Genome hexamer phasing is apparently

Genome hexamer phasing is apparently important not only for RdRp interactions during promoter recognition, but also during mRNA synthesis as the hexamer phase of the cis-acting mRNA signals (start, stop and editing) are conserved (Kolakofsky et al., 1998). The hexamer phases of the mRNA editing signals are the most strikingly conserved, even though the precise location of these P gene signals varies for each virus (Fig. 3). For example, that of rubulaviruses, 3′ A3 UUCUC4 (as minus-strand RNA) that directs the addition of two Gs to the mRNA at high frequency, is always 3′ NAAAUU CUCCCC (spacing shows hexamer phase); and for morbilliviruses that add one G at high frequency, the 3′ U5C3cis-acting sequence is always 3′ UUUUUC CCNNNN. The hexamer phase of the respirovirus editing signal, 3′ U6C3, is not conserved; and tellingly, nor is the pattern of G insertions. For SeV, which like morbilliviruses adds one G at high frequency, the phase is 3′ UUUUUU CCNNN (the highlighted C is the stutter site), whereas for human and bovine PIV3, which add one to six Gs at almost equal frequency, the phase is 3′ AUUUUU UCCCCN. When rSeV genomes are engineered to contain a model 3′ UAAU6C3, editing sequence within the 3′ UTR of the L gene (where its operation has little or no consequence for virus replication), this sequence directs the insertion of either one G at high frequency, or one to six Gs at almost equal frequency, and this choice remarkably depends on the hexamer phase of this cis-acting sequence (bottom panel, Fig. 4) (Iseni et al., 2002).
So, how can the hexamer phase of a sequence, which is ostensibly determined by its spacing within the N protomer cores to which it is bound, still affect mRNA synthesis when the N chain is separated from the genome RNA being copied within the RdRp? Editing of mRNA occurs when the transcriptase responds to a cis-acting sequence (e.g., 3′ UAAUUUUUUCC). Assuming a 7bp hybrid between the template and the nascent transcript (underlined), when the initial G has been added to the mRNA opposite the stutter site g protein coupled receptors (boxed in yellow Fig. 4), most of this sequence is contained within the dsRNA hybrid in the synthesis cavity, and the upstream remainder (3′ UAAU) in the template exit channel. The complement of this upstream sequence is presumably also present in the transcript exit channel (boxed in green Fig. 4).
The stuttering mechanism of this form of mRNA editing (top panel, Fig. 4) proposes that after the initial G is added at the stutter site, rather than ratchet down the template RNA by one position to continue transcript elongation, RdRp backtracks on the template, removing the recently formed transcript 3′ end from the NTPi site and into the NTP entry channel. Elongation thus pauses until the transcript 3′ OH end is back in place. This replacement can occur by RdRp simply reversing the backtracking. Or, during this pause, the nascent transcript can realign on the template U6C2 “slippery” sequence by shifting one position upstream, forming a single G: U base pair in the dsRNA hybrid (marked with an asterisk), and replacing the transcript 3′ end back in the NTPi site. This realignment allows a 2nd (now “pseudo-templated”) G to be incorporated opposite the stutter site cytosine. If RdRp now resumes normal elongation, only a single pseudo-templated G will have been inserted at the mRNA. Within the SeV P gene, a single cycle of RdRp backtracking and G insertion apparently occurs around 25% of the time, as 70% of the P mRNA is unedited, 25% has had one G inserted and mRNAs with two or more Gs inserted are rare in SeV-infected cells (bottom panel, Fig. 3). If, however, RdRp backtracks yet again before normal elongation resumes, the process can repeat itself; a 2nd G: U bp is added to the dsRNA hybrid and two Gs will have been inserted in the mRNA. For PIV3, this apparently happens up to 6 times, such that 1–6 Gs are added at almost equal frequency (Fig. 3). A possible explanation for why this hyper-editing appears to be limited to 6 insertions may be that the complement of the upstream part of the cis-acting sequence (5′ …AUUA…) eventually exits the transcript exit channel and can no longer interact with RdRp.

g protein coupled receptors Our prior work has focused on

Our prior work has focused on the development of viral gene therapy vectors where transgene expression is controlled by p53. As part of this work, we have previously described the construction of a chimeric p53-responsive promoter (PGTxβ, or simply PG) and reporter assays that demonstrate robust expression in a controlled manner, dependent on the activity of p53 (Bajgelman and Strauss, 2008) and have explored the use of such vectors in cancer models (Merkel et al., 2010; Strauss et al., 2005; Strauss and Costanzi-Strauss, 2004). In a separate study using the AAVPG vector, we have clearly shown its p53-responsiveness, including when g protein coupled receptors treated with doxorubicin, hypoxia or mechanical stress (Bajgelman et al., 2013). We hypothesized that the application of the AAVPG vector would be appropriate for cardiac hypertrophy since, according to the literature, the accumulation of p53 occurs in a transition phase, before the deterioration of cardiac function from a compensated to a non-compensated state (Das et al., 2010; Sano et al., 2007). Hence, the p53-responsive mechanism should provide virus expression during a critical phase of hypertrophy.
The properties of VEGF extend beyond the induction of angiogenesis. It has been shown that intracardiac injection of VEGF improves heart performance and inhibits cardiomyocyte apoptosis (Ruixing et al., 2007), induces the mitosis of adult cardiomyocytes (Laguens et al., 2002), induces hyperplasia of cardiomyocytes (Laguens et al., 2004) and it has been suggested that it also participates in the mobilization of stem cells for cardiac tissue repair (Ferrarini et al., 2006). Our observations are consistent with these reports since our experimental results in animals that underwent TAC and received the therapeutic AAVPG-VIG virus were associated with preservation of cardiac function that was correlated with preservation of capillary density and the lack of fibrosis, yet animals that did not receive gene therapy suffered loss of function, capillary rarefaction and induction of fibrosis.
The TAC procedure did not yield profound alterations in ejection fraction or fractional shortening when measured at base line (in the absence of additional hemodynamic stress). This result is not without precedent as it has been observed in several animal models (Smeets et al., 2008). Moreover, the concept of cardiac reserve, the g protein coupled receptors heart׳s ability to perform beyond its basal level, supports the notion that the impact of TAC may not be seen without added stress (dos Santos et al., 2010a). We do not rule out the possibility that a longer follow-up period may have revealed base line alterations due to our TAC procedure. Alternatively, a more acute model, such as induction of infarct, may be used in future studies in order to reveal pronounced changes in heart function. In the present study, the benefit of the AAVPG-VIG vector was revealed when sudden hemodynamic stress was applied. This method is sufficiently sensitive such that changes in cardiac function are revealed even in animals that had no visible changes under basal conditions (dos Santos et al., 2010a). Such an approach supports analysis of heart function at stages that precede obvious deterioration and it has been used to assess strategies to prevent cardiac deterioration post-myocardial infarction using transplantation of fibroblast cells genetically modified to express VEGF or adult stem cells of different origins (Danoviz et al., 2010; dos Santos et al., 2010b; Goncalves et al., 2010; Nakamuta et al., 2009).
The in vivo transduction efficiency seemed to be sufficient to bring about the desired functional result. Since we used a relatively low titer AAV2 as compared to examples from the literature (Aikawa et al., 2002; Kimura et al., 2001; Raake et al., 2008; Su et al., 2004), the transduction level may have been suboptimal, though this was not tested directly. Even though the use of serotypes 6 and 9 would be more appropriate in a cardiac model (Asokan et al., 2012), the functional benefit of our AAV2 vector was clearly evident. The use of AAV6 or 9 may facilitate intravenous administration of the vector, yet the functional results of this proof-of-concept study indicate that we encountered no shortcoming with the intramuscular delivery of AAV2.

g protein coupled receptors br Conflict of interest statement br Acknowledgements

Conflict of interest statement

The author wishes to thank Dr. Akos Pakozdy of the University of Veterinary Medicine, Vienna, Dr. Tarja S. Jokinen of the University of Helsinki, Dr. Fiona M.K. James of the University of Guelph, and Dr. Miyoko Saito of Azabu University, for their helpful information and discussions which aided in writing this manuscript. The author also would like to thank Dr. Kiyotaka Hashizume of Izumi Memorial Hospital, Dr. Hironaka Igarashi of Niigata University, Dr. Ichiro Takummi of Nippon Medical School, Dr. Nobukazu Nakasato of Tohoku University, Dr. Hiromitsu Orima (my past mentor) and Dr. Tatsuya Tanaka (the vice president of ILAE), respectively. They led me into epileptology. The author would like to thank all colleagues and students of the unit of neurology and neurosurgery, Nippon Veterinary and Life Science University, cooperating my studies. This review is financially supported by a Grant from The Science Research Promotion Fund administered by The Promotion and Mutual Corporation for Private Schools of Japan.

A quest for clean serum specimens to detect immunological responses to the presence of important pathogens has characterised the history of disease testing in veterinary medicine. Serum has traditionally been preferred over whole blood to decrease non-specific reactions, and give more accurate and reliable results. Assays such as the complement fixation test, dating from the very beginning of the last century (Bialynicki-Birula, 2008), agar gel immune-diffusion test (Ouchterlony, 1948), radioimmunoassay (Yalow and Berson, 1960) and immunofluorescence antibody testing (Voller, 1964) were some of the first tests used. In more recent years, ELISA (Engvall and Perlmann, 1971; Van Weemen and Schuurs, 1971) in its various permutations (direct, indirect, competitive, sandwich, capture) has been able to detect either antibody or antigen, and is popular because it is simple, inexpensive and rapid.
Newer technologies such as g protein coupled receptors and quantitative PCR (Mullis and Faloona, 1987) are pathogen detection methods and could readily be applied to a variety of a specimen types containing genetic material. Extraction and amplification clean-up steps now make PCR less prone to interference than earlier versions that relied on observation to detect lines or agglutination. Recent further developments in PCR technology, eliminating the need for expensive thermocyclers, have the potential to further revolutionise field diagnostics (Thekisoe et al., 2007).
In some parts of the world it is still difficult to get good quality specimens reliably to a diagnostic laboratory, either because the necessary transport infrastructure is absent or distances within the country or between countries to the laboratory infrastructure are too great. Similarly, convenience and cost-effectiveness can affect specimen collection, as obtaining a blood specimen is often the domain of veterinary or para-veterinary personnel, adding to the expense of diagnostic testing. Other readily obtainable body fluids, excretions or tissues could be obtained by less skilled personnel or animal owners to save on collection costs. In the human diagnostic field there is currently significant interest in exploring alternative specimen analysis such as dried blood spot testing for the diagnosis of hepatitis C (Coats and Dillon, 2015).

Milk can be a suitable medium for animal disease testing as it is generally easy to obtain (often without any specialised equipment), and in dairy cattle it is often available throughout the year. Using milk as a specimen, a wide range of animal diseases can be tested for in individual animals and in pooled specimens from herds. Tests for antibodies against the following pathogens are available: Brucella abortus (Nielsen and Gall, 2001), bovine viral diarrhoea virus (BVDV; Lanyon et al., 2014b), enzootic bovine leukosis (EBL) and bovine herpes virus 1 (BoHV1; Reber et al., 2012), Neospora caninum (Schares et al., 2004; Hall et al., 2006), liver fluke (Fasciola hepatica; Reichel et al., 2005), Johne\’s disease (Mycobacterium avium subspecies paratuberculosis; MAP; Collins et al., 2005) and Ostertagia ostertagi (Charlier et al., 2005; Forbes et al., 2008) in cattle. Single animal testing can be performed and tank milk from herds of dairy cows is a ready-made pool for testing groups of animals. Tank milk presents a natural pool of animal biological specimens that, with adequate test analytical sensitivity, enables the tester to screen large numbers of animals for the presence or absence of disease. While testing for EBL by antibody ELISA, milk pools rarely exceeded 100–200 cows (Ridge and Galvin, 2005), but PCR testing for BVDV is now routinely performed on pools in excess of g protein coupled receptors 400 because high test analytical sensitivity provides a very cost-effective way of screening large herds and, more broadly, whole dairy industry. Herds of >400 cows are typical for the New Zealand dairy industry (Hill et al., 2010).

Owing to the broad host

Owing to the broad host range and organ tropism of T. foetus, much emphasis has been placed on identifying the difference, if any, between host-specific isolates. Various molecular methods including multi-locus genotyping have grouped the porcine T. foetus isolate with bovine T. foetus isolates, whereas isolates of T. foetus from cats are genetically distinct from bovine isolates (Tachezy et al., 2002; Šlapeta et al., 2010; Šlapeta et al., 2012). In an accompanying study by Mueller et al. (2015), the porcine isolate PIG30/1 was shown to be identical to the ‘bovine genotype’ of T. foetus at 9 diagnostic molecular markers. Whole genome differences between the ‘feline genotype’ and the ‘bovine genotype’ are minute and analysis of 1511 orthologous protein-coding genes shared between a bovine (BP-4) and a feline (G10/1) isolate indicate little divergence, despite their vastly different origin (. at 9 diagnostic molecular markers. Morin-Adeline et al., 2014). In addition, differences in specific virulence factors have been investigated at a cell-wide level in an attempt to understand molecular mechanisms that govern host-specificity of the different isolates (Šlapeta et al., 2010, 2012; Morin-Adeline et al., 2014). In particular, the cysteine protease (CP) gene family have been used as they are regarded as a crucial aspect of T. foetus virulence involved in cleavage and inactivation of host protective g protein coupled receptors (Bastida-Corcuera et al., 2000). Distinct differences exist in the cysteine protease gene family between the bovine and feline genotype T. foetus and to date, 7CPs have been used to as molecular diagnostic markers to distinguish the ‘bovine genotype’ from the ‘feline genotype’ (Šlapeta et al., 2012). The most divergent of the family, CP2, confirmed that T. foetus isolate (PIG30/1) is ‘bovine genotype’ (Mueller et al., 2015).
The apparent identity of the porcine and bovine isolates of T. foetus compared to the feline isolate of T. foetus, together with their broad host range, presents an intriguing model to studying the factors that drive T. foetus host adaptation. Comparative analysis of transcriptomes offers an ideal method for whole-cell comparisons in the absence of a sequenced nuclear genome (Morin-Adeline et al., 2014a). In this study, RNA-sequencing (RNA-seq) was utilised to obtain whole-cell transcribed sequences of the novel PIG30/1 porcine T. foetus isolated by Mueller et al. (2015). The newly assembled transcriptome of PIG30/1 was then compared to the published bovine (BP-4) and feline (G10/1) T. foetus (Morin-Adeline et al., 2014).

Material and methods


The transcriptome library generated for PIG30/1 represents the first porcine T. foetus transcriptome and complements the published bovine (BP-4) and feline (G10/1) T. foetus RNA-seq data (Morin-Adeline et al., 2014). The cell-wide transcriptomic analysis confirms the closer similarity of the porcine isolate (PIG30/1) to the ‘bovine genotype’ T. foetus (PB-4) compared to the ‘feline genotype’ T. foetus (G10/1). Since the three-way reciprocal best hit (RBH) BLAST results were filtered to transcripts with at least 70% identity between the three T. foetus isolates in the current analysis, the 5-fold greater number of shared transcripts between the bovine and porcine T. foetus confirms the closeness of these two isolates compared with the feline T. foetus isolate.
The ability of T. foetus to adapt to the varied nutrient environment of its niche within the three hosts (cow, pig, cat) hints at the highly versatile metabolism this parasite possesses. In trichomonads, energy in the form of ATP is produced by fermentative glycolysis that occurs in the cytoplasm, as well as phosphate-level phosphorylation that occurs in the mitochondria-derived organelle, the hydrogenosome (Cerkasov et al., 1978; Lindmark et al., 1989). The end product of glycolysis is pyruvate which is the substrate for hydrogenosomal metabolism. Hydrogenosomes lack the necessary components for high energy yield by the Kreb’s cycle (Cerkasov et al., 1978; Lindmark et al., 1989), therefore, anaerobic protozoa must have efficient ATP yield from cytoplasmic fermentative metabolism. One way of achieving this is by the replacement of ATP-dependent glycolytic enzymes with pyrophosphate-dependent (PPi) enzymes as phosphate donors (Reeves et al., 1974; Mertens et al., 1989; Hrdy et al., 1993). For instance, the ubiquitous ATP-dependent pyruvate kinase (PK), an important enzyme responsible for catalysing the production of pyruvate from phosphoenoipyruvate (PEP), can also exist as the PPi phosphate dikinase (PPDK) (Mertens, 1993). Although PPKD catalyses the same reaction as PK, it offers biochemical versatility by permitting the reverse reaction to occur for the use of alternative metabolic pathways depending on nutrient availability (Mertens, 1993). In addition, a 1.5-fold increase in fermentative ATP yield is attained directly through the use of PPi-dependent glycolytic enzyme compared to the ATP-dependent enzyme (Mertens, 1993). Curiously in T. foetus, enzymatic activity of PK or PPDK has not been observed to date (Mertens et al., 1989; Muller, 1992; Hrdy et al., 1993; Slamovits and Keeling, 2006). Mapping the T. foetus PIG30/1 transcriptome with KEGG metabolic enzymes have revealed evidence of homologues to both PK and PPDK in T. foetus. The presence of both versions of this enzyme in anaerobic protozoa is not uncommon. In Entamoeba histolytica, only the activity of PPDK was originally detected and it was assumed to have replaced PK (Reeves et al., 1974). Later, identification of a PK homologue in the genome of E. histolytica led to work that confirmed PK activity similar in magnitude to PPDK activity in this parasite (Saavedra et al., 2004). In the human T. vaginalis, a similar situation exists where only the activity of PK has been detected to date, but genomic research demonstrated evidence of PPDK as well (Mertens et al., 1989; Carlton et al., 2007). PPi-dependent enzymes have previously been suggested as a drug target in protozoans as they are absent in mammalian eukaryotes (Verlinde et al., 2001). Possession of both PK and PPDK suggests that this strategy in T. foetus is not ideal as the parasite has the capacity to utilize PK as a substitute for PPDK. Furthermore, finding transcripts that correspond to both PK and PPDK not only close the gap in knowledge of T. foetus glycolytic metabolism, but implies that alternation between the two enzymes depending on the nutrient environment may facilitate the parasites survival within its three hosts.

br Validation of model br Conclusion br Introduction

Validation of model


In ancient period, the structures were constructed by using materials like lime, clay, mud, surkhi, wood, egg, jaggery, sugar, burnt coconut shells etc. Oral traditional sources tell us egg whites were used as ingredients of mortar, which were used to bind building materials for the ancient constructions. Egg whites were generally used as adhesive which is a g protein coupled receptors that adheres or bonds two items. Historically, they were also used to produce paint binder [1]. Among the ancient admixtures, jaggery and egg were widely used. Michelle had a research on existing historical buildings by collecting mortar samples and proved that egg was used in building constructions [1]. After invention of cement by Joseph Aspdin in 1824, cement has been widely used in construction. The major drawback of cement usage is liberation of huge amount of green house gas (CO2) emissions into environment which causes global warming. Recently, various supplementary cementitious materials such as fly ash, ground granulated blast furnace slag, rice husk ash etc., are being used as partial replacement of cement to reduce green house gas emissions. Several investigations are being done on historical constructions and concluded that lime, mud and surkhi were used as binders and starch, jaggery and egg were used as admixtures.
Jaya Sankar et al. used egg shell powder as partial replacement of cement in concrete and designed for M 20, M 25 & M 30 grade of concrete [2]. They concluded that the compressive strength and split tensile strength were decreased with the increasing replacement level of egg shell powder. Dhanalakshmi et al. also concluded that the compressive strength, workability and density of concrete were decreased with the increasing replacement of egg shell powder [3]. Hanifi Binici et al. concluded that replacement of egg shell powder in sand, the compressive strength and flexural strength of cement mortar were decreased. But it has higher resistance to radiation effect [4]. Ferhat and Ilhan concluded that class F fly ash can be replaced up to 55% to cement [5]. Siddique concluded that splitting tensile strength (STS) depends on compressive strength of concrete and age of concrete [6]. Guru Jawahar et al. concluded that the compressive strength of Class F g protein coupled receptors fly ash blended concrete was increased due to pozzolanic reaction of class Fly ash [7].
Ramesh Babu and Neeraja [8] have concluded that the Natural Admixture (NAD) acts as accelerator to enhance the hydration of binder, when testes added to binder. They were explained that the fresh properties of binder with and without NAD with standard consistency and initial setting time. At 0.25% NAD dosage the initial setting time of binder is less than that of without NAD and they were concluded that at this dosage the setting takes places very faster. The fresh properties of concrete were explained with workability of Conventional Concrete (CC) and Class C fly ash blended (FA) concrete was explained by slump cone test. They were concluded that, at 0.25% NAD the slump of concrete mixes were very less, because due to high viscosity of NAD the mix becomes homogenous and high bonding nature.
The mechanical properties of CC and FA blended concrete were explained and they were reported that 0.25% of NAD is concluded as optimum dosage [9]. And 25% Class C fly ash can be replaced with addition of 0.25% NAD to get designed strength.

Experimental study

Experimental procedure

Results and discussion

The following conclusions have been drawn based on the investigation studied on the influence of natural admixture (broiler hen egg) on mechanical properties of CC and FA blended concrete:

Precast concrete elements produced by steam-cured, such as sleepers, track slabs and pre-stressed concrete beams, are mainly used in railway engineering infrastructures. The advantages of steam-cured concrete are that it can rapidly improve strength of concrete in early ages and efficiency of template turnover [1,2]. However, compared to concrete cured at room temperature, there are some macroscopical and microcosmic disadvantages in steam-cured concrete, such as concrete brittleness developing, concrete surface layer micro cracks increasing and porosity enlarging [3–5]. High temperature and moist steam-cured process would give rise to great differences between surface and inner concrete due to temperature-stress difference, heat-mass transfer and non-uniform of hydration of cementitious, being the important reasons for arising SCHD. Studies show all these disadvantages resulting from the steam-cured process will put great side effects on concrete [6–8]. In this paper, these concrete disadvantages caused by steam-cured are called steam-cured heat damage (SCHD).

br Figure thinsp xA Representative sequential

Figure 2. Representative sequential chest radiographs and the graphs of excursion and peak motion of the diaphragms obtained by chest dynamic radiography (“dynamic X-ray phrenicography”). (a) Radiograph of the resting end-expiratory position. (b) Radiograph of the resting end-inspiratory position. (c) Graph showing the vertical excursions and the peak motion speeds of the bilateral diaphragm. A board-certified radiologist placed a point of interest (red point) on the highest point of each g protein coupled receptors on the radiograph at the resting end-expiratory position (a). These points were automatically traced by the template-matching technique throughout the respiratory phase (double arrows in b) (Supplementary Video S1); red double arrow indicates the vertical excursion of the right diaphragm and blue double arrow indicates that of the left diaphragm. Based on locations of the points on sequential radiographs, the vertical excursions and the peak motion speeds of the bilateral diaphragm were calculated (c). The lowest point (0 mm) of the excursion on the graph indicated that the highest point of each diaphragm was at the resting end-expiratory position (ie, null point was set at the end-expiratory phase) (c). (Color version of figure is available online.)Figure optionsDownload full-size imageDownload high-quality image (305 K)Download as PowerPoint slide

Pulmonary Function Tests

The pulmonary function tests were performed in all participants on the same day of the imaging study. Parameters of pulmonary function tests were measured according to the American Thoracic Society guidelines 20 ;  21 using a pulmonary function instrument with computer processing (DISCOM-21 FX, Chest MI Co, Tokyo, Japan).

Statistical Analysis

Descriptive statistics are expressed as mean ± standard deviation for continuous variables and as frequency and percentages for nominal variables. A paired t test was used to compare the excursion and peak motion speed between the right diaphragm and the left diaphragm. The associations between the excursions of the diaphragms and participants\’ characteristics were evaluated by means of the Pearson\’s correlation coefficient and a simple linear regression or Student\’s t test depending on the type of variable (ie, continuous or nominal variable). Continuous variables were height, weight, BMI, tidal volume, vital capacity (VC, %VC), forced expiratory volume (FEV1, FEV1%, and %FEV1), and nominal variables were gender and smoking history. The robustness of the results of the univariate analyses was assessed with multiple linear regression models. The significance level for all tests was 5% (two sided). All data were analyzed using a commercially available software program (JMP; version 12, SAS, Cary, NC, USA).


Participants\’ Characteristics

Table 1 shows the clinical characteristics of all the participants (n = 172).

Excursions and Peak Motion Speeds of the Bilateral Diaphragm

Univariate Analysis of Associations Between the Diaphragmatic Excursions and Participants\’ Demographics

Figure 3. Estimated regression line of the excursion of the diaphragm on BMI or tidal volume. (a) Association between BMI and excursion of the right diaphragm. (b) Association between BMI and excursion of the left diaphragm. (c) Association between tidal volume and excursion of the right diaphragm. (d) Association between tidal volume and excursion of the left diaphragm. Lines show estimated regression (a–d). All scatterplots show correlations (P < 0.05). BMI, body mass index.Figure optionsDownload full-size imageDownload high-quality image (226 K)Download as PowerPoint slide

Multivariate Analysis of Associations Between the Excursions and Participants\’ Demographics

Multiple linear regression analysis using all variables as factors (Model 1) demonstrated that weight, BMI, and tidal volume were independently associated with the bilateral excursion of the diaphragms (all P < 0.05) after adjusting for other clinical variables, including age, gender, smoking history, height, VC, %VC, FEV1, FEV1%, and %FEV1. There were no significant associations between the excursion of the diaphragms and variables including age, gender, smoking history, height, VC, %VC, FEV1, FEV1%, and %FEV1 (Table 4). Additionally, a multiple linear regression model using age, gender, BMI, tidal volume, VC, FEV1, and smoking history as factors (Model 2) was also fit as a sensitivity analysis, taking into account the correlation among variables (eg, BMI, height, and weight; VC and %VC; FEV1, FEV1%, and %FEV1). Model 2 (Supplementary Data S1) gave results consistent with Model 1 (Table 4): higher BMI and higher tidal volume were independently associated with the increased bilateral excursion of the diaphragms (all P < 0.05). The adjusted R2 in Model 1 was numerically higher than that in Model 2 (right, 0.19 vs. 0.16, respectively; left, 0.16 vs. 0.13, respectively).

Phylogenetic analyses Both molecular markers were analysed separately in

Phylogenetic analyses
Both molecular markers were analysed separately in a series of preliminary maximum likelihood phylogenetic analyses that produced trees virtually without conflict, mostly because of lack of support for most nodes. Therefore, the aligned cox1 and wnt data were concatenated in a single alignment that was used for subsequent phylogenetic analyses upon adding the homologous sequences from two outgroups in the subfamily Chrysomelinae: Calligrapha polyspila (Germar) [AM160984 (cox1) and LM644939 (wnt)] and Stilodes sp. [LM644645 (cox1) and LM644959 (wnt)]. The phylogenetic analysis on the combined matrix was under a maximum likelihood framework as implemented in RAxML 7.2.8 ( Stamatakis, 2006). We ran seven independent analyses under a general time-reversible substitution model with different combinations of data partitioning (each g protein coupled receptors position of each gene as a different partition, or first and second codon positions of each gene in the same partition), and inclusion of heterogeneity parameters in the probability of nucleotide changes (a CAT approximation, proportion of invariable sites [I] and/or the parameter gamma [Γ]). Specifically, the evolutionary models tested were: (1) GTR + Γ with two codon partitions per gene (12_3); (2) GTR + Γ + I with two codon partitions per gene; (3) GTR + Γ + I with three codon partitions per gene (1_2_3); (4) GTRCAT with two codon partitions; (5) CAT with three codon partitions; (6) GTRCAT + I with two codon partitions; and (7) GTRCAT + I with three codon partitions. Tree searches consisted in the search of the best-scoring maximum likelihood tree from the optimisation of 100 random starting trees. Moreover, bootstrap support was added to each optimal tree based on 100 data pseudo-replicates.
Molecular identification of host-plants
We investigated potential host-plant associations of galerucine beetles of interest following the strategy of Jurado-Rivera et al. (2009) and the protocols outlined in Papadopoulou et al. (2015) and De la Cadena et al. (2017). Specifically, we amplified short flanking regions and intergenic spacer of the cpDNA psbA-trnH locus. Primers and conditions used were the same as in De la Cadena et al. (2017). The psbA-trnH sequences obtained were submitted to the bioinformatic pipeline BAGpipe to assist taxonomic assignments based on joint analyses with publicly available and taxonomically labelled homologue sequences from public sequence databases ( Papadopoulou et al., 2015). Several criteria for taxonomic inference were used, including an assessment based on sequence similarity and genetic distances, and an assessment based on maximum likelihood phylogenetic analyses and clade support. The former consisted on the identification of GenBank sequences producing the best match as well as those below a 4% divergence threshold. The latter extracted the taxonomy of the supported clade including the psbA-trnH sequence from our Galerucinae specimen (inner taxon) and that of the closest supported node moving to the root of the tree (outer taxon).

These modes show a dependency in the number of

These modes show a dependency in the number of atoms as shown in Fig. 5a. This generally reflects a decrease to lower wavenumbers when the cluster is made up of a higher number of atoms. The intensity of the Raman RBM obtained from calculations of DFT is shown in the following Fig. 5b. There has been a significant decay in the intensity of the modes for clusters with size above (Lin, n > 15). We assume that for larger clusters the intensity of the RBM will show probably a decay in congruence with the non-detection of Raman bands in metal bulk. Due to the calculation time and availability in our equipment these calculations are not considered in this g protein coupled receptors work. Likewise, if we link this vibrational behaviour to the metallic nanoparticles synthesized in the Ofi extract, we can observe that the mode type tends to decrease to a particular particle size. In general terms, we can say that this type of mode can be found in lithium nanoparticles and possibly lithium nanoparticles with lower size than 10 nm.
Fig. 5. DFT vibrational calculations at the B3LYP approximation level (a) radial breathing modes (RBM) in small clusters of Li and (b) RBM Raman intensity in small clusters of Li.Figure optionsDownload full-size imageDownload as PowerPoint slide
Complementing the theoretical calculations and considering the structure of the unit cell type for bulk lithium (Fig. 6), the cubic structure was optimized by Hartree-Fock (HF) method and the approximation levels B3LYP, B3PW91 (Becke’;s, Burke and Ernzerhof functional) and LSDA (local spin density approximation) with the basis set LANL2DZ, SDD (Stuttgart/Dresden) and cc-pVTZ (Dunning correlation-consistent, polarized valence, triple-zeta basis). Subsequently, obtaining the Raman spectrum of that structure in each theory level. An RBM was detected in this structure for the levels of theory previously mentioned, located around 213 cm?1 for the approximation level B3LYP.
Fig. 6. Radial breathing mode (RBM) detected in a cubic structure FCC type by Hartree-Fock and several approximation levels of DFT.Figure optionsDownload full-size imageDownload as PowerPoint slide
Table 2 shows the vibrational results obtained by HF and the different approximation levels of DFT. Generally, due to the considered theories, we suppose that the RBM frequency is located between 193-220 cm?1. Even though we know that the vibrational mode technically depends on the length bond. As we know, HF overestimate the bond length and conversely, LSDA underestimate the bond length. As a result, HF shows the RMB to lower wavenumbers and LSDA to higher wavenumbers. For the last case studies (Lin > 15) the cluster size is close to 1 nm. Due to the cost of calculation that requires the use of the DFT, structures of hundreds of atoms have a great difficulty and cost of time to be studied. However, vibrational analysis and trend observed in small structural arrangements may become sufficient to describe a pattern of behaviour.
4. Conclusions