The 13-valent pneumococcal conjugate vaccine (PCV13) is the only licensed PCV with serotype 3 polysaccharide in its formulation. Postlicensure PCV13 effectiveness studies against serotype 3 invasive pneumococcal disease (IPD) in children have shown inconsistent results.  We performed a systematic review and meta-analysis of observational studies to assess PCV13 vaccine effectiveness (VE) for serotype 3 IPD in children. We systematically searched PubMed, Embase, and the Cochrane library for studies published before 14 August 2017. We identified 4 published studies and 2 conference posters that provided PCV13 VE estimates stratified by serotype. The pooled PCV13 VE against serotype 3 IPD from the random-effects meta-analysis was 63.5% . A sensitivity analysis including conference posters gave a pooled VE estimate of 72.4% . The pooled data from case-control studies with similar methodologies and high quality support direct PCV13 protection against serotype 3 IPD in children. Meta-analysis of published case-control studies of 13-valent pneumococcal conjugate vaccine gave a pooled vaccine effectiveness of 63.5% against serotype 3 invasive pneumococcal disease (IPD). The pooled data from case-control studies with similar methodologies and high quality support direct protection against serotype 3 IPD in children. Streptococcus pneumoniae remains a significant cause of infection associated with high mortality and morbidity in children and adults ..12].I2 statistic to estimate the percentage of between-study heterogeneity [Using Stata version 14.0 software , we calculated a combined VE estimate using the Stata command Metan to fit a random-effects model . Inverseogeneity , 22.The initial search identified 3016 publications as potentially relevant . After sThe 4 published studies were either matched case-control (studies 1 and 2) , 13 or iIn the matched case-control study in the United States (study 1) , VE was In the indirect cohort studies, cases of nonvaccine-type IPD were used as controls  and VE eTwo of the published studies provided information on the vaccination status of the serotype 3 cases. In study 3 from Germany , 11 seroStreptococcus pneumoniae Invasive Disease network (SpIDnet), which is funded by the European Centre for Disease Prevention and Control (ECDC) to perform active population-based surveillance of IPD in children in the European Union [The authors of the current manuscript identified 2 scientific posters  , 13–15. I2 = 15.7%, P = .313; I2 of 0%  with low heterogeneity , there was no significant difference in the serotype 3 geometric mean concentrations postbooster between schedules . In any Of the 6 studies identified, 3 used the indirect cohort method , 15, 24,Our analysis has limitations. First, statistical tests that are used to exclude the hypothesis of a heterogeneity of effect estimates in a meta-analysis (the null hypothesis being the absence of heterogeneity) are not sensitive when the number of studies under review is limited . Second,The incidence of disease at the population level is the product of both direct and indirect effects, and available surveillance data have shown a low incidence of serotype 3 IPD in children after PCV13 introduction. For example, in the United States, the incidence of serotype 3 IPD among children aged <5 years decreased in the years immediately following PCV13 introduction, dropping from 1.1 cases per 100 000 in 2010 to 0.25 in 2013 . The incOther findings are important to consider when interpreting data on PCV13 impact against serotype 3. First, PCV13 vaccination leads to a reduction in carriage acquisition and thus indirect protection against vaccine-type disease. PCV13 does not appear to prevent serotype 3 carriage acquisition to the same extent as other vaccine serotypes; however, studies of serotype 3 carriage in children are difficult to interpret due to relatively low carriage prevalence , 36. NonA coherent hypothesis for PCV13 impact on serotype 3 should take into account all of these data: substantial direct protection; evidence of overall reductions in population-based incidence in the early, but not later, years following introduction; and lower protection afforded by PCV13 against serotype 3 relative to other vaccine serotypes. A recent study has also suggested that there has been a genetic shift from a relatively low to a relatively high antibiotic-resistant serotype 3 clade that was not driven by PCV13 use . One posAdditional research is needed. First, it is unknown whether the direct protection against serotype 3 is of shorter duration than against other serotypes. Some prelicensure clinical trials showed that the immune response for serotype 3 following the booster dose was not increased above the levels seen after the infant vaccination series, suggesting potential hyporesponsiveness . DespiteThe data we present here support protection against serotype 3 IPD from direct vaccination of children with PCV13. This is a first step to understand the full impact of PCV13 in protecting against serotype 3 and the global host, environmental, and pathogen factors that determine serotype 3 epidemiology. This information will be critical for understanding the best public health use of the current vaccine and for developing better vaccines. For example, if PCV13 provides direct protection against serotype 3, but incomplete or no protection against carriage acquisition, it would imply a need for broader reliance on directly immunizing at-risk populations and less reliance on indirect protection from infant immunization programs.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciy920_suppl_Supplementary_Table_1Click here for additional data file.


In addition, the neurons secrete several peptide hormones that act on β-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet β-cells through actions on M ATP ion channels that are inactivated by the increased ATP/ADP ratio, and the resulting decrease in hyperpolarizing K+ current depolarizes the cell membrane i). This second messenger acts at several key points in the glucose transduction pathway that can influence islet oscillations, as well as stimulating insulin exocytosis [2+]i that promptly follows cholinergic stimulation in a glucose-rich environment [2+]i spike can potentially reset islet oscillators, and thereby play a key role in the synchronization of islet oscillations. In this study, we use mathematical modeling to demonstrate how this resetting can be achieved, even in a model where factors in addition to Ca2+ are involved in the production of islet oscillations.Acetylcholine and CCh bind to Mocytosis . Isolateironment ,41,44,45in vivo (~5 min) i for each islet using calibration constants i trace from a single group was background subtracted using a linear fit to the data and smoothed by inserting two data points between each pair of points using linear interpolation. A spectrogram of the background-subtracted data was then produced using a custom LabVIEW program  [2+]i data helps to quickly, yet quantitatively, determine the extent of synchronicity among islet groups as well as how the average islet [Ca2+]i oscillation period changes in response to CCh. All spectrograms shown throughout the Results are on the same intensity scale.For data analysis, the average islet i traces. The frequency with the highest magnitude was recorded before and after CCh pulses were applied and converted to an oscillation period (min). Calculated averages are reported with 1 SD in parentheses, unless otherwise noted.To supplement this analysis, the major oscillation period of each islet is plotted before and after the initiation of pulsing. To identify the major oscillation period, fast Fourier transforms were performed on the individual islet ) is then described by the following differential equation:IPamp3  = 0.35 μM when the 10 sec pulse was on, and 0 otherwise. The parameter 3 concentration changes. The code for the model is available for free from www.math.fsu.edu/~bertram/software/islet.The IOM was modified to simulate the effects of activation of muscarinic acetylcholine receptors by CCh. Activated receptors result in the production of IPm the ER . We inclThe microfluidic platform employed for this study facilitated precise and automated delivery of glucose and the various CCh profiles to groups of 3–4 islets. Multiple experiments were performed to investigate the effects of different CCh profiles. Although the layout of the microfluidic channels has been described previously, the use of the piezoelectric transducer in conjunction with the flow rate sensors was not. We found that this active flow rate control was much better at rapidly generating pulses and maintaining stable flow rates than passive methods. The reagents took 48 s (SD 1) to travel the 75 mm long channel from the inputs to the islet chamber. This dispersive flow resulted in attenuation of the 10 μM CCh pulses by 43.5% (SD 0.4). Thus, the concentration felt by the islets was ~5.7 μM. This value is lower than the those used in other reports ,26 altho2+ measurement from the 3 different experimental groups  tested. Any observed synchronization in the average Ca2+ trace was attributed to the small islet number within a single group.Control experiments were performed by continuously perfusing 3 groups of islets –S5 Figs 2+ oscillations of individual islets (gray traces) from one of the three groups as well as the mean of the four islets (black trace). Before CCh pulsing commenced, the Ca2+ oscillations were out of phase as shown by the minimal rhythmicity in the mean trace. The inset of 2+ oscillations were dominated by the effects of the cholinergic pulses; all four of the islets shifted their oscillation periods to 2.0 min indicating 1:1 entrainment by the periodic CCh pulses  with a train of twenty CCh pulses with R = 2 min, i.e., 2 min between each pulse. ctrogram  of the a2+ oscillations. Two of these islets had long natural oscillation periods of 6.8 and 10.2 min and the stimulus train made them oscillate faster, though the entrained period (~4 min) was still about twice that of the stimulus train. In contrast, for the remaining two islets, the entrained period was longer than that of their natural periods (~2.9 min). 2+]i trace in the representative experiment is shown in 2+]i peaks measured from this group. Nevertheless, it is clear from the analysis of the average [Ca2+]i trace and the change in oscillation periods of the individual islets that the islets were entrained 1:1. It is notable that the 5 min periodicity of the CCh pulse train, so effective at entraining the islets, is similar to that of blood insulin oscillations in healthy animals i when using a single pulse of ACh i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 1(PDF)Click here for additional data file.S4 Fignd negative control experiment with 4 islets perfused with 11 mM glucose is shown. Gray lines indicate the individual islets and the black line indicates the mean [Ca2+]i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 2(PDF)Click here for additional data file.S5 Figrd negative control experiment with 4 islets perfused with 11 mM glucose is shown. Gray lines indicate the individual islets and the black line indicates the mean [Ca2+]i. Neither the mean [Ca2+]i nor the spectrogram (B) of the mean trace show any clear evidence of synchronization for the 90 min measurement.(A) The 3(PDF)Click here for additional data file.S6 Fig2+]i traces (gray lines) of four islets and the average (black line) are shown when CCh pulses with rest durations of R = 2 min were applied. The timing of the CCh pulses is shown by the “x” at the top of the figure. The inset shows the 4 islets had natural periods ranging from 4–5 min prior to pulsing (open circles). During pulsing, oscillation periods transitioned to 2 min (filled circle) indicating 1:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The spectrogram of the average [Ca2+]i from the 4 islets in (A) is shown. A prominent oscillation period band emerges at 2 min for the 38 min long CCh pulsing duration indicating synchronization among this islet group. The spectrogram did not show robust oscillation period bands outside the pulsing duration. Any >10 min oscillation period band is an artifact from the STFT data analysis.(A) The [Ca(PDF)Click here for additional data file.S7 Fig2+]i traces (gray lines) of four islets and the average (black line) are shown when CCh pulses with rest durations of R = 2 min were applied. The timing of the CCh pulses is shown by the “x” at the top of the figure. The inset shows the 4 islets had natural periods ranging from ~3–10 min prior to pulsing (open circles). During pulsing, oscillation periods transitioned to ~4 min (filled circle) indicating 2:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The spectrogram of the average [Ca2+]i from the 4 islets in (A) is shown. An oscillation period band emerges at ~4–5 min after 20 minutes of CCh pulsing indicating some islet synchronization. The >10 min oscillation period band is an artifact from the STFT data analysis.(A) The [Ca(PDF)Click here for additional data file.S8 Fig2+]i from all 4 islets are shown in grey and the average is shown in black. The timing of the CCh pulses is shown by the “x”. The inset shows the natural period of these 4 islets ranged from 6.8–10.2 min (open circle), but all converged to 5 min during pulsing (filled circle) indicating 1:1 entrainment. (B) The synchronized response of the group is evident by the emergence of an albeit weaker oscillation period band at 5 min within the 55 min time span of pulsing in the spectrogram.(A) The individual [Ca(PDF)Click here for additional data file.S9 Fig2+]i from all 4 islets are shown in grey and the average is shown in black. The timing of the CCh pulses is shown by the “x”. The inset shows the natural period of these 4 islets ranged from 4.2–7.0 min (open circle), but all converged to 5 min during pulsing (filled circle) indicating 1:1 entrainment. (B) The synchronized response of the group is evident by the emergence of an oscillation period band at 5 min within the 55 min time span of pulsing in the spectrogram.(A) The individual [Ca(PDF)Click here for additional data file.S10 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 90 min are shown with an “x” noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods between 4.1 and 6.8 min (open circles) and transitioned to 5.0 min (1:2). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The 5 min period band in the spectrogram of the mean Ca2+ trace depicts the synchronicity among these islets during the 50 min pulsing duration. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S11 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 90 min are shown with an “x” noting the timing of each CCh pulse. The inset shows that 3/4 islets had natural oscillation periods between 5.1 and 6.8 min (open circles) and transitioned to 5.0 (1:2). The remaining islet had a >15 min natural oscillation period and transitioned into 1:1 entrainment. The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The prominent band at a period of 5 min in the spectrogram of the mean Ca2+ trace shows the high degree of synchronization among these islets during the 50 min pulsing duration. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S12 Fig2+]i traces of the four islets present (grey lines) and their average (black line) over 80 min are shown with an “x” noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods between 3.4 and 6.8 min (open circles) and transitioned to 5.0 (1:2) or 2.9 min oscillations (1:3) during pulsing (filled circles). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) As can be seen from the green band in the spectrogram of the average Ca2+ trace during the 50 min pulsing time span, these islets synchronized, albeit to a lesser degree than other islet groups in this experiment. After the final pulse at 70 min, oscillations diminished in magnitude.(A) The [Ca(PDF)Click here for additional data file.S13 Fig2+]i traces of the four islets present (grey lines) and their four islet average (black line) over 90 min are shown with an “x” noting the timing of each CCh pulse. The inset shows that the islets had natural oscillation periods of 6.8 or 10.2 min (open circles) and transitioned to 10.1 min oscillations (1:1) during pulsing (filled circles). The number of islets with identical oscillation periods before or during pulsing is indicated adjacent to the relevant circle. (B) The green 10 min oscillation period band in the spectrogram shows the synchronization of the doublet-like oscillations in among this islet group. The weaker ~5 min bands correspond to the intra doublet gaps in the average Ca2+ trace.(A) The i (black line). Each of the CCh pulses is shown by “x”. Although randomly spaced, the repeated CCh pulses produced a synchronized population of 4 islets as can be seen from the emergence of a green band in the spectrogram (B) of the 4 islet mean trace. The degree of synchronization rapidly diminished after the final pulse at 77 min.(A) The Ca(PDF)Click here for additional data file.S15 Fig2+ traces (grey lines) from a unique, representative experiment with a set of 4 islets are overlaid with the average [Ca2+]i (black line). Each of the CCh pulses is shown by “x”. Although randomly spaced, the repeated CCh pulses produced a synchronized population of 4 islets as can be seen from the emergence of a green band in the spectrogram (B) of the 4 islet mean trace.(A) The Ca(PDF)Click here for additional data file.


Rht) are known for yield gains due to lodging resistance and partitioning of assimilates into ear. The available and commercially exploited sources of dwarfism in Indian spring wheat are Rht1 and Rht2 genes inspite of availability of over 20 dwarfing genes. Rht8 a Gibberellic acid sensitive dwarfing gene is another reduced height gene commercially exploited in some Mediterranean countries. Two F2 populations segregating for Rht1 and Rht8 genes with each comprising 398 and 379 plants were developed by crossing European winter wheat cultivars Beauchamp and Capitole with Indian spring wheat cultivar PBW 621. Different genotypic combinations for Rht1 and Rht8 genes were selected from these populations through linked molecular markers and selected F3:4 lines were evaluated for various agronomic traits in a replicated trial. Reduction in plant height with Rht8 and Rht1 averaged 2.86% and 13.3% respectively as compared to the group of lines lacking dwarfing gene. Reduction was spread along all the internodes of wheat culm and reduction was lower as progress towards the lower internode. Grain number per spike and highest yield was observed in lines carrying only Rht1 gene. Reduction in plant biomass was observed with either of the dwarfing gene. Longest coleoptile length and seedling shoot length averaged 4.4 ± 0.09 cm and 19.5 ± 0.48, respectively was observed in lines lacking any of the dwarfing gene. Negligible reduction of 6.75% and 2.84% in coleoptile length and seedling shoot length, respectively was observed in lines carrying only Rht8 gene whereas F3:4 lines with Rht1 gene showed 21.64% and 23.35% reduction in coleoptile length and seedling shoot length, respectively. Additive effect of genes was observed as double dwarfs showed 43.31% and 43.34% reduction in coleoptile length and seedling shoot length.Optimizing wheat height to maximize yield has been an important aspect which is evident from a successful example of green revolution. Dwarfing genes ( Triticum aestivum 2n = 6x = 42) has been dramatically reduced. For example, the average heights of wheat varieties have dropped from 150 cm to 90 cm in UK [Rht1 and Rht2 dwarfing genes were transferred from Japan to USA, USA to CIMMYT, Mexico and from Mexico to all over the wheat cultivated area through Japanese variety Norin-10. Rht8 is mainly commercialized in Europe and transferred through Japanese variety Akakomugi from Japan to Italy and then from Italy to other parts of Europe [Rht1-B1b and Rht1-D1b dwarfing alleles of Rht1 and Rht2 not only reduce the plant stature, but also reduce the coleoptile length which is the deciding factor for early emergence under water deficit or stress conditions and hence, effect the crop stand. It has also been well documented that dwarfing genes Rht-B1b and Rht-D1b are associated with Type I susceptibility to Fusarium head blight in wheat and low anther extrusion [Rht genes have concluded that Rht8 gene reduces the plant height but it has negligible effect on coleoptile length [Rht8 gene and GA insensitive Rht1 and Rht2 genes have been conducted in Australian cultivars [Rht8 gene in combination with Rht1 gene in elite spring wheat background under Indian environmental conditions.To meet the escalating demand of fast growing human population, there is need of continuous increase in wheat grain production. Optimizing plant stature to enhance productivity under ever changing and unpredictable climate is one of the major strategy of plant breeders. Reduced height in wheat is often associated with increase in yield, probably, owing to reduced lodging and enhanced partitioning of assimilates to the grain . The concm in UK  and fromf Europe . Rht1-B1e length . The stuultivars  and in sRht8c) and genotype PBW 621 (having gene Rht1) in main season 2012–13. Beauchamp and Capitole are winter wheat varieties released in France in 1978 and 1964, respectively. These lines were selected from a diverse European winter wheat germplasm set of 376 cultivars that had been procured by Punjab Agricultural University, Ludhiana, Punjab, India from National Institute of Agricultural Botany, Cambridge, UK in 2010. PBW 621 is a spring wheat cultivar released in Indian in 2011 and widely grown in NWPZ of India. The F1 plants of these crosses were self-pollinated to generate F2 population. In wheat growing season 2014–15, 398 F2 plants from cross Beauchamp x PBW 621 and 379 F2 plants from Capitole x PBW 621 were space planted and plant height data was recorded. Further without any selection all of the F2 plants were allowed to self-pollinate to generate F3 progenies. The presence and absence of dwarfing allele(s) of both the Rht1 and Rht8 loci were determined by using linked molecular markers [Rht1 locus which leads to evolution of Rht-B1b allele  [3 progenies of Beauchamp x PBW 621 and Capitole x PBW 621 cross, respectively were identified as being double dwarfs (Rht1 + Rht8) or single dwarf (either Rht1 or Rht8) or tall . Two plants from each of these progenies were harvested separately and multiplied at Keylong, Himachal Pradesh  and 45 of these F3:4 lines with similar growth period were selected for agronomic evaluation in main season 2016–17 at experimental area, Punjab Agricultural University, Ludhiana.Two crosses (Beauchamp x PBW 621 and Capitole x PBW 621) were generated between cultivars Beauchamp and Capitole  procedure [Rht8 and gene specific dominant markers BF, WR1 and MR1 for Rht1 were used. The in-vitro amplification (PCR) and PCR product were resolved as per the protocol described [Rht1 gene [3:4 lines were also confirmed with KASP markers and genotypic composition of these groups are: both the dwarfing genes absent (r1r1r8r8), Rht1 absent and Rht8 present in homozygous form (r1r1R8R8), Rht1 in heterozygous form and Rht8 absent (R1r1r8r8), Rht1 in heterozygous and Rht8 present in homozygous condition (R1r1R8R8), Rht1 in homozygous form and Rht8 absent (R1R1r8r8) and both Rht1 and Rht8 in homozygous form (R1R1R8R8).Young leaves of parental lines and Frocedure . The SSRescribed ,13. Duriht1 gene . KASP is3:4 line with no physical damage and of uniform size were placed in the middle of moist germination paper. The seeds were first sterilized with the fungicide to avoid any infection and placed in wet paper with germ end down and then paper was rolled as a ‘cigar’. The cigars were then placed vertically into a container with 5–7cm of water at the bottom and then the container was placed in an incubator at 20°C for 10 days under dark conditions. After 10 days, cigars were unrolled and average coleoptile length of seedlings was recorded from the base of the seed to the coleoptile tip. Total shoot length of seedlings was also measured.For seedling traits, 12 seeds of each of the F2 individuals were space planted and plant height was recorded. During main season 2015–16, F3 progenies were planted as plant to progeny row. Selected F3 plants were harvested individually and planted at offseason 2016 for multiplication to conduct a replicated trial in coming main season. The experiment for certain agronomic traits was conducted for selected F3:4 lines in main growing season 2016–17. These lines along with parents were planted as 7 x 7 square lattice design with three replications. Three plants from internal rows of each plot were randomly selected for observations to eliminate the border effect. Final plant height and individual internodal length was measured in centimeter at plant maturity. Ear length was also recorded. The internode below the spike was designated as peduncle length and successive to peduncle length was defined as first internode, second internode and so on. Days to flowering, total tillers per meter row, spikelet per spike, grain per spike, grain yield per plot, 1000-grain weight, plant biomass and harvest index were also recorded.In 2014–15, F3:4 line was calculated with the Analysis of variance of square lattice design using SAS ver. 9.3. The relative effect of different gene combinations was compared as a percent change with respect to tall group.Adjusted mean of each of the FRht1, Rht2 and Rht8 confirmed that PBW 621 carries dwarfing allele of Rht1 and both winter wheat parents Beauchamp and Capitole carry dwarfing allele of Rht8 gene. Genotyping with WMS 261 marker showed a 192 bp band in Beauchamp and Capitole and approximately 165 bp band in PBW 621. Considering Norin-10, the source of dwarfism in Indian cultivar which carries both Rht1 and Rht2 gene [Rht1 and Rht2. Genotypes C-306 and C-518, the tall traditional varieties, widely grown before green revolution were used as a control. None of the parental genotype showed the presence of dwarfing allele of Rht2 gene with DF-WR2 primer combination. With BF-MR1 primer combination for Rht-B1a allele , all of the lines showed amplification but C-varieties and European winter wheat lines showed very high intensity PCR product whereas PBW 621 gave very faint band. For Rht-B1b allele  BF-MR1 primer combination was used and faint band was obtained for European winter wheat lines and C-varieties, whereas comparatively intense amplified product was obtained for PBW 621. From these results, it has been concluded that Rht1 gene is responsible for semi-dwarfing nature of spring wheat line used (PBW 621).PCR assay of parental genotypes for ht2 gene , parents3 progenies which seemed homozygous for plant height at early stages were chosen for molecular marker analysis for Rht1 and Rht8 genes. Leaves from all the plants of a progeny were bulked for DNA extraction so that a particular sample represent F2 genotype. For Beauchamp x PBW 621 cross, 85 F3 progenies were selected for molecular marker analysis. Out of 85, 19 progenies were observed to be positive for Rht8 gene, 49 were heterozygous and 16 progenies were negative for Rht8 gene with WMS 261 marker. Similarly, for cross Capitole x PBW 621, 75 progenies were selected for molecular marker analysis based on the phenotype of the F3 progeny. Among these 75 progenies, 15 were come to be positive, 41 were heterozygous and 15 were observed to be negative for Rht8 gene as revealed by marker WMS 261.F3:4 lines with similar growth period were selected for agronomic and seedling evaluation. Because of the uncertainty about Rht1 gene in the segregants, these 45 lines were genotyped with the KASP markers reported [Rht1 revealed that out of 45, 12 segregants were heterozygous for Rht1. So, based on the genotyping of selected F3:4 lines instead of four homozygous groups, these lines were categorized into six different genotypic classes i.e. both the dwarfing genes absent (r1r1r8r8), Rht1 absent and Rht8 present in homozygous form (r1r1R8R8), Rht1 in heterozygous form and Rht8 absent (R1r1r8r8), Rht1 in heterozygous and Rht8 present in homozygous condition (R1r1R8R8), Rht1 in homozygous form and Rht8 absent (R1R1r8r8) and both Rht1 and Rht8 in homozygous form (R1R1R8R8).Based on the molecular marker data and phenotype data, 45 Freported . KASP baColeoptile length (CL) of European wheat was marginally higher as compared to spring wheat cultivar PBW 621 whereas significant difference for seedling shoot length (SL) was observed between the European winter wheat lines and PBW 621. 3.6 cm and 3.5 cm of CL was recorded for Beauchamp and Capitole, respectively whereas PBW 621 was having 2.9 cm of CL. Total seedling shoot length for PBW 621 was 14.9 cm whereas for Beauchamp and Capitole it was 21.1 cm and 22.6 cm, respectively  than the tall category. Rht1 caused approximately 13.3% reduction in plant height when present in homozygous form which indicated that extent of height reduction was stronger with Rht1 as compare to Rht8. However, average plant height of lines with Rht1 in heterozygous form was 8.7 cm taller than the lines which carry Rht1 in homozygous form, suggesting the partial dominance nature of Rht1 gene  yet no significant variation between plant height of these lines were observed. Parental genotypes i.e. Beauchamp, Capitole and PBW 621 used in the study showed comparable plant height averaging 92 cm, 103 cm and 100 cm respectively. Whereas two populations developed from these parents were highly segregating for plant height. Wild type genotypic class (having no dwarfing gene) was taller among all with average plant height of 119.7 cm while both the dwarfing genes caused reduction in plant height with varying extent. Mean plant height of the lines with ht1 gene .Rht1 gene as compare to lines which carry Rht8 gene. Maximum reduction was observed in double dwarf group carry both dwarfing genes. Difference in the internodal length reduction was observed in the group carrying Rht1 in homozygous form and Rht1 in heterozygous form. The schematic representation of internode elongation pattern for different genotypic classes is shown in Individual internodal length was measured to know whether reduction was spread along all the internodes of wheat culm or due to a particular internode. No difference was observed in number of internodes among different genic groups. The reduction effect on wheat culm was spread along all the internodes, however, significant difference in internodal length was observed for peduncle length and first internode. As progress towards to the successive internodes lesser reduction was observed. Decrease in internodal length was larger in the lines which carry Rht8 gene alone and wild type group lacking dwarfing gene. Grain number per spike was recorded maximum in the lines which carries Rht1 gene either in heterozygous or homozygous form. No significant effect of different dwarfing genes was observed on thousand grain weight. However, slightly lower 1000-grain weight was recorded in the lines carry Rht8 gene as compare to tall lines. Group of lines which carry only Rht1 had comparable 1000-grain weight as that of tall group.Reduction in spike length was observed in lines with either single or double dwarfing gene, when the lines were classified into four homozygous groups. The effect on spikelet number per spike varied for different group. There was no significant difference for grain number per spike observed between the group with R1R1r8r8 category which carried dwarf allele of Rht1 gene and tall allele of Rht8 gene. It has been observed that Rht1 caused average yield increase of 10.77% when present in heterozygous form and 18.17% when present in homozygous form whereas Rht8 caused yield reduction of 18.27%. Decreased in plant biomass was observed in lines that carried either of the dwarfing gene or both dwarfing genes. These decrease in plant biomass and highest plot yield resulted in increased harvest index in the lines carry only Rht1 gene (Highest grain yield per plot was observed in ht1 gene .Rht8 gene in different studies [Rht1 or Rht2) have been reported [Rht8 gene [Rht8 [Rht8 as well as of Rht1 (13.3%) on plant height was observed. Our results also showed partial dominance behavior of Rht1 gene as the group with Rht1 in heterozygous form was little taller as compare to the group with Rht1 in homozygous. Additive effect of these genes was also observed, double dwarf group were smallest among all with 24.2% reduction in plant height.Effect of dwarfing genes on plant height and other traits were reported as highly varied with different genetic backgrounds and environmental conditions. 7–18% reduction in plant height was reported with  studies , 20, 21 reported ,18,19. 1ht8 gene  and 14% Rht8 gene and 25.4% reduction by Rht1 gene was reported previously [Rht1 and Rht8 gene together shortened CL by 28.4% whereas in the present investigation 42.94% reduction in CL has been observed. Less effect of Rht8 on coleoptile length and on early growth stages of wheat has been reported in many studies [Coleoptile is the pointed protective sheath that encases the emerging shoot as it grows from the seed to the soil surface and is one of the key trait for breeding wheat for drought tolerance. Emergence ability of seedling is highly influenced by coleoptile length. For a seed to emerge successfully, seed should not be planted deeper than its coleoptile length. Reduction in coleoptile length due to dwarfing genes as observed in present study, has also been reported in several other studies. For example, 6.2% reduction in coleoptile length in the wheat cultivar containing eviously  which ar studies , 9, 23.R1R1r8r8 category is consistent with the ‘tall dwarf’ model proposed [Rht alleles with other genes which increase both plant height and yield. Report of yield advantage with gibberellin insensitive Rht dwarfing genes over tall controls are also available [Rht1 or Rht2 and decrease by Rht8 have also been reported [Rht1 and Rht2, respectively and 21.0% and 5.3% yield reduction by Rht8 in two different genetic background.The observation of highest yield of proposed . They havailable . Yield ireported . HoweverRht1 gene out yielded as compared to the lines having Rht8 gene. However, for seedling traits lines with Rht8 were better performing than lines with Rht1 gene. Coleoptile length is one of the important trait for deep sowing under drought condition to capture moisture from deeper soil. For conservation agriculture, emergence under deep sowing is needed and cultivars with longer coleoptile can emerge quickly and can have better plant vigor. Hence, understanding of such pleiotropic interactions of dwarfing genes with different genetic background will facilitate effective deployment of dwarfing genes in modern varieties. The results of present study showed that lines which carry both Rht1 and Rht8 gene together has showed very high reduction in CL and SL. Such cultivars will not be suitable under water limiting conditions. Hence, considering the coleoptile length as an indicator for better emergence under drought conditions, it is suggested that in order to breed spring wheat cultivars for drought tolerance or water use efficiency, there is need to replace the residents dwarfing genes of Indian spring wheat cultivars which mainly consists of either Rht1 or Rht2. Wheat breeders in the NWPZ of India must adopt Rht8 as an environmentally adaptable alternative semi-dwarfing gene. The Rht8 gene can also be successfully combined with some Ppd and Vrn genes to offer height reduction and adaptability in the niche micro environments of region so as the duration of the crop can be extended to translate into yield enhancement.The present study revealed pleiotropic effect of dwarfing genes along with effect on plant height. Under favorable conditions, lines with Rht1 and Rht8 developed by European winter wheats and spring wheat cultivars allowed us to gain the understanding of potential of alternate dwarfing gene in Indian subcontinent. Lines with higher coleoptile length and seedling shoot length which is a deciding factor for crop establishment were identified in the study. This gave the candidate plant material to evaluate and select the material for conservation agriculture which is one of the new emerging breeding objective in light of climate change.Segregating population of S1 Table(XLSX)Click here for additional data file.


MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2. Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2. Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in  WWTR1 is the gene) and YAP (YAP1 is the gene) are developmentally important transcriptional coactivators benzamide (MS-275) in the same above three lines. Treatment with 1 μM MS-275 for 24 hours resulted in at least a 1.5 fold increase in expression of MST1 and MST2 in the three cell lines  ) was calculated by Spearman's correlation coefficient.Standard deviation for the quantitative western blots was calculated from fold change expression derived from different western blot experiments. For quantitative RT-PCR, standard deviation was calculated from fold change values from each triplicate. Correlation between fraction methylation of CpG islands and expression of the Hippo kinases (RSEM [log


We describe CD4 count recovery among HIV positive individuals who initiated antiretroviral therapy (ART) with and without severe immune suppression using complete laboratory data from South Africa’s national HIV treatment programme between 2010 and 2014 and discuss implications for CD4 count monitoring.Retrospective analysis of routinely collected laboratory data from South Africa’s National Health Laboratory Service (NHLS). A probabilistic record linkage algorithm was used to create a cohort of HIV positive individuals who initiated ART between 2010 and 2014 based on timing of CD4 count and viral load measurements. A CD4 count < 50 copies/μl at ART initiation was considered severe immunosuppression. A multivariable piecewise mixed-effects linear regression model adjusting for age, gender, year of starting ART, viral suppression in follow up and province was used to predict CD4 counts during follow up.1,070,900 individuals had evidence of starting ART during 2010–2014 and met the criteria for inclusion in the cohort -46.6% starting ART with CD4 < 200 cells/μl and 10.1% with CD4 < 50 cells/ μl. For individuals with CD4 counts < 200 cells/μl, predicted CD4 counts > 200 cells/μl, >350 cells/μl and >500 cells/μl corresponded with mean follow up durations of 1.5 years (standard deviation [s.d] 1.1), 1.9years (s.d 1.2) and 2.1 years (s.d 1.3 years). For those with CD4 counts < 50 cells/μl, predicted CD4 count above these threshold corresponded with mean follow up durations of 2.5 years (s.d 0.9 years), 4.4 years (s.d 0.4 years) and 5.0 years (s.d 0.1years) for recovery to the same thresholds. CD4 count recovery varied mostly with duration on ART, CD4 count at the start of ART and gender.For individuals starting with ART with severe immunosuppression, CD4 recovery to 200cells/μl did not occur or took longer than 12 month for significant proportions. CD4 monitoring and interventions recommended for advanced HIV disease should continue until full recovery. Antiretroviral therapy (ART) reduces mortality and morbidity among HIV positive individuals as well as the onward transmission of HIV. –3 UntreastJanuary 2010 and 31st December 2014 and followed up until March 2015. We discuss the implications of the findings on the continued use of CD4 count measurements to monitor the effectiveness of ART.Because the CD4 count is a good predictor of morbidity and mortality before and after ART start, HIV treatment guidelines in the past recommended CD4 count measurements for determining eligibility for HIV treatment and for monitoring its effectiveness. Poor or suboptimal CD4 recovery despite ART and viral suppression has been associated with increased risk of mortality and morbidity from tuberculosis and other AIDS related morbidities.–15 SinceSome of the methods of this paper have been described elsewhere . BrieflyRoutine specimen data from the laboratory management information system are archived in the Corporate Data Warehouse (CDW) of the NHLS and are available for analysis. However, the lack of a unique identifier for individuals enrolled into the CCMT programme and the inability to uniquely identify multiple tests over time and treatment sites has limited the use of these data in the past. As part of a broader evaluation of adherence and retention in HIV care within the South African National ART programme, the National Department of Health commissioned analyses of laboratory datasets using big data methods in 2015. Initial analyses of these data describing i) proportions of individuals who had CD4 recovery to given thresholds, ii) the time to recovery to these threshold and iii) speed of CD4 recovery in the first 12 months were conducted in 2015–2016 and have been published elsewhere.  The resuThe procedures for record linkage and creating a synthetic cohort of HIV positive individuals has also been described in detail elsewhere.  For thisThe date of ART start was defined as the date of the eligible baseline CD4 count described above. This date was taken to be the date of a CD4 count done three to 12 months before the first viral load date. Where multiple CD4 count test dates and results met this criterion, the earliest date was used. The CD4 count at ART start was defined as the result of this eligible baseline CD4 count while follow up CD4 counts were results from the CD4 count tests done at any point after the ART start date. The duration of follow up was defined as the time interval between the date of ART initiation and the date of the latest CD4 count and was considered to be the same as the duration on ART assuming that individuals remained on ART for the entire duration of follow up. A CD4 count of < 50 cells/μl at the start of ART was considered severe immunosuppression at ART start. Viral suppression was defined as having a viral load result of <400 copies/ml at any point during follow up while age referred to the individual’s age recorded at the eligible baseline CD4 count or determined from the recorded date of birth. Gender and year of ART initiation was taken to be the gender and year recorded at the eligible baseline CD4 count testing. Province was defined as the province where eligible baseline CD4 count testing was done regardless of whether or not the individual subsequently moved to other provinces. The main outcome of the analysis was the mean CD4 counts at given intervals post ART initiation. For individuals who had multiple CD4 counts done in the same interval of follow up, all measurements were used in determining the mean CD4 count in that interval.Once the data were linked to unique individuals using the linkage algorithm, it was exported to Stata 14.2  for analysis. The characteristics of eligible individuals at cohort entry were described using medians and interquartile ranges for continuous variables and as frequency and proportions for categorical data. A multivariable piecewise mixed-effects linear regression model—adjusting for age, gender, eligible baseline CD4 count, any virological suppression during follow up and province with splines at 3, 6, 12, 24, 36, 48 and 60 months post ART initiation—was used to predict the mean square root of CD4 counts during and at specified time intervals. The model was fit on the square root scale for the CD4 count outcome in order to normalise the distribution of the CD4 counts. The model allowed for the following interactions: i) age and gender, ii) eligible baseline CD4 count and duration on ART (as splines), iii) calendar year and duration on ART, iv) gender and duration on ART and v) province of entry into care and duration on ART. Predicted mean CD4 counts at different time intervals post ART start and for different combinations of age, gender and baseline CD4 count were obtained by squaring the predicted square roots of the CD4 counts. Likelihood ratio tests comparing i) the piecewise regression model with splines at 3, 6, 12, 24, 36, 48 and 60 months post ART initiation with no covariates (representing observed data) to the full model and ii)the same piecewise regression model but with no interaction terms to the full model were used to determine goodness of fit.This study was approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) and the Boston University School of Medicine IRB (H-31968.) Permissions were also obtained from the NHLS Research Office and the National Department of Health. Since this was a secondary analysis of routine laboratory data, no written informed consent was obtained from patients.From 3,977,761 unique individuals who had at least one viral load test and one CD4 count test record in the CDW database between January 2010 and March 2015, 1 070 900 (26.9%) individuals met the criteria for inclusion in the cohort. Compared to individuals included in the cohort, individuals excluded from the cohort were more likely to be male (33.3% vs. 30.1%), older (median 46 years [IQR 38–55 years] vs. 44 years [IQR 37–52 years]), had a higher median nadir (lowest ever) CD4 count (200 cells/μl vs. 167 cells/μl) and were less likely to be from KwaZulu-Natal Province (22% vs. 31.9%). Across all provinces the median CD4 counts at the start of ART were lower among males compared to females, decreased with increasing age from age category 25–34 years and increased with calendar years from 2010. Overall the observed CD4 counts increased rapidly from baseline to a mean of 319 cells/μl ) around 6 months post ART initiation. Beyond that, there were smaller but steady increases in the observed means to 358 cells/μl (s.d 36 cells/μl) at around 12 months, 409cells/μl (s.d 37 cells/μl) at 24 months, 434 cells/μl (s.d 40cells/μl) at 36 months, and 544 cells (s.d 44 cells/μl) by around 48 months post ART initiation. At all the time intervals, the observed CD4 count levels were lower among males compared to females see .Overall the predicted mean CD4 recovery was rapid during the first eight months on treatment before slowing down in the subsequent periods. The mean predicted CD4 counts during the first three months was 217 cells/μl . The mean predicted CD4 counts increased to 321 cells/μl  around 12 months; 396 cells/μl (95% CI 395–398 cells/μl) around 18 months; 413 cells/ μl (95% CI 411–415 cells/μl) around 24 months; 432 cells/μl (95% CI 430–435 cells/μl) around 30 months; 441 cells/μl (95% CI 437–444 cells/μl) around 36 months; 455 cells/ μl (95% CI 452–459 cells/μl) around 42 months; 458 cells/μl (95% CI 453–463 cells/μl) around 48 months and 473 cells/μl (95% CI 466–480 cells/μl) around 54 months. For individuals with CD4 counts < 200 cells/μl; predicted CD4 counts >200 cells, 350 cells/μl and 500 cells/μl corresponded with a mean ART durations of 1.5 years (s.d 1.1), 1.9 years (s.d 1.2) and 2.1 years (s.d 1.3 years) respectively. At all durations on ART, the predicted recovery was lower among males compared to females  and amonFor individuals who started ART with CD4 counts < 50 cells/ μl—severe immunosuppression, the mean predicted CD4 count at 12 months was 177 cells/μl (95% CI 175–179 cells/μl) overall. This mean was higher among females compared to males [192 cells/μl (95% CI 190–193 cells/μl) vs 160 cells/μl (95% CI 158–161 cells/μl)]. For both males and females, the mean predicted CD4 counts at 12 months were <200 cells, consistent with the observation that 71% of this population had not recovered to 200 cells/μl by 12 months of follow up with 39% not achieving CD4 recovery to 200cells/μl at all during follow up. Predicted CD4 counts >200 cells/μl, 350 cells/μl and 500 cells/μl corresponded with ART durations of 2.5 years (S.D 0.9 years), 4.4 years (S.D 0.4 years) and 5.0 years (S.D 0.1years) respectively. Because CD4 counts at the start of ART decreased with increasing age, the predicted CD4 count recovery among the oldest group of individuals was also determined at the given time points and for given baseline CD4 counts. Among the 324,667 individuals who were aged ≥50 years at the start of ART, the predicted CD4 counts at 12, 24, 36, 48 and 54 months increased with CD4 counts at the start of ART and were higher among females compared to males. For males with CD4 counts <50 cells/μl, the predicted mean of the CD4 count peaked at 48 months at 254 cells/μl while for females it continued to increase to 325 cells/μl by 54 months. For those with CD4 counts ≥500 cells/μl, the predicted CD4 count peaked at 54 months for males and 60 months among females at 612 cells/μl and 705 cells/μl respectively see . CompareTo compare the extent of CD4 count recovery across the nine provinces in the country, the predicted CD4 counts at 12, 36 and ≥54 months were determined for males and females aged 15–49 years with CD4 counts 50–199 cells/μl and ≥200 cells/μl at ART start. Among males, the mean predicted CD4 counts at 12, 36 and ≥54 months for individuals with CD4 counts of 50–199 cells/μl at ART start and for those with CD4 counts ≥ 200 cells/μl were highest in KwaZulu Natal, Free State and Gauteng provinces and lowest in the Eastern Cape, Northern Cape and North West provinces see . SimilarAs median CD4 counts at the start of ART increased with calendar year,  an analyWe used a multivariable piecewise mixed-effects linear regression model adjusting for age, gender, eligible baseline CD4 count, virological suppression during follow up and province to predict the extent and magnitude of CD4 count recovery among individuals aged >15 years old initiating ART. Our analyses found that across all ages, provinces and baseline CD4 counts, CD4 recovery was lower and slower among males compared to females even after adjusting for CD4 count at ART start as well as duration on ART. The analysis also found that among individuals with CD4 counts <50 cells/μl at the start of ART, the predicted mean CD4 count after 12 months post ART initiation remained below 200 cells/μl and that it took a mean of 2.5 years for recovery to 200 cells/μl in this group. Our analyses also consistently found greater CD4 count recovery in KwaZulu Natal, Gauteng and Free State provinces.Although this is the first ever national level analysis of CD4 count recovery data, this analysis confirms what smaller cohorts of up to 90 000 individuals from South Africa and other sub Saharan African settings have demonstrated. These studies demonstrated that i) the CD4 count at the start of ART was the strongest predictor of CD4 count recovery,  ii) CD4 Our study had some important strengths. Firstly this is a national level data set with representation of all provinces in the country. Second, there were long durations of follow up—up to five years for some eligible individuals. This meant the trajectories of CD4 count recovery could be described beyond one to two years. Our study also had a number of limitations: Firstly, this analysis used laboratory data linked to unique individuals through the use of a probabilistic matching algorithm to create a cohort and determine CD4 recovery outcomes. The accuracy of the cohort data depended on the performance of the algorithm used to link CD4 count and viral load data to unique individuals, and on the completeness of data in the database. The algorithm used to construct the cohort had a sensitivity of 89% and a positive predictive value of 85%, which means it matched correctly for the majority of individuals in the cohort.  It is thDespite these limitations, our findings have important implications for policy and practice. CD4 count measurement at entry or re-entry into care should be continued in order to identify individuals with advanced disease and to allow clinicians to define the appropriate package of care. However, ART initiation should proceed regardless of CD4, removing the risk of CD4 count measurement becoming a cause of pre-treatment loss-to-follow-up. The cost-effectiveness of different CD4 screening and monitoring strategies needed to be considered in order to ensure that CD4 guided HIV care is provided in the era of test and treat. Recent data from the South African care and treatment programme shows that as much as 10% of HIV positives who start ART do so with CD4 counts less than 100 cells/μl despite increase in the median CD4 counts at ART start.  For thosS1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Data from calculating the vertical electronic excitation of 20 excited states for each dye and from calculating excited state oxidation potential (ESOP) and Frontier HOMO/LUMO isosurfaces are also presented. This data is related to the article “Molecular and excited state properties of isomeric scarlet disperse dyes”  [1].X-ray crystallography and DFT calculations were used to characterize the molecular nature and excited state properties of isomeric photostable azo dyes for textile fibers undergoing extensive sunlight exposure. Structural data in CIF files arising from X-ray analysis are reported and the complete files are deposited with the Cambridge Crystallographic Data Centre as CCDC 1548989 ( The data are Supplementary material for the study describing the “Molecular and excited state properties of isomeric scarlet disperse dyes” The data arise from X-ray crystallographic analysis and computational methods in the characterization of isomeric monoazo dyes Sc2 and Sc3 is shown in Sc2 and 0.0001 for Sc3. Other key crystallographic data for the two dyes are summarized in θmax values, and bond lengths. The latter values are especially helpful in establishing the tautomeric form (azo vs. hydrazone) of the dyes analyzed   .Fig. 1ThSc2 shows intermolecular H-bond distances between the NH2 and CN groups (2.418 Å). Also seen are short contacts corresponding to intermolecular hydrogen bonds for Sc3, namely the NO2 and NH2 groups (2.188 Å), and the NH2 and CN groups (2.512 Å).Data for intermolecular H-bonding interactions between layers of molecules positioned parallel to each other are given in Sc2 and Sc3. From these data the excited state oxidation potential (ESOP) for each dye can be extracted.Calculation of vertical electronic excitation energies for 20 excited states along with the oscillator strength (f) and molecular orbitals involved for each dye led to the raw data shown in 2Single crystal X-ray diffraction analysis was conducted using a Bruker–Nonius X8 Apex2 diffractometer. The frame integration was performed with the program SAINT. The resulting raw data was scaled and absorption corrected using a multi-scan averaging of symmetry equivalent data using SIRPOW 2Cl2 solutions of Sc2 and Sc3 at room temperature gave thin plate-like single crystals that were suitable for X-ray crystallographic analysis. The equilibrium molecular geometries (EMGs) of Sc1, Sc2 and Sc3 were calculated in the neutral forms using density functional theory (DFT) employing the generalized gradient approximation (GGA) at the hybrid exchange-correlation energy functional 3-Parameter (Exchange), Lee et al. (B3LYP) Sc2 and Sc3 were superimposed on the corresponding calculated molecular geometries and the RMS was calculated in each case. The isosurfaces of the HOMO and LUMO were extracted for each dye from the corresponding checkpoint files. In addition, TD-DFT calculations were performed on the EMGs and the geometry of the excited state structure was calculated using single point energy calculations for each dye. Vertical electronic excitation energies for 20 excited states were calculated for each dye and the excited state oxidation potential (ESOP) for each dye was extracted.Slow evaporation of CH


In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes  Airn silences genes in the Igf2r imprinted cluster, focusing primarily on silencing of the distant imprinted gene Slc22a3. We found that Airn expression blocks chromosome interactions between the Slc22a3 promoter and the Airn gene locus. By making a large genomic deletion including the Airn gene we showed that these interactions are not essential enhancer/promoter interactions, but may help to guide the Airn RNA to target genes to recruit epigenetic silencing. Our study adds to the understanding of how lncRNAs may act to silence distant genes.Long non-coding (lnc) RNAs are numerous in the mammalian genome and many have been implicated in gene regulation. However, the vast majority are uncharacterised and of uncertain function making known functional lncRNAs valuable models for understanding their mechanism of action. One mode of lncRNA action is to recruit epigenetic silencing to target distant genes on the same chromosome. A well-characterized group of lncRNAs that act in this way to silence genes are imprinted lncRNAs. In this study we examined how the imprinted lncRNA  Long non-coding (lnc) RNAs are a diverse and numerous group of non-protein-coding RNA species longer than 200 nucleotides, some of which have been shown to be involved in gene regulation ,2. A groIgf2r cluster expands from 120kb in most embryonic and adult tissues to almost 10Mb in placenta, while the Kcnq1 cluster expands from 250kb in embryonic tissues to 690kb in VYS . By. ByAirn RSDel region is correct, deletion of these enhancers on the maternal allele where Airn is not expressed should prevent expression of Slc22a3 on this chromosome. Slc22a3 silencing should also be maintained on the paternal allele when these enhancers are deleted  to distant imprinted genes in extra-embryonic tissues [Igf2r cluster in extra-embryonic tissues. Therefore, we performed H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) on VYS endoderm from FVB x CAST reciprocal crosses to determine the allele-specific distribution of this mark in the Igf2r cluster and throughout the genome. Using the Allelome.PRO pipeline to analyze the data [Igf2r cluster, despite imprinted expression in VYS endoderm being limited to a 450kb region from Slc22a3 to Airn  is not the same as an interaction between the Airn genomic locus and the Slc22a3 promoter on the maternal allele (detectable by 3C).Previously it has been shown in placenta using a technique derived from RNA FISH called RNA TRAP (Tagging and Recovery of Associated Proteins) that promoter . SurprisSlc22a3 and Slc22a3 in the Igf2r cluster are covered by a broad enrichment of the PRC2 mark H3K27me3, as are imprinted genes in the Kcnq1 cluster and in other imprinted clusters. Imprinted lncRNAs including Airn, Kcnq1ot1 and Meg3 have been reported to directly interact with PRC2 and EHMT2 [Airn-PRC2 interaction was reported in embryonic stem (ES) cells where Airn is very lowly expressed and no genes in the Igf2r cluster show imprinted expression [Kcnq1 cluster that show extra-embryonic specific imprinted expression [Dlk1 by Meg3 requires PRC2 [Igf2r imprinted silencing by Airn does not require PRC2 or EHMT2 [Slc22a3 and other members of the Igf2r cluster has not been tested, loss of EHMT2 has been shown to lead to loss of imprinted silencing of Slc22a3 [Here we show in VYS endoderm that the repressed alleles of nd EHMT2 ,20,21, apression ,20. PRC1pression ,38, and res PRC2 . Igf2r ior EHMT2 , but whiAirn may silence distant imprinted genes like Slc22a2, Slc22a3 and Pde10a by recruiting and targeted PRC1, PRC2 and EHMT2 to these genes to deposit repressive chromatin modification and cause silencing. This indicates that Airn silences imprinted genes in the Igf2r cluster by two different mechanisms: Airn transcription silences Igf2r by transcription interference that does not require repressive chromatin modifying complexes [Airn RNA product recruits repressive chromatin modifying complexes and targets them to distant, non-overlapped genes like Slc22a2, Slc22a3 and Pde10a to cause silencing. Importantly, the mechanism for targeting silencing remains unknown.Although it is technically difficult to exclude a role for transcription of the lncRNA versus the RNA product, together these results indicate that imprinted lncRNAs like omplexes , and theAirn gene body and the Slc22a3 promoter are enriched on the maternal allele because Airn expression represses these interactions on the paternal allele. We showed that these interactions are not required to upregulate Slc22a3 expression on the maternal allele, indicating that they are not essential promoter-enhancer interactions, but they may serve to place the Airn locus and Slc22a3 promoter in close proximity in the nuclear space. Chromosome interactions can exist in the ground state or be formed during development [Airn locus and the promoter of Slc22a3 , are present on both alleles  Protocol on the Protection and Welfare of Animals. The Animal Research is covered by Federal Austrian legislation  and the overriding EU and international legislation and codes of conduct. No experimental procedures were performed on the animals so no extra permissions were required.Thp) mouse (EM:09898) contains an minimum 5.56Mb deletion on chromosome 17 that includes the Igf2r imprinted cluster allowing parental allele-specific analysis [AirnT) mouse (EM:09895) has a polyadenylation cassette inserted into the Airn gene, 3kb downstream from its start site causing it to be truncated and non-functional [R2Δ) mouse (EM:09897) has a deletion that includes the Airn promoter and the imprint control element (ICE) of the Igf2r imprinted cluster [Thp, AirnT and R2Δ mice have been cryopreserved by the EMMA mouse repository (EMMA ID indicated). The Sod2-flox mice contain loxP sites flanking the exon 3 of Sod2 and were made in a 129 ES cell line [FVB/NJ (FVB) mice were obtained from Charles River. CAST/EiJ (CAST) mice were obtained from the Jackson Laboratory. The FVB.AK-Del(17)T<hp>  was isolated from E9.5 and E12.5 embryos under a dissection microscope. The whole VYS was used for the chromosome conformation capture (3C experiments), while for RNA isolation and for chromatin immunoprecipitation (ChIP) the VYS endoderm was mechanically separated away from the rest of the VYS after 1–2 hours of DispaseII digestion at 4°C, as previously described .Igf2r imprinted locus we used reciprocal crosses of Thp and FVB mice [Airn on interactions we used Thp x AirnT cross, where AirnT mice have a truncated and non-functional Airn [Igf2r imprint control element (ICE). Around 28 VYS were then pooled per genotype and thawed on ice and then incubated with 8ml lysis buffer for 15 minutes on ice (1 tab Complete Protease Inhibitor (Roche) per 25ml lysis buffer ). The samples were then dounced in a 15ml glass dounce (Wheaton) with a loose pestle about 30 times, and then 30 times with a tight pestle, before centrifuging for 5 minutes at 2000g at 4°C. The supernatant was then removed leaving a nuclear pellet, which was then resuspended in 1 x EcoRI buffer (Fermentas). Samples were then subject to EcoRI digestion and ligation as previously described [Chromosome Conformation Capture (3C) was performed following established protocols with minor modifications ,49. To aFVB mice . To detenal Airn . We collnal Airn . Brieflyescribed . The forSlc22a3 promoter) and a “prey” primer was designed near the EcoRI site for fragments in the target region (e.g. Airn gene body). All 3C interactions detected in the Igf2r imprinted cluster (Thp deletion region on chromosome 17) were normalized by dividing by the mean of 2 interactions with the H19/Igf2 ICE, an independent locus on chromosome 7. To correct for technical and biological variation between experiments the highest interaction level was then set to 1. The primer/probe combinations used are given in We detected 3C interactions by Taqman quantitative PCR following a previously published protocol with minor modifications , and by Tissue from VYS endoderm or placenta was collected and homogenized in TRI reagent, and total RNA isolated according to the manufacturers protocol (Sigma-Aldrich).RSDel x FVB reciprocal crosses was DNase treated using the DNA-free kit (ThermoFisher Scientific), and then converted to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was then conducted using either a Taqman or SYBR Green system using the standard curve method to analysis the data, and normalization to a house keeping gene (cyclophillin A). The RT-qPCR results in Total RNA from E9.5 VYS endoderm and E12.5 placenta collected from RSDel x CAST reciprocal crosses using the TruSeq RNA Sample Prep Kit v2 (Illumina) modified as previously described [RSDel x CAST, maternal deletion cross) and 3x WT and 3x Del . Native ChIP was performed using an H3K27me3 antibody  on E12.5 VYS endoderm from FVB x CAST reciprocal crosses  as previously described [Strand-specific polyA enriched RNA-seq libraries were generated from E12.5 placenta and VYS endoderm from escribed . For eacescribed . ChIP-seescribed .RSDel mouse was backcrossed to FVB, but the region around the Igf2r cluster is likely to have a 129 background as both the R2Δ and Sod2Δ alleles from which the RSDel mouse is derived were made in 129 ES cells [Allele-specific expression and histone modification enrichment was detected from RNA-seq and ChIP-seq data using the Allelome.PRO program . The SNPES cells ,30. TherWe used the following Allelome.PRO parameters for our analysis:RNA-seq: minread 2 , RefSeq annotation.H3K27me3 ChIP-seq enrichment: FDR 1%, allelic ratio cutoff 0.7, minread 1, 20Kb sliding windows.Note: minread = minimum number of reads that must cover a SNP for it to be included in the analysis.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S4 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S5 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file.S6 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file.


Sir,in vitro and evaluating the efficacy of type I interferon pathway blockade for therapeutic purposes in adults with dermatomyositis  inhibitor. Finally, four adults with refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I IFN levels and IFN-inducible genes scores (We read with interest the recent article by Ladislau and colleagues reporting on the pathogenicity of type I interferon (IFN) et al. now suggests that JAK inhibition may also be of relevance to the treatment of non-genetic IFN-mediated diseases such as adult dermatomyositis (Juvenile dermatomyositis (JDM) is a multi-systemic autoimmune disease characterized predominantly by progressive proximal muscle weakness and pathognomonic skin rashes . Even thThe following standardized measures were used to assess JDM disease activity and response to treatment in this case (2 weekly). Over the next 6 months he developed extensive ulcerative skin disease (modified skin DAS range 5/5) at which point intravenous cyclophosphamide  treatment was initiated with some improvement in CMAS 44/52 and modified skin DAS 3/5. Persistence of cutaneous disease over time and development of calcinosis over his elbow joints, right ear lobe required sequential treatment with azathioprine, mycophenolate mofetil, infliximab, adalimumab, rituximab, tacrolimus and ciclosporin, intravenous immunoglobulin (IVIG) over the next 7 years. He remained symptomatic and steroid dependent (2 mg/kg/day of prednisolone). Genetic testing was performed using version 2 (VIP2) of our recently developed targeted gene panel for Vasculitis and AutoInflammation Panel (VIP), which contains 166 genes, to exclude known monogenic interferonopathies that may mimic JDM, such as the proteasome associated autoinflammatory diseases  and characteristic heliotrope rash, Gottron’s papules over the small and large joints of his hands (skin DAS of 4/5). He had raised creatine phosphokinase  and lactate dehydrogenase ; modestly elevated erythrocyte sedimentation rate  and normal C-reactive protein . His disease was complicated by pharyngeal involvement; but there was no evidence of interstitial lung disease, gastrointestinal or cardiac involvement. Myositis specific autoantibody detection showed positive anti-TIF1γ and anti-Ro-52 antibodies; antinuclear antibodies were positive with a titre 1:640, negative for dsDNA antibodies, complement function studies were normal and C1q, C3 and C4 levels were all within normal limits. He was initially treated with corticosteroids (30 mg/kg of intravenous methylprednisolone over 3 days followed by 2–3 mg/kg/day with intention to wean over 4–5 months) and subcutaneous methotrexate (15 mg/mTreatment with baricitinib (a selective JAK1/JAK2 inhibitor) was initiated at age 11.5 years because of ongoing severe skin disease activity and progressive calcinosis (expanded access protocol NCT01724580). He underwent dose escalation until he reached optimal tolerated treatment doses adjusted to his weight and renal function (6 mg twice a day). Clinical symptoms improved upon treatment with baricitinib at this dose with CMAS improving from 46/52 at time of start of therapy to 50/52 at 6 months of treatment; MMT-8 improving from 59/80 at baseline to 70/80 at 6 months; modified skin DAS 5/5 at baseline to 1/5 at 6 months; physician’s VAS from 4.3/10 to 1.5/10 . CPK remAt 12 months of treatment he stopped taking all his medication against medical advice and this led to a significant flare of his symptoms within 6 weeks with significant deterioration of his skin rash (modified skin DAS 5/5), worsening myalgia and arthralgia, CMAS decrease to 46/52 and MMT-8 to 59/80, physician’s VAS 6/10 , CPK eleBiomarkers of IFN signalling, and the type 1 IFN induced gene expression that were modestly elevated at time of start of treatment (note patient was receiving high doses of corticosteroids at that time point) significantly decreased during treatment with baricitinib at 6 months and 18 months with upregulation at 12 months when all treatment had been stopped due to lack of compliance C–E. We mIn line with the findings from The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary MaterialClick here for additional data file.


The authors wish to make the following corrections to this paper :Abstract: The sentence “The parameter rm2 penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test.” should read as: The parameter rm2 penalizes a model for large differences between observed and predicted values of the compounds of the whole set (considering both training and test sets) while the parameter Rp2 penalizes model R2 for a small difference between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test.Section 2.3.2.4: The sentence “We have used a parameter Rp2 [32] in the present paper, which penalizes the model R2 for the difference between squared mean correlation coefficient (Rr2) of randomized models and squared correlation coefficient (R2) of the non-randomized model.” should read as:p2 [32] in the present paper, which penalizes the model R2 for a small difference between squared mean correlation coefficient (Rr2) of randomized models and squared correlation coefficient (R2) of the non-randomized model.We have used a parameter RConclusions: The sentence “The parameter Rp2 penalizes model R2 for large differences between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test and thus confirms whether a model has been obtained by chance or not.” should read as: The parameter Rp2 penalizes model R2 for a small difference between determination coefficient of nonrandom model and square of mean correlation coefficient of random models in case of a randomization test and thus confirms whether a model has been obtained by chance or not.


A non-hydrolytic condensation route allows for precise control over the size distribution of HfO2 nanocrystals and enables the stabilization of the tetragonal phase of HfO2. 2 since it is predicted to outperform the thermodynamically stable lower-symmetry monoclinic phase for almost every application where HfO2 has found use by dint of its higher dielectric constant, bandgap, and hardness. However, the monoclinic phase is much more thermodynamically stable and the tetragonal phase of HfO2 is generally accessible only at temperatures above 1720 °C. Classical models comparing the competing influences of bulk free energy and specific surface energy predict that the tetragonal phase of HfO2 ought to be stable at ultra-small dimensions below 4 nm; however, these size regimes have been difficult to access in the absence of synthetic methods that yield well-defined and monodisperse nanocrystals with precise control over size. In this work, we have developed a modified non-hydrolytic condensation method to precisely control the size of HfO2 nanocrystals with low concentrations of dopants by suppressing the kinetics of particle growth by cross-condensation with less-reactive precursors. This synthetic method enables us to stabilize tetragonal HfO2 while evaluating ideas for critical size at which surface energy considerations surpass the bulk free energy stabilization. The phase assignment has been verified by atomic resolution high angle annular dark field images acquired for individual nanocrystals.There has been intense interest in stabilizing the tetragonal phase of HfO Precise control of the size of HfO2 nanocrystals enables unequivocal determination of the critical size required to stabilize the tetragonal phase and provides access to well-defined crystals of this technologically important metastable structure that is otherwise stable only above ca. 1720 °C.16A particularly powerful aspect of nanoscience that enables much new functionality derives from the greatly altered phase equilibria obtained upon confining materials to nanoscale dimensions.2 has been widely accessible for several decades, even without incorporation of dopant atoms, since the critical size at which the surface free energy terms overcome bulk free energy considerations is relatively large, variously estimated to be ca. 15–30 nm.2 is much more challenging since: (a) the bulk free energy stabilization of the monoclinic phase over the tetragonal phase is almost 40% greater for HfO2 as compared to ZrO2; and (b) the volume expansion accompanying the tetragonal to monoclinic phase transition is much smaller for HfO2 (ca. 2.7%) as compared to ZrO2 (4.0%).2 vary widely from about 2 to 10 nm but it is clear that this value is substantially smaller than the critical size for ZrO2.2 report the stabilization of at least some fraction of tetragonal HfO2; for instance, signatures of the tetragonal phase have been identified in particles obtained by the thermal decomposition of pure Hf(OH)4,2 prepared by methods such as atomic layer deposition in a low-oxygen environment,Tetragonal ZrO2 is not just an academic curiosity. Indeed, the tetragonal (space group P42/nmc) phase of HfO2 is anticipated to outperform the thermodynamically stable lower-symmetry monoclinic phase (space group P21/c) for almost every application where HfO2 has found use. Perhaps the most important application of HfO2 is in gate dielectric stacks as a high-κ dielectric because of its resistance to silicidation and silicate formation and its much greater dielectric constant as compared to SiO2.2, which is far surpassed by the value of almost 70 predicted for tetragonal HfO2.ca. 6.11 eV) as compared to the monoclinic phase (5.78 eV).27The stabilization of tetragonal HfO2 nanocrystals.2 nanocrystals, again crystallized in the monoclinic phase.2 nanocrystals as per:In comparison to aqueous methods, non-hydrolytic methods, often based on formation of oxo-bridges by elimination of small molecules, provide considerable control of the kinetics of condensation.2 and solid-solution HfxZrx1–O2 nanocrystals and established considerable control over the relative Hf : Zr concentrations.2 and ZrO2 nanocrystals as well as the relative Hf : Zr ratios of HfxZrx1–O2 nanocrystals.4 and MX4 species, substantial ligand exchange takes place to stabilize haloalkoxides such as M(OR)3Cl, M(OR)2Cl2, and M(OR)Cl3.Brus and co-workers extended this method to prepare HfOWhen two different metal precursors are reacted, the composition of the product depends on the relative condensation rates and reactivities. For metals exhibiting comparable reactivity, such as Hf and Zr, the heterocondensation reaction yields solid-solution nanocrystals and can be written as follows:tBu)4 or La(OiPr)3, greatly modifies the concentration of the active monomer depicted in 2 nanocrystals with only minimal incorporation of La and Ce. This unprecedented control of nanocrystal size allows us to explore ultra-small dimensions and determine the critical size for stabilizing the tetragonal phase of HfO2.However, if the rates of homo- and hetero-condensation are vastly different, the products of the faster reaction are obtained preferentially. In this work, we find that adding a much less reactive precursor, either Ce(Oiv) chloride, cerium(iii) chloride, lanthanum(iii) chloride, lanthanum(iii) iso-propoxide, and tri-n-octylphosphine oxide (TOPO) were purchased from Strem and used without any further purification. Hafnium(iv) tert-butoxide was purchased from Alfa Aesar and used as received. Cerium(iv) tert-butoxide was purchased from Gelest and used without further purification. To synthesize the metal oxide nanocrystals, a metal halide and metal alkoxide are mixed in equimolar amounts with ca. 10 g of TOPO in a three-neck round bottom flask under an Ar ambient within a glovebox. The total concentration of metal precursors is maintained at 4 mmol in all of the reactions. In all of the reactions, 2 mmol of HfCl4 is used as the chloride precursor, whereas the alkoxide precursor is x mmol of La(OiPr)3 or Ce(OtBu)4 and 4 – x mmol of Hf(OtBu)4. CeO2 nanocrystals were obtained by the reaction of equimolar amounts of CeCl3 and Ce(OtBu)4 as reported in our previous work.Hafnium, were used for characterization of the samples by powder X-ray diffraction. The crystallinity and size distribution of the nanocrystals was evaluated by high-resolution transmission electron microscopy (HRTEM) using JEOL-2010 and FEI Tecnai G2 F20 ST electron microscopes at an operating voltage of 200 kV. Samples for HRTEM analysis were dispersed in hexanes, drop-cast onto 400-mesh carbon-coated copper grids, and allowed to dry in air. The stoichiometry of the nanocrystals was determined by X-ray fluorescence (XRF) measurements, conducted by Oneida Research Services, Inc. in Whitesboro, NY, by inductively coupled plasma-mass spectrometry (ICP-MS) using a Perkin Elmer DRCII instrument, and by energy dispersive X-ray spectroscopy (EDX) on a JEOL JSM-7500F field emission scanning electron microscope (FE-SEM) operated at an accelerating voltage of 20 kV. Samples for ICP-MS analysis were prepared by acid digestion in an aqueous solution of 67% metals grade HNO3. Further characterization was performed using atomic resolution high-angle annular dark-field (HAADF) imaging. A probe-corrected JEOL JEM-ARM200CF instrument equipped with a cold field-emission gun operated at 200 kV was used with a convergence angle of 22 mrad. The HAADF inner detector angle was 90 mrad.A Rigaku Ultima IV diffractometer with a graphite monochromator and a Bruker-AXS D8 Advanced Bragg–Brentano X-ray powder diffractometer, both using Cu Kα radiation (4 with varying proportions of Hf(OtBu)4 and Ce(OtBu)4. x1–CexO2 nanocrystals and provides good agreement with the hafnium and cerium concentrations derived by elemental analysis 4 and HfCl4 are clearly monoclinic (P21/c) as delineated by the appearance of sharp (200) and (220) reflections (Joint Committee on Powder Diffraction Standards (JCPDS) 78-0050) indexed in 2 nanocrystals crystallize in a cubic fluorite structure (Fm3[combining macron]m) with reflections that can be indexed to JCPDS# 34-0394 4 to Hf(OtBu)4 is increased, the reflections are broadened indicating a pronounced diminution in size even though elemental analysis data presented in 4 and 2 mmol of Ce(OtBu)4 preponderantly yields the tetragonal phase of HfO2 characterized by a pronounced (101) reflection centered at 2θ = 30.3° 4, indicates the appearance of a reflection at 2θ = 31° that can be attributed to the (101) reflection of the tetragonal phase and suggests that this set of precursors yields a mixture of tetragonal and monoclinic phases. A reflection at 2θ = ca. 41° in  = 30.3° . The simtBu)4 in the reaction mixture, the morphology of the obtained nanostructures evolves from elongated nanorods to quasi-spherical particles. The nanorods preferentially grow along the [100] direction of monoclinic HfO2.x1–CexO2 nanorods are monotonically diminished with reduced concentration of Hf(OtBu)4 with respect to Ce(OtBu)4. The average length is decreased from 12.9 ± 1.8 nm for monoclinic nanocrystals grown using only Hf(OtBu)4 to 3.1 ± 0.4 nm for tetragonal nanocrystals grown using Ce(OtBu)4 as the only alkoxide precursor. The widths of the Hfx1–CexO2 nanocrystals are not substantially altered and remain around 2.5 ± 0.4 nm. The lattice-resolved HRTEM image in 2  imaging has been performed using an aberration corrected microscope. To verify the phase assignment of Hf4 with varying proportions of Hf(OtBu)4 and La(OiPr)3. x1–LaxO2 nanocrystals and the determined hafnium and lanthanum concentrations are in good agreement with the elemental analysis results 3 as listed in 3 and La(OiPr)3 yields LaOCl nanocrystals crystallized in the matlockite PbFCl-type phase.tBu)4, with increasing concentration of La(OiPr)3 in the reaction mixture, the (100) and (200) reflections indexed to the monoclinic phase of pure HfO2 are broadened and diminished in intensity suggesting a pronounced diminution in size. Again, although the powder XRD patterns are substantially altered, the elemental analysis results listed in 2. For the sample prepared by the reaction of HfCl4 and La(OiPr)3, the (100) monoclinic reflection is no longer observed and the (101) tetragonal reflection becomes the most prominent feature in the powder XRD pattern suggesting stabilization of the tetragonal phase of HfO2 with modest La doping.Analogous to the above discussion for Ce(OiPr)3, the Hfx1–LaxO2 nanocrystals again evolve from elongated nanorods to quasi-spherical nanocrystals. The width of the Hfx1–LaxO2 nanocrystals is relatively unchanged at ca. 2.5 ± 0.4 nm for the entire set of samples. However, the length of the nanorods is diminished from 12.9 ± 1.8 nm for monoclinic nanocrystals grown using only Hf(OtBu)4 to 3.3 ± 0.4 nm for Hfx1–LaxO2 tetragonal nanocrystals grown using La(OiPr)3 as the only alkoxide precursor.HRTEM images and size distribution histograms of this set of nanocrystals are shown in x1–LaxO2 nanocrystals as being tetragonal, atomic-resolution HAADF STEM images and fast Fourier transforms (FFT) of the HAADF images were acquired and are depicted in To confirm the phase assignment of HftBu)4 and La(OiPr)3 leads to a substantial and monotonic diminution of nanocrystal size even though elemental analysis results indicate that relatively low concentrations of Ce and La are actually incorporated within the lattice. At the smallest dimensions, the tetragonal phase of HfO2 is stabilized over the monoclinic phase. These results lead to two fundamental questions regarding the mechanistic basis for the size control achieved by addition of La and Ce precursors and the origin of the altered phase stability.The preceding discussion illustrates that the addition of Ce(OtBu)4 and La(OiPr)3, it is important to consider the overall equation depicted in eqn (1), which results in formation of an oxo-bridge with the elimination of an alkyl halide, as well as the catalytic scheme proposed by Vioux shown in To understand the origin of the size control achieved by the addition of Ce(O4 and Zr(OR)4 and ZrCl4 and Hf(OR)4 yield exactly the same compositions of solid-solution HfxZrx1–O2 nanocrystals suggesting that ligand scrambling precedes condensation;xZrx1–O2 and LaxCex1–O2, respectively, albeit with compositions that reflect the relative reactivities of the two metal precursors as modified by the alkoxide ligands.2 with very low dopant concentrations is obtained as the exclusive product upon the reaction of HfCl4 with mixtures of Hf(OtBu)4 and Ce(OtBu)4/La(OiPr)3. The relatively low incorporation of Ce and La within the doped HfO2 lattice, very much lower than the precursor concentrations, corroborates the idea of the lower reactivity of these precursors. tBu)4 and La(OiPr)3 allows for substantial tunability of the size of the obtained doped HfO2 nanocrystals. In 3OR species (derived from ligand exchange as per eqn (3)) mediates conversion of the alkoxo-bridge to an oxo-bridge with elimination of an alkyl halide. Indeed, Vioux has shown that the presence of MX3OR species is imperative for condensation and that MX2(OR)2 and MX(OR)3 species are much less reactive.3OR species formed by ligand exchange serves as the catalyst. The retention of equimolar amounts of halide and alkoxide precursors ensures that the amount of catalyst is kept essentially the same for all of the reactions listed in 4 and Ce(OtBu)4/La(OiPr)3 are much less reactive as compared to those prepared from HfCl4 and Hf(OtBu)4, the addition of cerium and lanthanum precursors essentially reduces the concentration of the active monomer. In other words, the reaction rate is diminished as a result of the lower reactivity of mixed metal Lewis acid–base adducts towards formation of oxo-bridges. As further corroboration for this idea, 2 nanocrystals that are 12.9 ± 1.8 nm. In contrast, the homocondensation of cerium precursors yields CeO2 nanocrystals that are 1.5 ± 0.5 nm indicating much slower kinetics of growth 4 when ramped at a rate of 15 °C to 500 °C during thermal analysis is monoclinic HfO2, the thermodynamically stable phase, along with an oxygen-deficient orthorhombic HfO2 phase. The inclusion of TOPO still yields monoclinic HfO2, albeit with a smaller size. These results illustrate that direct reaction of the precursors cannot yield the metastable, kinetically trapped phase. The incorporation of the La and Ce alkoxides is imperative to slow the kinetics of crystal growth. ca. 3.6–3.8 nm is the tetragonal phase stabilized. Since 3+ cations (110 pm for seven-coordinated and 116 pm for eight-coordinated sites) as compared to Ce3+ (107 pm for seven-coordinated and 114.3 pm for eight-coordinated) or Ce4+ (97 pm for eight-coordinated),x1–LaxO2 nanocrystals remain monoclinic even upon incorporation of 3.54 at% of La).As a next point, we discuss the origins of the stabilization of the tetragonal phase of HfO2 lattice will result in creation of half an oxygen vacancy to maintain electrostatic neutrality. The Ce4+/Ce3+ redox couple is readily accessible but since we start with tetravalent cerium precursors, the vacancy concentration generated upon cerium-incorporation is likely to be lower than upon lanthanum incorporation. Again, if oxygen vacancies were to provide the driving force for stabilization of the tetragonal phase, one would expect that lanthanum-incorporation should more readily bring about stabilization of the tetragonal phase as compared to cerium incorporation. Again, x1–LaxO2 nanocrystals with 3.54 at% La are monoclinic, whereas Hfx1–CexO2 nanocrystals with only 2.36 at% Ce are tetragonal. The preceding discussion and –2).2 nanocrystals becomes such that the surface energy term exceeds the bulk energy component of the free energy term (the difference between the two phases is 196 meV for pure HfO2),The second scenario pertains to the potential role of oxygen vacancies. The substitutional incorporation of trivalent lanthanum cations in the HfOd can be expressed as48G is the volume free energy change across the phase transformation, V is the strain energy, γ is the specific surface energy, and ρ represents the density. Hunter et al. used this expression to predict a critical size for stabilization of tetragonal HfO2 of d = 3.6 nm.2 nanocrystals prepared by aqueous methods is the almost 20–30% diminution of surface energy as a result of surface hydroxylation, which in this case further dilutes the influence of the surface energy contribution.202, which is in remarkably good agreement with predictions from this thermodynamic model. The origin of the stability of the tetragonal phase at these dimensions can thus be attributed to the substantially lower surface energies of this phase, which renders this phase energetically stable under conditions of constrained equilibrium.In 1972, Bailey and co-workers developed an expression for calculating the critical size for stabilizing a metastable state based on a classical thermodynamic treatment of the phase transformation and the competing bulk and surface energy terms. In this formulation, the critical size, 2 has been far more challenging as compared to ZrO2 given the smaller volume expansion accompanying the tetragonal → monoclinic transformation and the relatively greater stabilization enjoyed by the monoclinic phase. Classical thermodynamic models predict that the tetragonal phase should be stable at dimensions smaller than ca. 3.6 nm but the validity of these predictions have been thus far impossible to determine in the absence of synthetic approaches that can access such ultra-small dimensions with precise control of particle size. In this work, we have developed a non-hydrolytic condensation route wherein the concentration of the active monomer is precisely modulated by replacing Hf(OtBu)4 with less reactive Ce(OtBu)4 or La(OiPr)3 precursors. The latter alkoxides exhibit much slower kinetics of condensation and thus are incorporated within the doped HfO2 lattice only to small extents but play a significant role in modifying the growth kinetics by suppressing the concentration of the active monomer. This approach enables precise control of size in ultra-small dimensions and allows for systematic evaluation of the size-dependence of phase stabilities in this system. The much desired metastable tetragonal phase is stabilized at dimensions less than 3.6–3.8 nm, which is in good accord with predictions of thermodynamic models that take into account the competing influences of bulk free energy and specific surface energy.Stabilization of the tetragonal phase of HfOThe authors declare no competing financial interest.Supplementary informationClick here for additional data file.