Tumor targeting is of high clinical and biological relevance, and major efforts have been made to develop molecular imaging technologies for visualization of the disease markers in tissue. Of particular interest is apoptosis which has a profound role within tumor development and has significant effect on cancer malignancy.Herein, we report on targeting of phosphatidylserine-exposing cells within live tumor allograft models using a synthetic near infrared zinc(II)-dipicolylamine probe. Visualization of the probe biodistribution is performed with whole body multispectral optoacoustic tomography (MSOT) system and subsequently compared to results attained by planar and tomographic fluorescence imaging systems.Compared to whole body optical visualization methods, MSOT attains remarkably better imaging capacity by delivering high-resolution scans of both disease morphology and molecular function in real time. Enhanced resolution of MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that its tumor selectivity is gained by targeting the phosphatidylserine exposed on the surface of tumor vessels.The current study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development. Some earlier attempts to specifically image apoptotic responses in whole living animals using fluorescence molecular tomography attained generally low spatial resolution and imaging speed [A versatile pool of optical reporter agents and imaging methods for enhancement and probing of anatomical features and molecular function of tumors has been recently developed ,2. The cng speed ,14. As aMore recently, multispectral optoacoustic tomography (MSOT) has been emerging as a promising tool for high-resolution volumetric imaging of optical contrast in tissues , capablein vivo with high resolution using MSOT.Recently, a novel optical probe, comprised of a synthetic zinc(II)-dipicolylamine complex appended to a near-IR (NIR) carbocyanine fluorophore , has been shown to selectively target anionic membrane-bound phosphatidylserine (PS) exposed by dead and dying cells within xenograft tumors in rat and mouse models . Usually2 in order to meet the laser safety standards. Signal collection is based on a custom-made 64-element focused transducer array  covering a solid angle of 172° around the imaged object, while the detection plane coincided with the center of the illumination ring. The individual detection elements are manufactured using piezocomposite technology with central frequency of 5 MHz, bandwidth of more than 50%, and sensitivity of approximately 18 μV/Pa and are shaped to create a cylindrical focus at 40 mm. According to ultrasonic diffraction limit and focal width at the central frequency, the effective spatial resolution of the system is estimated at 150 μm (in-plane) and 800 μm . The detected signals are digitized at 60 megasamples per second frequency by eight multichannel analog to digital converters  with noise floor of approximately in vivo measurements, the animals were wrapped by a water-impenetrable transparent membrane that averts animal contact with water inside the imaging chamber. A linear stage  allows linear translation of the animal holder in the axial z direction for the acquisition of three-dimensional data sets.A real-time MSOT scanner, similar to the one described by Razansky et al. , was uti5 4T1 cells subcutaneously into the mouse's neck where they were allowed to grow for 7 days, reaching a diameter of 0.4-0.5 cm. The PSS-794 imaging probe was prepared as previously reported [All procedures involving animals and their care were conducted in full agreement with the institutional guidelines, complying with national and international laws and regulations. For imaging, four female 8-week old athymic CD-1 nude mice containing 4T1 tumor allografts were used. The tumors were obtained by injecting 0.8 × 10reported . The firreported . The micReconstruction of single-wavelength optoacoustic images was done with interpolated matrix model inversion method . Prior ta priori known spectra to the data, the ICA algorithm has more degrees of freedom which reduces the cross talk and leads to generally better performance.This technique is based on the assumption that the source components of the mixed multispectral dataset are statistically independent. In order to identify these source components, the algorithm seeks a transformation of the dependant multispectral dataset into a set of independent variables which is accomplished by maximizing their non-Gaussianity because, according to the central limit-theorem, statistical independent variables are less Gaussian than their mixed dependent counterparts. The measured absorption spectra of the probe and oxygenized and deoxygenized blood were fed to the algorithm as a starting point for a guided un-mixing. The probe component was identified by comparing the resulting spectra from the un-mixing algorithm with the measured probe spectra. In contrast to other methods based on least-square un-mixing, which try to fit an ex vivo with a newly developed 360° free-space FMT-XCT system which combines fluorescence molecular tomography (FMT) with small animal X-ray CT (XCT) into a hybrid imaging device [in vivo. For volumetric FMT reconstructions, 12 angular projections distributed over full 360° range were acquired close to the excitation and emission wavelengths of PSS-794. Following the FMT-XCT measurement, the euthanized mice were cooled to -80°C. For further verification of the in vivo MSOT and FMT images, cross-sectional color photographs (RGB) and fluorescence images were also obtained using a cryotome supplemented by a home-built multispectral epi-fluorescence system [For validation purposes, one mouse was also imaged g device . The syse system . The sysAfter cryo-slicing, selective tissue samples in the tumor area were used for histological validation for highly specific detection of apoptosis using commercially available annexin V antibody . The appearance of PS residues  on the surface of the cell is an early parameter of apoptosis, which can also be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong Ca2+-dependent affinity for PS and, therefore, can be used as a probe for detecting apoptosis.Figure in vivo 24 h after injections. The single wavelength optoacoustic images acquired at 900 nm are shown in gray scale, while the spectrally unmixed MSOT signals are superimposed in color. A transparency threshold of 65% was applied to the unmixed data when superimposed to the single wavelength background image. The boundary of the tumor is delineated in yellow. Figure Figure ex vivo FMT measurement, shown in Figure On the other hand, reconstruction of the subsequent in vivo MSOT and the ex vivo epi-fluorescence images clearly reveal that the fluorescence signal of the PSS-794 probe is localized in the blood vessels surrounding the tumor. This observation agrees well with literature which shows that, once there is only a small amount of apoptotic tissue in this tumor cell line, PS-targeting probes will be only observed in the tumor blood vessels [In summary, both the  vessels .Previous studies have shown that the fluorescent PS-targeting probe, PSS-794, can be used to optically image apoptotic/necrotic tissues often found inside tumors . Here, iThe lack of extravasation is not surprising in this case since both probes are known to associate with serum proteins, and it is hard for the large protein/dye complex to leak from the neovasculature, especially if there is a high interstitial pressure in the tumor . On the ®  is especially effective at targeting this class of exposed PS, and it induces impressive antitumor activity in a wide array of tumors with no evidence of toxicity [The PS-binding antibody Bavituximabtoxicity ,38. Thisin vivo over strong background absorption of blood with spatial resolution on the order of 150 μm, attaining unprecedented image quality for deep-tissue imaging of optical contrast. The enhanced resolution of the MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that the probe gains its tumor selectivity by targeting the PS that is exposed on the surface of the tumor blood vessels. The MSOT results further correlated well with cryo-slices and epi-fluorescence images of ex vivo specimens. Overall, this study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development.In conclusion, we used MSOT to accurately detect the targeted PSS-794 probe The authors declare that they have no competing interests.AB, EH, VN and DR designed the experiments. AB and EH performed the data analysis. AA did the FMT-XCT imaging. AB, DR and BDS have written the manuscript. BDS provided the PSS-794 contrast agent. DR and VN provided conceptual input and supervised the research. All authors read and approved the final manuscript.


Acartia spp., Temora longicornis, and Pseudocalanus acuspes, three dominant zooplankton species inhabiting different pelagic habitats in the Central Baltic Sea (CBS). We use the three copepods as indicator species for changes in the CBS zooplankton community and apply non-linear statistical modeling techniques to compare spatial population trends and to identify their drivers. We demonstrate that effects of climate variability and change depend strongly on species-specific habitat utilization, being more direct and pronounced at the upper water layer. We propose that the differential functional response to climate-related drivers in relation to strong habitat segregation is due to alterations of the species’ environmental niches. We stress the importance of understanding how anticipated climate change will affect ecological niches and habitats in order to project spatio-temporal changes in species abundance and distribution.Understanding and predicting species distribution in space and time and consequently community structure and dynamics is an important issue in ecology, and particularly in climate change research. A crucial factor determining the composition and dynamics of animal populations is habitat heterogeneity, i.e., the number of structural elements in a given locality. In the marine pelagic environment habitat heterogeneity is represented by the distribution of physical oceanographic parameters such as temperature, salinity and oxygen that are closely linked to atmospheric conditions. Little attention has been given, however, to the role of habitat heterogeneity in modulating the response of animal communities to external climate forcing. Here we investigate the long-term dynamics of  Understanding and predicting species distribution in space and time and consequently community structure and dynamics is an important issue in ecology, and particularly in climate change research. Animal populations in terrestrial and aquatic environments shift their geographical range or show strong fluctuations in abundance in response to climate-induced changes in, e.g., temperature, precipitation or ocean circulation A crucial factor determining the composition and dynamics of animal populations is habitat heterogeneity, i.e., the number of structural elements in a given locality The pelagic of the Central Baltic Sea (CBS) is characterized by spatial hydrographic gradients that create a number of different habitats for local animal populations. Horizontally, both salinity and temperature generally decline from West to North-East . Even moAcartia spp., Temora longicornis, and Pseudocalanus acuspes, three dominant zooplankton species that make up to 60% of the spring biomass in the CBS and distribute differently according to these habitats. Acartia spp. mainly inhabits the surface water, T. longicornis the mid-water and P. acuspes the deeper, saline layer (Clupea harengus) and sprat (Sprattus sprattus) feed mainly in deeper waters The CBS zooplankton community is composed of six major taxonomic groups , dominated by very few species (often 1 to 3) ne layer  [29], [3Here we use the three copepods as indicator species for changes in the CBS zooplankton community, and specifically test (i) whether our target zooplankton species display different population trajectories in space and time, and (ii) if hydro-climatic factors and predation pressure differentially affect the long-term development of the individual specieś populations. We apply statistical modeling techniques to compare species-, area-, and season-specific long-term population trends and to identify their main drivers.et al.For our study we have, for the first time, combined data sets from three long-term zooplankton monitoring programs in the Central Baltic Sea CBS; . With th−3) for Acartia spp., Temora longicornis and Pseudocalanus acuspses, we merged the three data sets according to the following procedure:In order to construct basin-specific time series on spring (sampling mainly in May) and summer (sampling mainly in August) biomass  transformation, we combined the three data sets by calculating annual means for each species, basin and season weighted by the sampling number of each individual sampling program.sbBM(y) is the mean biomass of a species in season s and basin b in a particular year y, Subsequently, we accounted for differences between sampling programs due to gear-specific capture efficiencies and different spatial and temporal resolutions following the approach suggested by Mackas and Beaugrand Using log-transformed anomalies accounts for any potential sampling bias that is inherent in each annual mean value as well as in the overall time series mean Acartia spp., T. longicornis and P. acuspes are water temperature and salinity Acartia spp. and T. longicornis respectively  and characteristic stage-distributions in each season P. acuspes population in summer. Hydrographic data were retrieved from the International Council for the Exploration of the Sea (ICES) website (http://www.ices.dk/ocean/aspx/HydChem/HydChem.aspx).We included several hydro-climatic variables and predation pressure as covariates in our statistical analyses. The main hydrographic variables affecting vely see . P. acusAs an indicator for the overall climate condition we used the Baltic Sea Index (BSI) provided by the GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany . This loSprattus sprattus) is the dominant planktivore in the Baltic Sea basins and, at times, appears capable of exerting top-down control of P. acuspes population Gadus morhua). Eastern Baltic cod is the major piscivore in the Baltic Sea and its stock size is inversely related to the stock size of sprat et al. Sprat  techniques f and bf are the single and respectively basin-specific thin plate regression spline functions of the variable time. α is the intercept and εby the random noise terms assumed to be normally distributed with zero mean and finite variance. To focus only on the major trends and avoid over-parameterization, we limited the effective degrees of freedom (edf) to a maximum of 3. For each of the species- and season-specific comparisons between models including a spatially uniform or basin-specific smoothing functions, we applied an F-ratio test In a first analysis we tested for spatial differences in temporal trends in species biomass between the three basins. We modeled the log-transformed spring or summer biomass anomalies of each species either as a single function of time (year), i.e., for the entire CBS area,b as the intercept at basin b and bf, bg, bi, and bj as basin-specific thin plate regression spline functions (maximum edf  =  4) describing the effect of the locale climate index BSI, depth-specific temperature (T) and salinity (S), and predation (PI) respectively. Prior to the analysis we tested for collinearity among the covariates using Pearson correlation coefficient and a variance inflation factor (VIF) of 3 as exclusion criterion Acartia spp. residuals showed temporal autocorrelation of lag 2, hence we extended the GAM to a Generalized Additive Mixed Model (GAMM) by including a correlation structure. Such an extension allows a more complex stochastic model structure and implies that the single elements of the response variable are not independent anymore In a second analysis we used the non-linear regression technique to identify the role of climate, hydrography and predation pressure on the seasonal long-term trends of each copepod. Each species' spring and summer biomass was then modeled by basin-specific smoothing functions of the different environmental variables, independent of whether long-term trend were significantly different between basins. The following model was used:Acartia spp., Temora longicornis, and Pseudocalanus acuspes in the Central Baltic Sea (CBS). We found T. longicornis and P. acuspes long-term trends to be spatially homogeneous, while long-term dynamics of Acartia spp. significantly differed between the three basins  where population sizes increased linearly with time  to be the best predictor of annual variation in Acartia spp. biomass. For spring biomass the BSI effects were positive and linear, but of basin-specific strength  and, thus, is not presented here.Our statistical analyses for  effects . The mosn effect . Hence wP. acuspes revealed a particularly strong response to external drivers for summer biomass  and no basin-specific relationships . This finding agrees with previous studies reporting that changes in zooplankton phenology and biomass levels were particularly sensitive to variability in climate-driven physical factors during the beginning of the productive season Acartia spp. compared to spring temperature suggests that other factors related to the BSI are of importance, such as vertical mixing intensity affecting the timing of the spring bloom and phytoplankton development Acartia spp. Acartia spp. shows maximum egg production rates at Chl a concentrations of > 14–20 µg L−1Chl a L−1) Chl a as proxy) but also food quality can be important for growth and reproduction . The main trend of the spring and summer temperature and salinity in different water layers (see yers see  are give(EPS)Click here for additional data file.Figure S2Time series of local climate and fish variability. (A) Winter anomalies of the Baltic Sea Index (BSI) (Dec-March). (B) Predation index (PI) based on cod spawning stock biomass (SSB) (in black circles) in comparison to the shorter sprat SSB time series (in blue triangles). Both, BSI and PI have dimensionless units.(EPS)Click here for additional data file.Figure S3Partial plot of the BSI effect in the alternative spring model of Acartia spp. Values on the y-axis indicate the effect that the term on the x-axis has on the biomass anomaly. The number in parentheses on the y-axis indicates the estimated degrees of freedom. The solid line indicates the smoothed parametric trend, shaded areas indicate the pointwise 95% CI.(EPS)Click here for additional data file.Table S1Overview of zooplankton datasets and their respective monitoring programs.(DOCX)Click here for additional data file.Table S2List of environmental variables used as covariates in the Generalized Additive Models (GAM) of at least one copepod species together with the definition and data source of each variable.(DOCX)Click here for additional data file.Table S3Summary of Generalized Additive Models (GAM) that performed best (based on F-ratio tests) in modeling seasonal biomass anomalies of each of our model species as a function of time.(DOCX)Click here for additional data file.


We proposed that the strong binding between Cd2+ and halide ions reduced the reactivity of the precursors, decreased the nuclei formed in the nucleation stage, and led to the high concentration of precursor in the growth stage, resulting in the increase of size and phase transformation of CdS nanocrystals. In addition, it was found that the multi-armed CdS nanocrystals lost quantum confinement effect because of the increase of the size with the increase of the concentration of CTAC.Monodispersed CdS nanocrystals with controllable shape and phase have been successfully synthesized in this study by adding cationic surfactant in noninjection synthesis system. With the increase of the amount of cetyltrimethylammonium chloride (CTAC) added, the shape of the CdS nanocrystals changed from spherical to multi-armed, and the phase changed from zinc-blende to wurtzite. It was found that halide ion Cl Colloidal semiconductor nanocrystals have received considerable attention because of their size-dependent properties and applications in fields such as optoelectronic devices -3 and biFor nanocrystal photovoltaic application, semiconductor nanorods or branched nanocrystals are preferable to quantum dots because they naturally provide direct electron transport path which can enhance charge collection efficiency . Peng anRecently, we have proposed a noninjection method to synthesize high-quality size-controllable CdS quantum dots in noncoordinating solvents without nucleation initiators . All the- plays a key role in the transformation, and other halide ions such as Br- can also induce similar transformation. Compared with alkylphosphonic acid and oleylamine, CTAC is quite economic. Therefore, we believe that this approach is suitable for large-scale production of branched CdS nanocrystals for photovoltaic application.In this article, we propose the shape and phase control of CdS nanocrystals based on a noninjection method. We found that addition of cationic surfactant cetyltrimethylammonium chloride (CTAC) could induce the phase transformation of the CdS nanocrystals from cubic to hexagonal structure, resulting in the formation of multi-armed CdS nanocrystals. It was found that halide ion ClCadmium oxide  and sulfur (99.5%) were purchased from Shanghai Chemical Reagent Ltd.(Shanghai China). OA (tech. 90%), 1-octadecene , and cetyltrimethylammonium hydroxide (CTAOH) were purchased from Aldrich . CTAC and cetyltrimethylammonium bromide (CTAB) were purchased from Aladdin Chemistry (Shanghai China). All the chemicals were used as received without any further purification.A mixture of CdO (1 mmol), OA (6 mmol), and 15 mL ODE was degassed at room temperature for 15 min in a three-necked flask and then heated to 280°C under Ar flow. After the CdO was totally dissolved and the solution turned clear to form a Cd-oleate precursor solution, the temperature of the solution was lowered to about 30°C. 0.5 mmol S and cationic surfactant  with different amounts were added into the flask. Then, the mixture was heated to 240°C at a rate of approx. 20°C/min and reacted at this temperature for 60 min under a flow of argon gas. The resultant nanocrystals were collected by precipitation with excess ethanol followed by centrifugation, and the product could be redispersed in hexane for characterization.Transmission electron microscopy (TEM) images were collected using a Philips CM200 transmission electron microscope operating at 160 kV. High-resolution TEM (HRTEM) measurements were carried out using a JEOL 2010F equipment operating at 200 kV. A sample for TEM analysis was prepared by drying a drop of nanocrystal hexane solution on a carbon-coated copper grid and letting it dry in air. X-ray powder diffraction (XRD) was conducted on an X'Pert pro X-ray diffractometer. The samples were prepared by drop casting the nanocrystal hexane solution on glass slides. Ultraviolet-visible (UV-Vis) absorption spectra were recorded on a U-4100 Spectrophotometer.Selecting proper surfactants has been found to be the key to control the shape and phase of colloidal nanocrystals ,21. In oTo investigate the morphology transformation induced by the addition of CTAC, XRD analysis was employed for determining the crystal structure of the CdS nanocrystals. The XRD patterns shown in Figure For a more detailed structural characterization of the multi-armed CdS nanocrystals, HRTEM images of the sample with 0.5 mmol CTAC added were recorded. Figure Compared to our previous study , the onl- and the surfactants OA and oleylamine. Tai et al. [- favors the formation of wurtzite CdS. According to the "hard-soft acid-base model," [2+ is a soft acid, and Cl- is a soft base. Soft acids bind strongly to soft base, so Cl- binds strongly to Cd2+, which reduces the reactivity of the precursors. As a result, the nuclei formed in the nucleation stage decrease, and the precursors remained for the growth of the nuclei increase, leading to the formation of larger CdS nanocrystals. Earlier study has proven that the structure of CdS nanocrystals is size dependent [Recently, other research groups also found that halide ions could influence the growth of CdS nanocrystals significantly. Saruyama et al.  reportedi et al.  showed t model,"  Cd2+ is ependent -33. The ependent . Howeverependent . UnfortuFigure - must be responsible for the phase transformation from zinc blende to wurtzite, and other halide ions such as Br- can also induce similar transformation. It is believed that this scheme can be extended to synthesize other semiconductor nanocrystals with complex structure.We report the successful shape and phase control of CdS nanocrystals using cationic surfactant CTAC in noninjection synthesis. Multi-armed CdS nanocrystals with zinc-blende cores and wurtzite arms are synthesized economically, which are suitable for nanocrystal photovoltaic application. It is confirmed that ClCTAB: cetyltrimethylammonium bromide; CTAC: cetyltrimethylammonium chloride; CTAOH: cetyltrimethylammonium hydroxide; HRTEM: high-resolution TEM; OA: oleic acid; ODE: 1-octadecene; TEM: transmission electron microscopy; UV-Vis: ultraviolet-visible; XRD: X-ray powder diffraction.The authors declare that they have no competing interests.ZY carried out the experiments and drafted the manuscript. LD participated in the discussion of the results and in revising the manuscript. YD supervised the work and revised the manuscript. All authors read and approved the final version of the manuscript.


Single visit scale and polish is frequently carried out in dental practices however there is little evidence to support (or refute) its clinical effectiveness. The purpose of this research was to compare patient-reported outcomes between groups receiving a scale and polish at 6-, 12-, and 24-month intervals. Outcomes recorded included participants’ subjective assessment of their oral cleanliness; the perceived importance of scale and polish for oral health and aesthetics; and frequency at which this treatment is required.A practice-based randomised control trial was undertaken, with a 24-month follow-up period. Participants were healthy adults with no significant periodontal disease (BPE codes <3) randomly allocated to three groups to receive scale and polish at 6-, 12-, or 24-month intervals. Patient-reported outcomes were recorded at baseline and follow-up. Oral cleanliness was reported using a 5-point scale and recorded by examiners blinded to trial group allocation. A self-completed questionnaire enabled participants to report perceived importance of scale and polish , and required frequency of treatment . The main hypothesis was that participants receiving 6-monthly scale and polish would report higher levels of oral cleanliness compared to participants receiving scale and polish at 12- and 24-month intervals.369 participants were randomised: 125 to the 6-month group; 122 to the 12-month group; and 122 to the 24-month group. Complete data set analysis was carried out to include 107 (6-month group), 100 (12-month group) and 100 (24-month group) participants. Multiple imputation analyses were conducted where follow-up data was missing. The difference in the proportions of participants reporting a 'high’ level of oral cleanliness at follow-up was significant (Chi-squared P = 0.003): 52.3% (6-month group), 47.0% (12-month group) and 30.0% (24-month group). Scale and polish was thought to be important by the majority in each group for keeping mouths clean and gums healthy, whitening teeth, and preventing bad breath and tooth decay; there were no statistically significant differences between groups at follow-up. Most participants at follow-up thought that the frequency of scale and polish should be “every 6 months” or more frequently: 77.9% (6-month group), 64.6% (12-month group), 71.7% (24-month group); differences between groups were not statistically significant (Chi squared P = 0.126). The results suggest that participants in the 24-month trial group were more likely to choose a scale and polish interval of “once a year” or less frequently .The majority of healthy adults regarded 6-monthly single-visit scale and polish as being beneficial for their oral health. Receiving the treatment at different frequencies did not alter this belief; and those with the longest interval between scale and polish provision perceived that their mouth was less clean. In the absence of a strong evidence base to support (or refute) the effectiveness of single-visit scale and polish, the beliefs and preferences of patients regarding scale and polish may be influential drivers for maintaining provision of this treatment. Patients attending primary care dental practices may receive 'scale and polish’  as part of their dental care; the treatment is traditionally linked to a routine, usually 6-monthly, dental check-up . This isA recent Cochrane systematic review investigated 'routine scale and polish for adult periodontal health’ and was unable to identify strong evidence to support (or refute) beneficial (or harmful) effects of the treatment; furthermore, the authors were unable to conclude the optimal frequency at which scale and polish should be provided . Given tA practice-based randomised controlled trial (RCT) was carried out in the North West of England (during the period February 2006 - September 2009) to investigate the health outcomes of single-visit scale and polish when this is provided at different frequencies. The objective clinical findings  have been published separately ; this paThe trial protocol was reviewed and approved by Cheshire Local Research Ethics Committee (reference Q/1506/100.) Trial registration was with UKCRN (ID 5101); and ISRCTN (ISRCTN56889016). Core funding of the trial was provided by The Oral Health Unit of the National Primary Care Research and Development Centre at The University of Manchester. Cheshire and Merseyside Comprehensive Research Network  awarded research support costs.The study was a randomised 3-armed parallel trial with an allocation ratio of 1. Recruited dental practice patients were recalled for routine dental check-ups and provision of scale and polish  over a period of 2 years; this 24-month follow-up equates to the maximum period advised between routine dental recall appointments .Participants were recruited from three large family dental practices in North West England. Patients, aged 18–60 years, scheduled for a routine dental check-up were sent an information leaflet about the trial and allocated an appointment during a specified trial recruitment session. Individuals who were interested in trial participation were screened by independent trial examiners to ensure that they fulfilled the eligibility criteria , was carried out. In order to detect clinically significant differences in proportions of participants with gingival bleeding, and allowing for 20% loss to follow-up, 369 patients were required to consent to participate in the trial.Participants were stratified according to the presence or absence of supragingival calculus deposits at baseline and the trial manager used minimisation ,14 to geet al.[All participants received a single-visit scale and polish at baseline. This was carried out by one of nine practice hygienists and therapists, all of whom were appropriately qualified, registered with the regulatory body in the UK  and experienced in delivery of scale and polish. The definition of single-visit scale and polish used by Beirne et al. was usedet al..Participants were recalled for dental check-ups with their family dentist at 6-monthly intervals, which included monitoring their periodontal health using BPE. Immediately after their dental appointment they attended a hygienist or therapist for standardised oral hygiene instruction  and thei“How clean does your mouth feel on a scale of 1 to 5, where 1 is the least clean you could imagine and 5 is the cleanest you could imagine?” Data were recorded on specially designed paper forms in the surgery.Measurements were taken at baseline and 24-month follow-up. Independent trial examiners asked patients to assess their oral cleanliness on a five-point scale in response to the question, A short, self-completed questionnaire was delivered in the practice waiting area prior to patients undergoing their baseline and follow-up examinations. Participants were asked to rate the importance of scale and polish  for oral cleanliness; maintenance of gum health; prevention of halitosis; prevention of dental decay; and “whiteness” of teeth. Scores ranged from '1′ indicating 'of no importance at all’ to '5’ indicating 'extremely important’.The groups were labelled to conceal the identity of the intervention and permit blind statistical analysis. PASW Statistics 18  and STATologit) [A Kruskal-Wallis test was used to compare ordinal outcomes between groups at follow-up. This data included subjectively-reported levels of oral cleanliness, and questionnaire responses. The subjectively-reported oral cleanliness scores were dichotomised: participants scoring 4, or 5 were defined as reporting a 'High’ level of oral cleanliness. Similarly, the questionnaire data regarding frequency of scale and polish was dichotomised: 'Every six months or more frequently’; and 'Every year or less frequently’. A chi-squared test was used to compare the binary outcomes between treatment groups at follow-up. Logistic regression, adjusted for the baseline values of each outcome, was used to calculate the odds ratios for binary outcomes. An ordered logit model (procedure ologit)  was usedmi logit procedure for binary outcomes and mi ologit procedure for ordinal outcomes [Multiple imputation (n = 100 imputations)  was perfoutcomes  with theBoth complete dataset and multiple imputation analyses are presented in Tables A CONSORT diagrammatical representation of participant flow  is preseAt follow-up Table , particiThe distributions of patient-reported scores for importance of scale and polish with respect to oral health  were not significantly different between groups at follow-up Table .The majority of participants at follow-up  reported that they required a scale and polish every 6 months or more frequently  the scale and polish intervention or the frequency of its provision often 6-monthly) to maintain or improve periodontal health -monthly . Whilst In this trial, participants in the 24-month group were more likely to feel they needed scale and polish every year or less frequently. It is possible that receiving a less frequent scale and polish regime 'enforced’ as a result of trial participation influenced participants in the 24-month group to feel that they needed this treatment less frequently. However, it must be remembered these patients were recalled at 6-monthly intervals during the trial for a dental check-up with their 'family dentist’  In England, NHS non-exempt dental patients currently pay the same treatment charge for their dental check-up whether or not they have a scale and polish; so under the current contract, there appears to be little financial benefit to the NHS if intervals between scale and polish for low risk patients are extended but the dental recall period remains 6-monthly. Had dental recall frequency been adjusted in accordance with trial scale and polish provision, patient-reported findings may have been different. However, modifying recall intervals (and remuneration for treatment provision), and economic analysis of treatment frequency was not a feature of this preliminary trial; further research including investigation of these issues is required before confident conclusions can be drawn.Evidence-based practice requires clinicians to make decisions which are underpinned by a combination of available evidence, their own experience and patients’ wishes . Given aThis trial raises important issues regarding patient perceptions of scale and polish and the required frequency of its provision.Scale and polish appears held in high regard by regularly-attending healthy adult patients; and receiving the treatment at different frequencies during the trial did not change this. However, groups who had longer intervals between scale and polish imposed upon them appeared to perceive that the treatment could reasonably be provided annually or less frequently.Currently, in the absence of a strong evidence base to support (or refute) the clinical effectiveness of single-visit scale and polish, the beliefs and preferences of patients regarding scale and polish are likely to be influential drivers for treatment provision.A requirement for further research investigating different aspects of scale and polish provision has been previously highlighted. In addition, the effects of various methods of oral hygiene instruction on oral health, as adjuncts to the professional clinical intervention, require further investigation .As the evidence base for scale and polish develops to a stage at which clear guidelines can be developed, it is important that policy-makers engage appropriately with dental professionals and patients as part of the decision-making process. A combination of appropriate communication, support, and professional incentives will be required to overcome barriers and facilitate any future proposed changes to primary care based (state-funded) scale and polish provision.The authors declare that they have no competing interests.Martin Tickle and Keith Milson were responsible for the initial research question. Clare Jones, Martin Tickle, Keith Milson, and Tatiana Macfarlane drafted the study protocol which detailed the trial methodology. The delivery of the trial within the practices, and the collection and management of trial data was the responsibility of Clare Jones. Philip Ratcliffe and Annette Wyllie both contributed clinical and practical information for the development of the trial protocol; and both assisted in the operational management of the study. Tatiana Macfarlane, the trial statistician, was responsible for the design and conduct of the analyses. This paper was initially drafted by Clare Jones and Martin Tickle following which all the authors provided comments. All have read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/13/50/prepub


The corpus callosum is hypothesized to play a fundamental role in integrating information and mediating complex behaviors. Here, we demonstrate that lack of normal callosal development can lead to deficits in functional connectivity that are related to impairments in specific cognitive domains. We examined resting-state functional connectivity in individuals with agenesis of the corpus callosum (AgCC) and matched controls using magnetoencephalographic imaging (MEG-I) of coherence in the alpha (8–12 Hz), beta (12–30 Hz) and gamma (30–55 Hz) bands. Global connectivity (GC) was defined as synchronization between a region and the rest of the brain. In AgCC individuals, alpha band GC was significantly reduced in the dorsolateral pre-frontal (DLPFC), posterior parietal (PPC) and parieto-occipital cortices (PO). No significant differences in GC were seen in either the beta or gamma bands. We also explored the hypothesis that, in AgCC, this regional reduction in functional connectivity is explained primarily by a specific reduction in interhemispheric connectivity. However, our data suggest that reduced connectivity in these regions is driven by faulty coupling in both inter- and intrahemispheric connectivity. We also assessed whether the degree of connectivity correlated with behavioral performance, focusing on cognitive measures known to be impaired in AgCC individuals. Neuropsychological measures of verbal processing speed were significantly correlated with resting-state functional connectivity of the left medial and superior temporal lobe in AgCC participants. Connectivity of DLPFC correlated strongly with performance on the Tower of London in the AgCC cohort. These findings indicate that the abnormal callosal development produces salient but selective  resting-state functional connectivity disruptions that correlate with cognitive impairment. Understanding the relationship between impoverished functional connectivity and cognition is a key step in identifying the neural mechanisms of language and executive dysfunction in common neurodevelopmental and psychiatric disorders where disruptions of callosal development are consistently identified. Consisting of over 190 million axons, the corpus callosum is the largest white matter tract and is hypothesized to play a primary role in cognition Individuals with agenesis of the corpus callosum (AgCC), including those with complete (cAgCC) and partial (pAgCC) absence of the corpus callosum, present a unique opportunity for understanding the relationship between the development of callosal integrity and cognitive processing. For instance, there is emerging evidence indicating that even in individuals in the normal IQ range, anatomically overt abnormalities of callosal development (including its complete absence) lead to deficits in cognitive abilities. Primarily, individuals with AgCC show deficits in both problem solving abilities Collectively, these findings suggest that an intact corpus callosum contributes significantly to efficient executive function, or the execution and maintenance more generally of complex cognitive abilities To address these questions, we used magnetoencephalography (MEG) to examine resting state functional connectivity in AgCC individuals and age- and IQ-matched healthy controls. MEG permits the imaging of brain activity in the sub-millisecond time-scale by measuring magnetic field changes on or near the scalp surface. Whole brain activity can be estimated from MEG sensor data using source reconstruction algorithms that allow one to overlay cortical oscillatory activity onto subjects' anatomical images in order to localize changes in cortical oscillations to specific brain regions. This process is referred to as magnetoencephalographic imaging (MEG-I). Advances in statistical signal processing have improved the spatiotemporal resolution of MEG-I and have also more recently been adapted to interrogate functional interactions in cortical oscillatory activity across brain regions and frequency ranges In the present study, we measured MEG-I resting-state connectivity to test the hypothesis that AgCC individuals have decreased functional connectivity in comparison to age and IQ-matched controls. It has been proposed that ipsilaterally projecting fibers that develop in callosal agenesis patients (i.e. Probst bundles) This study was approved by the UCSF Committee on Human Research, and all experiments were conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent to participate. Additional written consent was obtained from the parents/guardians of the participants involved who were under the age of 18.This study involved 18 patients with agenesis of the corpus callosum (AgCC) aged 17–57 years  recruited through the UCSF Comprehensive Center for Brain Development  and 2. Sa priori for correlations with the functional connectivity data: the Combined Color Naming and Word Reading verbal processing speed (VPS) measure from the Color-Word Interference Test (CWIT-VPS) and the Total Achievement problem-solving (PS) measure from the Tower Test (TT-TA). Although a full D-KEFS was obtained in each participant, we chose these measures as they probe domains of cognitive function that, based on ongoing work in our laboratory, appear to be central to observed behavioral impairments in patients with AgCC. The CWIT-VPS score is representative of basic processing speed (CWIT) Both individuals with AgCC and healthy controls were evaluated with either the Wechsler Adult Intelligence Scale-III (WAIS-III) The D-KEFS behavioral measures were analyzed with STATA version 10 using a one-way MANOVA with corpus callosum status  as the independent multi-level variable and the participants' score on the TT-TA and CWIT-VPS as the continuous dependent measures. The one-way MANOVA comparing an overall effect of group  by test (TT-TA and CWIT-VPS) revealed no statistical group differences  = 0.42, p = 0.7). The group means were lower and variance was greater in the AgCC cohort for both TT-TA  and CWIT-VPS  scores when compared to healthy controls. Although we do not observe significant differences in these measures within our sample, in a similar analysis with a larger cohort that included subjects from this study, there are indeed significant differences in problem solving and processing speed in AgCC Structural (T1-weighted) anatomical images were acquired for source space reconstruction, data visualization and second-level group analyses. Scanning was performed using a 3.0T GE Trio scanner installed at Surbeck Lab for Advanced Imaging at the UCSF China Basin campus. For each subject, a 3D-FSPGR high-resolution MRI was acquired .Data were collected from patients and controls using a whole-head biomagnetometer  containing 275 axial gradiometers at a sampling rate of 1200Hz. For registration, coils were placed at the nasion and 1 cm rostral to the left and right preauricular points angled toward the nasion in order to localize the position of the head relative to the sensor array. These points were later co-registered to a high-resolution structural MR image through a linear affine transformation between fiducial points, MEG device coordinates and MRI coordinates . Scan sessions where head movement exceeded 0.5 cm within a run were discarded and repeated. Participants were lying in a supine position and instructed to remain awake with their eyes closed during a four-minute continuous recording session.http://nutmeg.berkeley.edu). In each subject, a local sphere was generated based on registration between each MEG sensor and the head surface obtained from the subject's T1-weighted structural MRI, resulting in a multiple local-sphere head model From the four minute spontaneous (awake) recording session, a single, contiguous 60s artifact-free segment of the MEG data was selected for analysis. The full recording session was not used as nearly all participants had at least one noisy, transient (<60s) segment of the data that was excluded from the analysis. Furthermore, previous investigations have determined that this 60s window provides reliable, consistent power for reconstruction of brain activity from the resting-state MEG data MEG sensor data were downsampled to 600Hz, bandpass filtered (fourth-order Butterworth filter) and reconstructed in source space using a minimum-variance adaptive spatial filtering technique For each subject, power spectrograms of this 60s epoch of the sensor data were generated using FFT  in order to select a subject-specific alpha band (identified as the greatest power density within the 8–12 Hz range). A peak in the alpha band was easily identifiable from this amount of data in both AgCC patients and controls . Visual http://www.fil.ion.ucl.ac.uk/spm/software/spm2). This process created a non-linear transformation matrix for each subject that, was then applied to each individual subject's GC map Functional connectivity was estimated using imaginary coherence (IC), a metric that overcomes estimation biases in MEG source data by isolating non-zero time-lagged interactions A comparison between the AgCC connectivity maps relative to HC connectivity was performed using a non-parametric unpaired two-tailed t-test A post-hoc analysis to assess within intrahemispheric connectivity and interhemispheric connectivity was conducted using region of interest (ROI) analysis. The ROIs were defined based on the difference in the GC maps of the total AgCC group vs. HC. The euclidian midpoint or center of mass was computed from each significantly different cluster and a seed voxel (20 mm) was generated at this location for each subject. Global functional connectivity  was averaged across each subject for the connections of the ROI within  and across  hemispheres. This type of data reduction gives us a single point value (GC average) for a single seed of its connections either within the ipsilateral hemipshere (intrahemispheric connectivity) or between that seed and all other voxels in the contralateral hemisphere (interhemipsheric connectivity). Differences between and within groups were assessed using a 2×2×2 mixed-effects ANOVA varying group (HC vs. AgCC), hemisphere (left vs. right) and connection type (intrahemispheric vs. interhemispheric). Given the reduced sample size (n = 9 in each group) when the AgCC cohort is split into pAgCC and cAgCC subtypes, we also conducted an exploratory planned comparison directly comparing the intra- and interhemsipheric connections between the two groups using two-tailed unpaired t-tests.In order to derive an estimate of within-session reliability of resting-state functional connectivity analyses using IC, we calculated the intra-class correlation coefficient ICC;  between r correlation between GC maps and age-scaled scores from the D-KEFS battery. Data was collapsed across pAgCC and cAgCC groups to maximize power in our analyses.To explore the relationship between connectivity and cognition, we did a voxel-wise Pearson's For all voxelwise fcMEG-I analyses  we performed a multiple comparisons correction using a False Discovery Rate (FDR) set at 10%. When effects were significant, we explored how robust these effects were by applying more stringent thresholds, including a lower FDR q-value (either 5% or 1%), or a Family-Wise Error (FWE) rate. We report the most conservative multiple comparisons correction (either FDR or FWE) still producing significant effects here.We hypothesize that congenital absence of the corpus callosum will alter interhemispheric functional connectivity throughout the cortex. Furthermore, given our prior findings of reduced volume and FA of the ventral cingulum in AgCC individuals In the alpha (8–12 Hz) range, greatest global connectivity values were present across the same regions of parietal, temporal and occipital cortices in both the AgCC and HC groups . SignifiIn the frontal lobe, a cluster of voxels over the middle frontal gyri in the left and right dorsolateral prefrontal cortices (DLPFC) were significantly . Likewise, no significant differences were identified for a group comparison between HC and AgCC for the resting-state gamma reconstructions .In AgCC individuals, neuroanatomical changes extend beyond just an impairment or absence of callosal development. In many individuals in this study, the commissural axons that did not cross the midline remained within the same hemisphere, forming Probst bundles For the DLPFC seeds, we were unable to identify a main effect of connection type or hemisphere, and there were no significant two- or three-way interactions ; Table 4In order to determine if connectivity of cortical fields outside of the three ROI definitions  were missed in our analysis of intra- and interhemispheric connectivity, we conducted a secondary analysis splitting these two connection types across all voxels. Here, connectivity maps were generated by collapsing across all IC values of a single voxel either with every voxel within the same hemisphere (intra-hemispheric connectivity) or with every voxel in the contralateral hemisphere (inter-hemispheric connectivity). Comparisons between the HC and AgCC groups were then performed using non-parametric unpaired t-tests. For intrahemispheric connections, significant reductions in coherence were observed over DLPFC, PPC and PO bilaterally  with no In order to examine potential differences between patients with complete callosal absence (cAgCC) and those with remnant callosal fibers (pAgCC), we also conducted a planned comparison between these two groups for the connection types of each ROI. For intrahemispheric connections, no significant differences , functional connectivity of two regions in pre-frontal cortex (PFC) positively correlated with TT-TA scores in individuals with AgCC. The first area, in an anterior region of medial PFC mPFC;  was posiThis study provides compelling evidence that the absence of a fully developed corpus callosum impacts functional interactions both within each hemisphere and between the two hemispheres in the resting alpha frequency range (8–12 Hz). This impairment of functional interactions appears to be most evident in regions within the frontal, parietal and occipital cortices that are implicated in cognitive domains known to be impaired in individuals with AgCC. We also found that the degree of diminished connectivity in specific cortical regions was directly correlated with verbal processing speed and executive performance in these individuals. In our sample, there was substantial anatomical  and behavioral (processing speed and/or problem solving impairment) variability, similar to what has been identified in the general AgCC population The posterior resting-state alpha network itself is not grossly redistributed in individuals with AgCC . InsteadPrevious studies using fMRI or EEG have suggested decreased connectivity in individuals with AgCC While previous studies have suggested that, in agenesis of the corpus callosum, interhemispheric interactions are severed while intrahemispheric connections are relatively spared  and gamma (30–55 Hz) frequency bands were reconstructed and identified in both the HC and AgCC groups, no significant group differences in functional connectivity were found in either frequency band. Although some signal is present in the resting-state MEG recordings in these higher frequency bands , it is cThe examination of spontaneous brain activity and, more specifically, interregional interactions using neuroimaging has recently emerged as a powerful research approach in determining both standard and pathological brain states, as it does not rely on patient compliance or the need to average data across multiple trials for sufficient power Poor performance on both tests of verbal processing speed and problem solving was found to be related to low levels of functional connectivity in specific regions of the brain. For the verbal processing speed score, two regions of the left temporal lobe  were positively and significantly correlated with this measure . These rr scores . While nIt is also possible that concomitant anatomical tracts that develop specifically in agenesis patients such as Probst and heterotopic bundles In conclusion, MEG-I can be used to identify how alterations in neuroanatomical connectivity  translate into faulty functional coupling in a discrete frequency range  both between and within the cerebral hemispheres, even within the brain's “resting state”. In AgCC, the degree of disconnection that occurs when callosal fibers fail to develop is directly related to processing speed and problem solving, indicating that these measures represent real-time brain interactions. These findings also serve to validate MEG-I as a tool to understand the impact of compromised cortical interactions on essential cognitive abilities


We studied the cytotoxicity of acadesine(5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) for tumor and normalcells of various species and tissue origin. In tumor cells, acadesine triggerednon-apoptotic death; the potency of the compound to normal cells wassubstantially lower. Acadesine was toxic for tumor cells with multidrugresistant phenotypes caused by the transmembrane transporter Р-glycoprotein orlack of proapoptotic p53. Activity of adenosine receptors was required foracadesine-induced cell death, whereas functioning of АМР-dependent proteinkinase was not required. A more pronounced cytotoxicity for tumor cells, aswell as the non-canonical death mechanism(s), makes acadesine a promisingcandidate for antitumor therapy. Acadesine obtained according to the authors’procedure was used for further experiments. Table 1that P388 (murineleukemia) and Sp2/0 (murine myeloma) cells exhibit the highest sensitivity toacadesine: ~ 1/3 of the cell population survives at an acadesine concentrationof 0.125 mM. Sub-millimolar concentrations of acadesine also cause the death ofother transformed cell lines. It is important to note that the acadesinecytotoxicities are almost identical for the K562 leukemia cell line and itssubline with Pgp-mediated MDR (K562/4). This is also valid for a MCF-7 breastadenocarcinoma cell line and the MDR subline (Table 1). Thecomparison of the cytotoxicities of acadesine for a HCT 116 line and HCT116p53KO subline (resistant to a number of DNAdamaging anticancer drugs) [It follows from the data listed in r drugs)  has demoTable 1).Thus, acadesine primarily causes the death of transformed cells , including the sublines resistant to other anticancer drugs.Nontumor cells are damaged by acadesine to a significantly lesser extent. Thesefeatures speak to the potential of using acadesine as an antitumor agent.However, the mechanisms that underline the toxicity of acadesine for tumorcells need to be understood.The considerably higher survival rate of nontumor cells in the presence ofacadesine is also important: death of the donor’s lymphocytes andnon-transformed fibroblasts was virtually absent even when continuously exposedto acadesine at millimolar concentrations for 72 h  were observed 24 and 48 h,respectively, after the introduction of acadesine (0.25 mM). Accumulation offragmented DNA can be indicative of apoptotic cell death if the DNA is split ininternucleosomal regions, which can be seen from the formation of a number of140- to 170-bp-long fragments during electrophoresis. In order to verify thisidea, DNA integrity in acadesine-treated HCT 116 cells was determined. Itturned out that acadesine, as opposed to the control compound .The effect of acadesine on ploidy distribution in a HCT 116 large intestineadenocarcinoma cell line was studied by flow cytofluorometry. Arrest in the Sphase and massive cell death   argue in favor of a nonapoptotic mechanism of deathof НСТ116 cells under the action of acadesine. Annexin V binds tophosphatidylserine on the plasma membrane (translocation of phosphatidylserinefrom the inner lipid layer of the membrane to the outer one is considered to bea sign of apoptosis). Propidium iodide is capable of penetrating into cellsundergoing necrosis (disrupting the integrity of the plasma membrane).Acadesine-treated НСТ116 cells  were not stained with AnnexinV-FITC ; contrariwise, the cells accumulated propidium iodid , which allows one to hypothesize about a necrotic component of thecell death mechanism. Similar results were obtained when necrotic cells werevisualized using trypan blue (data not shown). The disruption of the integrityof the plasma membrane is presumably a late event during acadesine-induced celldeath. The control agent, alkyl cationic glycerolipid, caused an increase inthe number of Annexin V-positive cells, which is typical of apoptosis. Since apoptotic cell death assumes that caspasesplay a significant role in it, the effect of the pan-caspase inhibitor zVAD-fmkon acadesine cytotoxicity was studied. HCT 116 cells were incubated with 200 μMzVAD-fmk for 30 min; acadesine was then introduced in the cultures, and theincubation was carried out for an additional 24 h. The presence of zVAD-fmk didnot reduce cell death, which supports the conclusion about the non-apoptoticmechanism of the cytotoxicity of acadesine.The results of staining cells with Annexin V-FITC and propidium iodide(Interaction with the adenosine receptors is required to ensure the deathof tumor cells under the action of acadesineTable 2).Acadesine can be transferred from the extracellular space into the cells byadenosine transporters . We haveTable 2). Hence, cell death inresponse to acadesine is not caused by the formation of ZMP or activation ofAMPK.In order to shed light on the role of the metabolic pathway acadesine–ZMP–AMPKin the cytotoxicity of acadesine (its phosphorylation by adenosine kinaseyielding ZMP and activation by AMPK), cells were incubated with acadesine andthe adenosine kinase inhibitor 5-iodotubercidine. The inhibitor had no effecton acadesine cytotoxicity (Thus, the investigation into the mechanisms of acadesime cytotoxicity hasrevealed a number of features indicating a nontrivial nature of thepharmacological effects of this compound. Acadesine triggers death in culturedtumor cells, while its effect on nontumor cells is pronounced to a considerablylesser extent. Acadesine is toxic for cells with molecular determinants of drugresistance: Pgp expression and non-functional p53. It is important to emphasizethe non-apoptotic character of the tumor cell death induced by acadesine. Theseresults provide grounds for regarding acadesine as a crucial agent for studyingthe mechanisms of tumor cell death and a promising drug candidate.The question regarding the intracellular target of acadesine, the interactionwith which causes tumor cell death, remains open. We have demonstrated that thefunction of adenosine transporters is the criterion for cell death, while noactivation of AMPK is required. It is reasonable to assume that tumorsexpressing the aforementioned adenosine transporters and receptors will exhibitthe highest sensitivity to acadesine. The role of the purine nucleotidetransport in cell death has yet to be studied sufficiently; an analysis of thedifferential expression of adenosine carriers and receptors in different typesof tumors is required. The enhanced expression of these molecules may turn outto be a novel molecular marker of tumor sensitivity to acadesine and acriterion for the selection of patients for proper therapy.


As an obligate intracellular parasite, Apicomplexa interacts with the host in the special living environment, competing for energy and nutrients from the host cells by manipulating the host metabolism. Previous studies of host-parasite interaction mainly focused on using cellular and biochemical methods to investigate molecular functions in metabolic pathways of parasite infected hosts. Computational approaches taking advantage of high-throughput biological data and topology of metabolic pathways have a great potential in revealing the details and mechanism of parasites-to-host interactions. A new analytical method was designed in this work to study host-parasite interactions in human cells infected with Plasmodium falciparum and Cryptosporidium parvum.We introduced a new method that analyzes the host metabolic pathways in divided parts: host specific subpathways and host-parasite common subpathways. Upon analysis on gene expression data from cells infected by Plasmodium falciparum or Cryptosporidium parvum, we found: (i) six host-parasite common subpathways and four host specific subpathways were significantly altered in plasmodium infected human cells; (ii) plasmodium utilized fatty acid biosynthesis and elongation, and Pantothenate and CoA biosynthesis to obtain nutrients from host environment; (iii) in Cryptosporidium parvum infected cells, most of the host-parasite common enzymes were down-regulated, whereas the host specific enzymes up-regulated; (iv) the down-regulation of common subpathways in host cells might be caused by competition for the substrates and up-regulation of host specific subpathways may be stimulated by parasite infection.Results demonstrated a significantly coordinated expression pattern between the two groups of subpathways. The method helped expose the impact of parasite infection on host cell metabolism, which was previously concealed in the pathway enrichment analysis. Our approach revealed detailed subpathways and metabolic information are important to the symbiosis in two kinds of the apicomplex parasites, and highlighted its significance in research and understanding of parasite-host interactions. Living organisms gain energy from and discharge wastes to their environment. In the case of obligate intracellular parasites, living environment changes with the internal biochemical state of hosts. The need of intracellular parasites to retrieve nutrients and fulfill their energy requirements is achieved by manipulating the host metabolism. Such parasitic relationships, as well as other forms of species-to-species interactions, set the selective pressures to shape the evolution of each species . TherefoMany different approaches have been developed to study the possible interactions of host and parasite in metabolic pathways. Some traditional cellular and biochemical methods investigating protein or molecular functions in metabolic pathways were used in previous studies ,5. CompuA study on malaria previously reported by Ockenhouse, C. F. et al. provided the expression data of epithelial blood cell from acute infected patients and uninfected persons. The data was obtained from the GEO (GDS2362). Time series data were as retrieved from a work on the expression of Cryptosporidium parvum infected HCT-8 ileocecal epithelial cells at various time points, including 6 time points, up to 72 hours after infection with Cryptosporidium parvum (GDS2240). KEGG orthology (KO) information and pathway information of both host’s and parasite’s genes were downloaded from the Kyoto Encyclopedia of Genes and Genomes (KEGG) . The gen2-fold changes greater than 1.5 and a P value less than 0.05 were considered significantly differentially expressed genes. A list of differentially expressed genes, their fold changes, and P values of t-test at different time points is provided in the additional files 1,2,3.The dataset of GDS2362 was obtained from Affymetrix Human Genome U95 Version 2 Array (GPL8300), and that of GDS2362 from Affymetrix Human Genome U133A Array (GPL96). We used the BioConductor package ‘affy’ to perform the data preprocessing and normalization[http://www.genome.jp/kegg/). We identified the host-parasite ortholog gene pairs as: genes from the genomes of both host and parasite that belong to a group of ortholog genes in KEGG (one KO group).Original KEGG-defined ortholog genes were used in our analysis without any changes. KEGG provides a list of ortholog genes, which were downloaded from KEGG web site , which may reveal significance of gene-set wise alternation[Firstly, we tried to find which genes are differentially expressed after infection. Using false discovery rate (FDR) less than 0.01, we found a group of genes whose expressions were significantly altered when comparing the epithelial blood expression profile from acutely infected patient with from uninfected people. After that, we performed enrichment analysis to detect significantly altered expression pathways using the hyper geometric method (see ternation. The resThen we separated pathways into host specific pathways and host-parasite common pathways. According to the KEGG orthology (KO) information, we first identified 1357 genes for which host-parasite ortholog pairs exist, and 8092 genes which were found only in host. We defined a subpathway consisting of ortholog pairs as host-parasite common subpathway, and a subpathway consisting of host only genes as a host specific subpathway. We subsequently performed enrichment test on each subpathway. Filtering out the subpathway s that contain only one gene, the result showed 6 host-parasite common subpathways and 4 host specific subpathways were significantly altered . P. falciparum requires an extracellular supply of pantothenate to support its proliferation during the erythrocytic stage of its development in humans . We founOther detected pathways that have their subpathways up or down-regulated also show important aspects of plasmodium during its development. The host-parasite common subpathway of Linoleic acid metabolism (KEGG ID hsa00591) was gene enriched and showed a down-regulation pattern. Linoleic acid is well known for its promotion of beta-hematin formation in vitro. Previous study suggests that linoleic acid or diglyceride containing dilinoleolyglycerol plays the critical role in promoting FP polymerization in malaria parasites.Method), based on data from a previous study on human cell expression response[We use the same method as above analysis to detect DEGs . Here we did not include the dataset of infection after 6 hours which is still in the invasion stage, and different from others.Something interesting in the cells infected by the Plasmodium is that the number of pathways having a down-regulated pattern on ortholog genes grew with time, while the same manner appeared in the pathways with a significant alteration in ortholog genes. We excluded the result of 6 hours after infection which was still in the invasion stage, and Spearman correlation test showed positive correlation , indicating that as infection progressed, more host-parasite common subpathways were impacted, which displayed down-regulation pattern on enzymes or compounds in the corresponding subpathways. However, more samples at different times after infection are needed to further test and verify this trend.One factor that caused the down-regulation of host-parasite common subpathways in the metabolic pathways was competition of metabolites by the parasite. The gene in fatty acid metabolism pathway (KEGG ID hsa00071) which encodes the acyl-CoA synthetase long-chain family member 1 EC: 6.2.1.3) was significantly down-regulated. Previous study suggested that the enzymes encoded by ortholog of plasmodium, long chain fatty acid ligase (EC: 6.2.1.3), would enter the host cytoplasm through the apicomplex and was involved in the formation of Hexadecanoyl-CoA ,5. The a.1.3 was The host-specific subpathways might tend to have a up-regulate pattern because of the interference of parasite enzyme in the host-parasite common subpathways, as was shown in the numbers of host-specific subpathways in Plasmodium infected cells: 12 subpathways were enriched with up-regulated genes and 4 were enriched with down-regulated genes. This assumption could not be fully proved from the analysis of C. parvum . The expression pattern of host specific subpathways was rather complex as genes in these pathways were under complicated regulations. The topology of these subpathways may also contribute to the complexity: some of them are up-stream of the host-parasite common subpathways and some have parallel host-specific subpathways. Another reason for the variation to predict nutrition relationship between host and parasite might be due to the incompleteness of the pathways from which we retrieve the topological information. Because the KEGG pathway was structured mainly on the genome annotation from the sequence data of a few species, unavailablely there might be inaccuracy associated with some particular pathway. There would be gene merely similar at the sequence level, but it may not necessarily have the same function as the orthologs in other species. According to the gene annotation of KEGG database, a large number of putative genes were found in the KEGG metabolic pathway of these two species: Plasmodium falciparum and Cryptosporidium parvum. Care must be taken in metabolic pathways analysis, and more study needs to be done with updated pathway information.In this study, we introduced a new method for exposing the interactions between infected host cells and parasites: Plasmodium falciparum and Cryptosporidium parvum. We divided the host metabolic pathway into two parts: host specific subpathways and host-parasite common subpathways according to ortholog gene information of host and parasite. We performed analysis on the subpathways separately, and the results showed different character between these two groups subpathways. With this method, we revealed the impact of parasite infection on host cell gene expression, which was previously concealed in the pathway enrichment analysis. We provided the results from statistical analyses and visualized them by mapping the data to the KEGG pathway. Our approach revealed detailed subpathways and metabolic information that are important to the symbiosis in two kinds of the apicomplex parasites. The results highlight the significance of our approach in research and understanding of the interactions between parasites and host cells.The authors declare that they have no competing interests.TX ,PJ and PH conceived the study. TX and YY performed statistical analyses for the study, and drafted the manuscript. FY and YT advised on the analytical methods. XL directed the study, and reviewed and finalized the revised manuscript. All authors approved the final manuscript.Analysis results of gene expression data of Plasmodium infected human cell.The file “Additional file_Plasmodium.xls” contains expression level of genes and used 0/1 to indicate if they were DEGs. The table also shows the significantly up- or down- regulated host-parasite common or host specific subpathways according to the enrichment result.Click here for fileAnalysis results of gene expression data of C.parvum infected human cell.The file “Additional file_Cparvum.xls” contains differentially expressed gene list and the corresponding expression level of the genes. It also shows significantly up- or down-regulated host-parasite common or host specific subpathways in different time points.Click here for fileSignificantly altered pathways in C.Parvum infected human cell.It contains the significantly altered pathways after infection of C.Parvum.Click here for file


Hypoglycaemia and the fear of hypoglycaemia are barriers to achieving normoglycaemia with insulin. Insulin degludec (IDeg) has an ultra-long and stable glucose-lowering effect, with low day-to-day variability. This pre-planned meta-analysis aimed to demonstrate the superiority of IDeg over insulin glargine (IGlar) in terms of fewer hypoglycaemic episodes at equivalent HbA1c in type 2 and type 1 diabetes mellitus (T2DM/T1DM).Pooled patient-level data for self-reported hypoglycaemia from all seven (five in T2DM and two in T1DM) randomized, controlled, phase 3a, treat-to-target trials in the IDeg clinical development programme comparing IDeg once-daily (OD) vs. IGlar OD were analysed.95%CI, RR:0.6495%CI and RR:0.1495%CI. In the overall T2DM population, significantly lower rates of overall confirmed and nocturnal confirmed episodes were reported with IDeg vs. IGlar 95%CI and RR:0.6895%CI). In the T1DM population, the rate of nocturnal confirmed episodes was significantly lower with IDeg vs. IGlar during maintenance treatment . Reduction in hypoglycaemia with IDeg vs. IGlar was more pronounced during maintenance treatment in all populations.Four thousand three hundred and thirty subjects (2899 IDeg OD vs. 1431 IGlar OD) were analysed. Among insulin-naïve T2DM subjects, significantly lower rates of overall confirmed, nocturnal confirmed and severe hypoglycaemic episodes were reported with IDeg vs. IGlar: estimated rate ratio (RR):0.83The limitations of this study include the open-label design and exclusion of subjects with recurrent severe hypoglycaemia. This meta-analysis confirms that similar improvements in HbA1c can be achieved with fewer hypoglycaemic episodes, particularly nocturnal episodes, with IDeg vs. IGlar across a broad spectrum of patients with diabetes. Improving glycaemic control through timely and intensive diabetes treatment reduces the risk of developing complications in patients with diabetes Insulin degludec (IDeg) is an ultra-long-acting basal insulin in clinical development for the treatment of type 2 and type 1 diabetes mellitus (T2DM and T1DM). Soluble multi-hexamers are formed in the subcutaneous tissue upon injection, creating a depot from which monomers are slowly and continuously absorbed into the circulation The aim of this pre-planned meta-analysis was to show the superiority of IDeg over IGlar in terms of hypoglycaemic episodes at equivalent HbA1c using pooled, individual patient-level data from all phase 3 trials in the IDeg clinical development programme, as discussed in advance with the regulatory authorities. The treat-to-target trial design was adopted for all of the trials included in this meta-analysis and comparable titration algorithms were applied in an attempt to achieve similar glycaemic goals.This pre-planned meta-analysis included pooled patient-level data from all seven trials in the IDeg development programme that compared IDeg once daily (OD) vs. IGlar OD  Appendix  14–21. IAll trials were randomized, controlled, open-label, multicentre, phase 3a, treat-to-target trials of 26 or 52 week's duration Appendix . Key recThe meta-analysis plan was based on prior discussions with and subsequent review by regulatory authorities prior to the unblinding of data for the individual trials. The reviewer's comments were addressed with additional analyses, including analyses for hypoglycaemia during the maintenance phase (after stable glycaemic control and stable insulin dose had been achieved following active titration). Data for insulin-naïve T2DM subjects, all T2DM subjects and T1DM subjects were combined in the primary analysis and further analysed as separate populations. All randomized subjects were analysed following the intent-to-treat principle.The primary endpoint for this meta-analysis was overall confirmed hypoglycaemia , baseline characteristics and demographics were similar between groups Appendix . Among pInsulin-naïve subjects experienced significantly lower rates of overall confirmed episodes  and nocturnal confirmed episodes  with IDeg than with IGlar across the entire treatment period  and 3. TSignificantly lower rates of overall confirmed and nocturnal confirmed episodes were reported with IDeg than with IGlar in the overall T2DM population across the entire treatment period, and these differences were more apparent during the maintenance phase  and 3. TThe majority of the overall confirmed and nocturnal confirmed episodes in T1DM subjects were symptomatic . There was no significant difference between treatments in the rate of overall confirmed episodes across the entire treatment period, although it was slightly higher with IDeg than with IGlar  and 3. TSubjects in the pooled population experienced significantly lower rates of overall confirmed episodes  and nocturnal confirmed episodes  with IDeg than with IGlar across the entire treatment period  was demonstrated in the Forest Plots, both among the five trials involving T2DM subjects and, separately, between the two trials involving T1DM subjects . A high Two major strengths of this meta-analysis are the inclusion of all phase 3 trials comparing IDeg OD to IGlar OD, and its pre-planned design. The results of this meta-analysis further contribute to the evaluation of the risk-benefit profile of IDeg The lower rate of hypoglycaemia, particularly nocturnal hypoglycaemia, observed with IDeg across trials is likely a consequence of its ultra-long and stable pharmacokinetic profile, and lower day-to-day variability in glucose-lowering action Hypoglycaemia stems from an excess of insulin action relative to endogenous and exogenous glucose supply. Current basal–bolus therapy consists of rapid-acting bolus insulin that provides mealtime coverage, and basal therapy that provides coverage in the post-absorptive and fasting states. Nocturnal hypoglycaemic episodes are typically unrelated to the use of bolus insulin; hence, the rate of nocturnal episodes provides the most relevant standard of comparison for basal insulin preparations. Therefore, it is of interest to observe that the largest and most consistent differences in the rate of hypoglycaemia between IDeg and IGlar were observed during the nocturnal period, when glycaemic control is primarily affected by basal insulin and less so by bolus insulin . This observation is further supported by the higher rates of overall confirmed hypoglycaemia observed with both IDeg and IGlar in the trials that used the basal–bolus treatment  compared with trials that used the basal-only therapy .Although insulin-naïve T2DM subjects reported the fewest hypoglycaemic episodes, the lower rate of hypoglycaemia for IDeg compared with IGlar is noteworthy because the fear of hypoglycaemia is a major impediment to initiating the insulin treatment that will ultimately be required by many T2DM patients as the disease progresses The corresponding numbers for confirmed hypoglycaemia in T2DM subjects on a basal–bolus regimen are much higher: for every 100 subjects treated with IDeg instead of IGlar for 1 year, a total of 326 overall confirmed hypoglycaemic episodes, of which 71 are nocturnal confirmed episodes, will be avoided [calculated using the observed rates with IGlar in the maintenance period  may be unsuitable candidates for treatment to the glycaemic levels recommended for the general diabetes population. Further, inclusion of these patients could have introduced heterogeneity to this meta-analysis.In conclusion, hypoglycaemia is widely acknowledged as the main limiting factor to achieving tight glycaemic control. This pre-planned meta-analysis shows that similar improvements in HbA1c can be achieved with fewer hypoglycaemic episodes, particularly nocturnal episodes, with IDeg than with IGlar across a broad spectrum of patients with diabetes (particularly T2DM patients), and insulin regimens. The benefits of lower rates of nocturnal hypoglycaemia with IDeg over that of IGlar are most clearly demonstrated for both T1DM and T2DM patients during the maintenance phase of their treatment once the optimal dosage has been determined. The lower rates of hypoglycaemia observed with IDeg than with IGlar across individual trials, and the support of these findings in this meta-analysis, provide strong evidence of the benefits of IDeg in reducing the risk of hypoglycaemia in patients with diabetes, thus allowing safer and more intensive insulin titration to minimize diabetes complications.