP3 dissociation constants, and KCaA is the dissociation constant for IP3R channel activation, gives a much stronger framework for molecular considerations. For example, a consequence of the new Γ formulation is that the initialized index-register qubit becomes 3R binding subunits for each IP3R monomer (Mn) primed for channel activation and Ca2+ conductance. The first, second, and third vector columns of |100〉 represent the respective (saturated) occupied IP3-biding subunit , the unoccupied high-affinity Ca2+-binding subunit , and the unoccupied low-affinity Ca2+-binding subunit . When multiqubit-controlled function θ(x) = 1 = |110〉, the high-affinity subunit binds Ca2+, making the IP3R permeable to cations. The IP3R Grover's operator, also acting as a multiqubit operator through coligand channel gating kinetics, as earlier conjectured, then labels |x〉 by flipping the final two or all column values in conjunction with saturating- or subsaturating-IP3, Ca2+-dependent channel inactivation. The operators's rate and probability of flipping between 0 and 1 for each column and of finding target solution m is expectedly proportional to percent saturation of [Ca2+]C for high- and low-affinity Ca2+-binding subunits and, therefore, proportional to index ΓC, DC, and vC.where 3R-based Grover's quantum algorithm of CICR behavior and Ca2+-mediated cellular response regulation thus imparts greater comprehensiveness than possible with traditional single receptor, intracluster, and intercluster models. For instance, above quantum depictions of the IP3R conformational state vector |x〉 uniquely permits study of computationally efficient subcellular superdense coding  saturation on coligand-dependent IP3R allostery ! = 4 superposed combinations of: (1) initial activation-primed conformational patterns |100〉 for about 19 ms or less to finish θ (x)-labeling before IP3 dissociates from its IP3R binding subunit, (2) θ(x)-transformed conformational patterns |110〉 for about 600 ms or less to finish the Oracle call before Ca2+ dissociates from its high-affinity IP3R binding subunits, and (3) Oracle-transformed conformational patterns |101〉 or |001〉 for about 5 s or less to finish the algorithm's final phase shift before Ca2+ dissociates from its low-affinity IP3R binding subunits  relativistic quantum physicochemistry theory to aptly match IP3R protein structure and function with the workspaces and operators of Grover's quantum algorithm, and (2) fire-diffuse-fire or lattice-percolation mathematical treatments of intracluster IP3R activity and CICR dynamics to fully address aspects of reaction-diffusion stochasticity and cytosolic Ca2+ buffering 1/2 and wave velocity of vC = v2S to be realized. However, whether it is activated by stochastic blips of high Ca2+-conductance or deterministic cellular Ca2+ loading, a neuronal version of Grover's quantum algorithm, just like that proposed for other eukaryotic cells, figures to promote advantageous subcellular superdense coding  CICR models, agrees with expression of three-agent quantum teleportation over communication channels transmitting classical bits via the information content of Γ, a circumstance, as described in the above section, that exposes superdense coding and quantum molecular memory for IP3R systems. Limited evidence suggests digital representation of cellular processing is encoded, transmitted, and stored by free intracellular Ca2+ , large temporary elevation of Ca2+ microdomain concentrations following post-synaptic Ca2+ entry through activated NMDARs initiates widespread and differential response regulation in dendritic, somal, and axonal compartments. The amount and spread of inward Ca2+ current is too small to effect most transduction processes. Instead, NMDAR-mediated Ca2+ entry stimulates secondary Ca2+ release from intracellular stores. Information processed by NMDAR-dependent Ca2+ cascades is augmented by glutamate diffusion to extrasynaptic GPCRs responsible for intracellular IP3 generation. Compared to RyRs, IP3Rs tend to have greatest density in ER membranes located in the soma and dendritic shafts of neurons, where coligand IP3 may exert maximum influence  gene transcription and protein synthesis accompanying longer-term structural plasticity, chiefly that of LTP (cf. Fitzjohn and Collingridge, 2+ diffusion rates, stable continuous Ca2+ wave modes, and, consequently, activation of kinase and other messenger pathways via detectable free cytosolic Ca2+ concentrations and properties of IP3R channel conductance.Equally significant, the quantum computational value of a receptor-scale Grover's quantum algorithm also can be expected to contribute to surprising classical information processing over much longer intracellular distances and times common to global, multicompartmental Ca Plieth, , CaMKII  Plieth, , and oths Clark, . Unlike Trypanosoma parasites, that selfishly coopt neuron intracellular Ca2+ systems can use the algorithm to optimize timing and effectiveness of infection stages against barriers to invasion, pathogenesis, proliferation, and release. While most pathogens deploy well-timed Ca2+-dependent trophic and deleterious strategies, such as genomically encoded proteins and lipopolysacchrides, to exploit host-cell physiology, infected neurons are unusually susceptible to metabolic distress, apoptosis, and additional harmful effects leading to host cognitive impairments (Clark, 2+ concentrations are known to commence and accelerate synaptic loss and amyloid plaque disposition, manifest pathocytological characteristics of the disease. While the excitotoxic nature of Ca2+ dysregulation in Alzheimer's disease is well understood, the exact causes are not (Stutzmann and Mattson, 3R activation by presenilins, protein products of autosomal inherited mutated genes PS1 and PS2 linked to early onset Alzheimer's disease, may be a major contributing factor. The corresponding heightened IP3R-mediated Ca2+ mobilization means implementation of IP3R Grover's quantum algorithm drives diseased, injured, and/or aging neurons toward faster catastrophic failure than would be otherwise possible with classical response regulation. Hence, the delicate balance between beneficial and detrimental uses of a neuronal version of Grover's quantum algorithm demands thorough theoretical and empirical scrutiny of cellular conditions governing the algorithm's application in both healthy and diseased states.However, the appearance of a quantum-efficient search algorithm in the function of neurons or, for that matter, any other cell type need not necessarily guarantee evolutionary and/or ecological benefit for the cell that implements the algorithm and the host to which the cell belongs. Indeed, viral, bacterial, fungal, and protozoal infectious agents, including, among other pathogens, Human Immunodeficiency Virus type 1 and s Clark, . Similars Clark, . In the The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility  for assessing effects of varying residues at selected positions on proteins’ structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations’ effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits. There are several important considerations when designing randomized libraries of protein variants. The mutagenesis method used should produce the required kind and range of variability. It should also be sufficiently reliable to enable robust predictions of the libraries’ composition and completeness, which are required to optimize screening efforts. For oligonucleotide-directed mutagenesis, equations have been derived to predict how complete a given library is likely to be, and the size of a library required for a given probability of 100% coverage, or other selected threshold In practice, every method for generating protein variants has strengths, weaknesses and biases. Thus, ideally parameters describing the performance of mutagenesis methods should be empirically estimated and incorporated into the formulas to avoid underestimation of required screening efforts Frequently, the most important objective is to avoid missing rare “positive hits”. However, in some cases (such as the one described here) there may be numerous “positive hits”. Thus, retaining variability may be more important and this may have significant implications for mutation generation and screening strategies.8 proteins, is an active site formed by four variable loops extending from the conserved structure of the protein. A key structural determinant of substrate specificity in Zm-p60.1 – repeatedly confirmed in structural, docking and kinetic studies – is the F193–F200–W373–F461 cluster β-glucosidase Zm-p60.1 is an enzyme that was originally identified in maize, where it catalyzes the release of active cytokinin from glucoside transport and storage forms, thereby playing a key role in hormonal regulation Brassica napus, severely impairs catalytic activity Brassicaceae consensus cluster (F193A/F200K/W373K/F461L) leads to extensive structural alterations and almost complete abolition of enzymatic activity One of the findings of the cited studies is that W373 plays an important role in stabilizing the aglycone part of the substrate in the entrance of the active site In the presented study we further explored the effects of varying position W373, and unexpectedly found that numerous generated variants had enzymatic activity. This finding led to a detailed consideration of the optimal combination of rational and random strategies for constructing β-glucosidase Zm-p60.1 protein libraries for further structural-functional analyses.Published data Several codon randomization schemes NNK randomization is the most widely used procedure for saturation mutagenesis because it provides all amino acids and only one stop codon. Thus, it generates significantly smaller libraries for sequencing than NNN randomization. Theoretically, there will be 95% probability that an NNK-generated library includes all possible amino acid variants when it contains 171 clones.E. coli clones However, in our Zm-p60.1 engineering project we initially used NNM  codon randomization and functional screening of On the basis of our previous work we expected to find one or a few functional variants of the weakly catalyzing mutant W373K Theoretically, every codon has an equal chance of being present in any clone, resulting in a 30/32 probability of coding codons and 2/32 probability of stop codons at the varied position. However, amino acids in NNM randomization are coded by different numbers of codons, so the probabilities of occurrence at a varied position in the final library are: 1/32 for Cys, Asp, Glu, Phe, His, Lys, Asn, Gln and Tyr; 2/32 for Ala, Gly, Ile, Pro, Thr and Val; 3/32 for Leu, Arg and Ser; 0 for Met and Trp.21 shows that it is highly improbable that all of the positive clones we obtained represented a single variant, and even the negative clone could have had a stop codon at the varied site. Thus, the identification of new active (and inactive variants) of Zm-p60.1 seemed highly feasible. Furthermore, even with such a small set of variants useful predictions can be obtained of possible library compositions and likely relative proportions of positive hits due to new variants and random multiple occurrences of the same variant.A simple calculation of the combined probability even for the most frequent amino acids (3/32)Thanks to the small size of the library we sequenced all 21 clones. We identified 10 variants at the 373 position: Leu (five copies); Pro and Arg (each in three copies); Ser, Ala and Gly (each in two copies); His, Thr, Asp and Phe . Thus, even in this small sample there was a clear correlation between the prevalence of amino acids at the varied site and the numbers of corresponding codons used in the randomization procedure.The clone with no glucosidase activity proved to be a proline variant with an additional stop codon introduced at position 206 by an unknown error in the mutagenesis procedure.The results show that there are at least 10 active variants of the β-glucosidase, and raise the question whether there are other active variants at position W373 or all variants are active. The simplest procedure for finding another active variant would have been to broaden the screening and identify positive clones, but this procedure would have been more likely to find variants that had already been identified. When screening libraries with hundreds or thousands of variants and only a few clones have been identified (a much more common scenario) this is not an issue. However, when there are high fractions of positive clones and high numbers of identified mutants, attempting to complete the library by extending the random mutagenesis and screening could be unnecessarily expensive. To demonstrate this problem, we determined theoretical average numbers of clones needed to find new variants and complete the library in computer simulations .The simulations show that there is a sharp increase in numbers of clones needed for the last quarter of library construction, and increases in the variability of clone numbers, using all three codon randomization procedures . These results are reflected in the costs of library creation by saturation mutagenesis. Because of the high probability of finding new variants in the beginning of the screening we propose that an ideal strategy should combine random mutagenesis with site-directed mutagenesis (SDM) to generate missing variants .Thus, in the mutagenesis of Zm-p60.1 we initially used NNM codon randomization and after finding 10 variants completed the library by using SDM to obtain missing variants.E. coli expression strain, all 20 variants at position 373 were expressed, purified to homogeneity and tested for catalytic effect. Our results show that in surprising contrast to the high conservation of Trp it is possible to construct variants that have residual catalytic activity with all amino acid residues at this position. The residual hydrolytic activities of purified enzymes towards the natural substrate trans-zeatin-O-β-D-glucopyranoside (tZOG) and artificial substrate p-nitrophenyl β-D-glucopyranoside (pNPG) were also measured. The tZOG hydrolytic rate constant was low for all mutants, but slightly higher for W373H W373F, W373M, W373Y mutants than for the others. W373H also had slightly higher activity than the other variants towards pNPG  in relation to pH. We found no significant difference between the W373K mutant and WT pH profiles, but the pH optimum of the W373H mutant shifted from 5.5 to 6.0 . The presented study illustrates the efficiency of NNM codon degeneration and finalization by SDM for saturation mutagenesis of β-glucosidase Zm-p60.1.E. coli (or other) expression strains, variations in expression levels of protein variants and mixing of clones when picking colonies. Given all these considerations, a combination of randomized codon-based saturation mutagenesis and standard SDM could be beneficial, especially when a complete library is needed.Randomized codon-based saturation mutagenesis becomes inefficient for finding new variants beyond coverage thresholds that are strongly influenced by differences in frequencies of amino acid substitutions in generated libraries. These differences can be at least partly predicted and included in the library design. However, the reliability and other mutagenesis parameters strongly depend on the procedure used, so observed and predicted frequencies of substitutions may differ due to random errors and false negative clones in the screening. The screening could also be adversely affected by various factors, including low competency of the tZOG and artificial substrate pNPG. In surprising contrast to the high conservation at this position all enzyme variants were able to hydrolyze the substrates, albeit at reduced levels  The carbohydrate-active enzymes database  Δbgl pLysS-T1R . All wild-type and engineered variants of Zm-p60.r were subcloned into the pRSET A vector , in which expression of recombinant protein is under control of the phage T7 promoter and all recombinant enzymes are His-tagged at their N-terminus ends.Zm-p60.r construct as a template. Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit . A library of Zm-p60.1 mutants with variations at the Trp373 position was created using the following set of primers introducing NNM degeneracy:For randomized codon-based saturation mutagenesis the “QuikChange” Site-Directed Mutagenesis protocol  was applied, with minor adaptations, using the pRSET A::5′-cctcctatgggaaatccaNNMatctacatgtaccctgagggc-3′5′-gccctcagggtacatgtagatKNNtggatttcccataggagg-3′Dpn I restriction was then applied to avoid amplification of the template. Competent E. coli cells were transformed, the transformants were grown and selected. The number of colonies was low due to the low transformation efficiency of the engineered E. coli strain. Finally, to complete the library we created missing variants using the QuikChange kit again in individual reactions with the same template and following primers –Mutant strands were synthesized using a 2400 GeneAmp PCR system , with 60 cycles of denaturation at 95°C for 2 min, annealing at 55°C for 20 s and extension at 65°C for 2 min 18 s. Cys (5′-gccctcagggtacatgtagatGCAtggatttcccataggagg-3′)Glu (5′-gccctcagggtacatgtagatTTCtggatttcccataggagg-3′)Ile (5′-gccctcagggtacatgtagatAATtggatttcccataggagg-3′)Met (5′-gccctcagggtacatgtagatCATtggatttcccataggagg-3′)Asn (5′-gccctcagggtacatgtagatGTTtggatttcccataggagg-3′)Gln (5′-gccctcagggtacatgtagatCTGtggatttcccataggagg-3′)Val (5′-gccctcagggtacatgtagatCACtggatttcccataggagg-3′)Tyr (5′-gccctcagggtacatgtagatATAtggatttcccataggagg-3′).Due to expression problems with Leu and Ser variants their codons were changed to TCC for Ser and CTG for Leu. W373K was obtained previously −1 dose of XGlc was found to be sufficient and cultivation at 37°C for 12 h, followed by 12 h at 16°C, gave the best results.Positive clones were screened for activity directly on Petri dishes by a procedure developed to identify clones with β-glucosidase activity using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-O-β-D-glucopyranoside , which yields a blue reaction product trans-zeatin-O-β-D-glucoside  at a concentration of 1 mM. An enzyme reaction with β-glucosidase was performed in triplicate at 30°C, in 50 mM citrate-phosphate buffer . Amounts of glucose released in 25 µl portions of the enzymatic reaction mixtures were measured using an Amplex Red Glucose/Glucose Oxidase Assay Kit  and a Tecan Infinite 200 PRO fluorescent reader . Hydrolysis rates for each variant were then calculated using a glucose standard calibration curve. The procedures applied for determining enzyme activity, using 4-nitrophenyl-O-β-D-glucoside , have been previously described The protein variants were expressed and purified as previously reported File S1The pH optimum analysis of the most and least active variants (W373H and W373K).(DOC)Click here for additional data file.File S2Multiple sequence alignment ((PDF)Click here for additional data file.File S3Multiple sequence alignment ((PDF)Click here for additional data file.File S4The protein variants expression and purification details.(DOC)Click here for additional data file.File S5Determination of enzymes activity, using 4-nitrophenyl-O-β-D-glucoside.(XLS)Click here for additional data file.File S6The simulation used to calculate theoretical average numbers of clones that needed to be screened for a new hit based on Monte Carlo method.(ZIP)Click here for additional data file.