Amaranthus, commonly known as pigweeds, and considered some of the most problematic weeds in agricultural systems. In a greenhouse experiment that allowed controlled plant growth conditions and a high number of individual plant specimens to analyze for statistical robustness (n = 8 per species), we show that specific bacterial assemblages form in the rhizospheres of A. retroflexus L. (redroot pigweed), A. palmeri S. Watson , and A. tuberculatus (Moq.) J. D. Sauer (waterhemp). Using a relatively rapid and easy approach of T-RFLP community profiling of the 16S rRNA genes, distinct assemblages corresponded to plant species , and further within each species, similar communities  were associated with three rhizosphere soil fractions taken in increasing distances away from the root tissue. These results provide the first solid basis for distinct plant-microbe relationships within three closely related Amaranthus species, warranting closer examination of the identities and function of the microorganisms that appear to be selectively recruited from the extant soil community. More intensive efforts to obtain the microbial taxonomic identities via sequencing are underway that can lead to further detailed studies to elucidate important functional plant-microbe interactions that may associate with weed success. Such data provides underlying key information that may ultimately exploit weed-microbe interactions in development of new integrated weed control tactics.Weed management remains a major challenge in cropping systems worldwide, with rising interest in ecological based approaches that can be integrated with herbicide use. Soil microbial communities may play important, yet undiscovered, roles in weed success. Little is known about the rhizosphere communities associated with weeds like  The drive toward increasing the development of integrated weed management (IWM) practices in agriculture is considered one of the grand challenges in modern cropping systems worldwide , 2. WeedAmaranthus species are small-seeded broadleaf annual weeds that are difficult to manage in cropping systems due to their wide distribution, extended range of seed germination times, high seed production, and long-lived seed banks [Amaranthus species can coexist in agronomic fields, and genetic outcrossing between species is highly possible for some species in natural environments [Amaranthus species are important agronomic weeds of concern and significant attention has been paid to their phenotype and growth characteristics, competition with crops, and their acquisition and transmission of herbicide resistances, little information about the microbial ecology associated with these plants is available.ed banks . Commonled banks –6. Despironments . While AMicroorganisms have long been known for their many important roles in ecosystem services that benefit plants, soil quality, and human health. Contemporary studies in microbial ecology are now just starting to reveal the incredible diversity present in microbial taxa and their activities –10, espeAmaranthus species considered some of the most problematic weeds in U.S. cropping systems. The microbial communities in the rhizospheres of three species, A. retroflexus L. (redroot pigweed), A. palmeri S. Watson , and A. tuberculatus (Moq.) J. D. Sauer (waterhemp) were compared to determine if plant species formed distinct bacterial assemblages recruited from the extant populations in the soil in which they were grown. Here, we used a relatively rapid qualitative method of microbial community profiling, namely terminal restriction fragment length polymorphism (T-RFLP) following amplification of the 16S rRNA genes present in the DNA retrieved from the loose soil immediately surrounding the root zone (rhizosphere bulk soil), root-adhered soil (rhizosphere soil), and root-bound (rhizoplane) microbial communities in plants grown under a low stress greenhouse environment. The conditions imposed in the greenhouse study described here were designed to mimic summer annual cropping systems commonly seen for commercial corn and soybean production in the U.S. Midwest region, namely non-limiting N and diurnal air and soil temperature cycles similar to the period of seasonal Amaranthus emergence and growth. Anticipating interest in pursuit of similar research with other weed species and continued study with Amaranthus species, we also provide detailed methodological description to aid future molecular-based studies involving microbial community analyses.In this study, we sought to provide a novel basis for examining microbial assemblages associated with three A. palmeri and A. retroflexus, Azlin Seed Service, Leland, MS) or from plants collected around agricultural fields located in Illinois  were used to generate seed lines for experimental use in this study. Soil used for plant growth was collected by bulk (0–10 cm depth) in Fall 2020 from the SoyFACE (Soybean Free Air Concentration Enrichment) facility , a site belonging to the University of Illinois crop research program freely accessible to university researchers and their collaborators. The SoyFACE (SF) soil was sieved (4 mm) to remove large debris and plant material, mixed by hand to homogenize, and stored at 25 °C prior to use. General soil characteristics are described in Initial seed lots of three pigweed species obtained commercially (Amaranthus species was grown independently to avoid propagation of genetic hybrids between species. Soil (~2 kg) was placed in 14 cm2 polypropylene pots and 20 seeds of Amaranthus species were sprinkled on the surface and lightly misted with distilled (DI) water, then pressed lightly into soil but not covering. DI water (50–75 ml) was used to evenly moisten soil before placing into a controlled environment growth chamber (Conviron) programmed to cycle 16 h day length at 28 °C and 8 h night length at 22 °C, with plant watering occurring when needed. Soil moisture varied in accordance to natural cycles of wetting and drainage under the temperature conditions of the incubation chamber or daily greenhouse environment. Emergence of seedlings fully occurred after 7 d, and at 28 d plants were thinned to four plants per pot and transferred to a greenhouse  fertilization regimen starting at 38 d and consisted of twice weekly addition (10 ml) of a mixture of urea (26.7 mM) and NH4NO3 (20.0 mM). No additional amendments were needed  as the ambient soil concentrations were considered adequate for plant growth . On average, soil moisture by gravimetric measurement was maintained between 15–17% (w/w). Eight individual plants served as replicate units per plant species, with nine plant-free pots in total used as control replicates. Plants were grown to maturity and then harvested to obtain rhizosphere fractions as described below. Homogenized SF soil used for plant growth also served as the reservoir for microbial recruitment to the plant rhizosphere and seven samples in total taken from the bulk homogenate were used to represent the baseline microbial community characteristics.All A. palmeri and A. tuberculatus included distinction of male and female plants among the eight replicates for each species. For A. palmeri male (n = 3) and female (n = 5), and from A. tuberculatus male (n = 4) and female (n = 4).Following daily watering and drainage on the day of sampling, plants were carefully removed from pots, allowing loose soil to fall free. Soil moisture was measured gravimetrically using ~5 g soil taken from the loose soil volume. Following, three rhizosphere fractions were separated in sequence from each plant harvested and defined as follows: 1) rhizosphere bulk (RB) soil–soil that remained loosely root bound after plant was pulled from the pot, followed by 2) rhizosphere (R) soil–soil removed from roots following mechanical shaking using an orbital shaker  into 150 ml phosphate buffered saline , and finally, 3) rhizoplane (RP) sample–soil and associated biomass bound tightly to root surfaces removed during 30 s sonication (Bransonic ultrasonicator) in 150 ml PBS buffer containing TWEEN 80 surfactant (0.01% v/v). RB soil was removed by placing the root mass on a sterile metal tray and loose soil was carefully removed from the root mass using a sterile metal spatula and transferred to sterile plastic bags. A subsample of RB soil (0.5 g dry wt) was directly transferred into 2.0 ml bead tubes  containing (800 μl) to use for DNA extraction (see below) and the remaining RB soil was stored frozen (-20 °C). The remaining root mass was transferred to a 250 ml flask containing 150 ml PBS buffer followed by gentle shaking for10 min at 300 rpm using an orbital shaker for recovery of R soil fractions. Each R soil suspension was centrifuged (3220 x g) at 4 °C for 10 min, the supernatant was removed, and the soil pellet was resuspended in PBS buffer (800 μl) before transfer to 2.0 ml bead tubes. Following, each RP fraction was recovered by placing the root mass into a fresh flask containing PBS buffer and TWEEN 80 and shaken for10 min at 300 rpm on an orbital shaker before undergoing 30 s in a sonication bath. Each RP suspension was centrifuged (3220 x g) at 4 °C for 10 min, the supernatant was removed, and the pellet was resuspended in PBS buffer (800 μl), and transferred to 2.0 ml bead tubes. Following each plant growth phase, parallel-run plant-free control soils were mixed thoroughly by hand using sterile spatulas while in pots to obtain a representative homogenate of soil before subsamples of ~0.5 g (dry wt.) were removed into 2 ml bead tubes containing PBS buffer. Replicates of field SF soil used for plant propagation was sampled (~0.5 g dry wt.) and frozen on day 1 of the experimental set up. An additional attribute for each of the rhizosphere fractions obtained from -1 stock). The nucleic acids were precipitated by addition of 1 volume polyethylene glycol (PEG) 6000 (stock 20% PEG 6000 made in 2 M NaCl). The nucleic acids were allowed to precipitate for 2 h at -20 °C, followed by centrifugation  for 15 min. The supernatant was discarded and the pellet was washed using 200 μl ice cold ethanol (70% stock), then centrifuged  for 5 min. The ethanol was removed by pipet and the pellet was air dried before resuspending in 50–200 μl nuclease-free water. The crude nucleic acid extract was transferred to a clean 0.5 ml microfuge tube and stored at -20 °C until further use. One sample was selected randomly to provide three technical replicates for an internal assessment of variability due to extraction efficiencies and subsequent PCR gene amplification for the soil type used in this study.The extraction method was developed in-house by modification of standard phenol-chloroform extraction methods optimized here for recovery of both DNA and RNA and detailed as follows. Only DNA was targeted for analyses in this experiment; for future reference, RNA fractions can be recovered from the nucleic acid pool following standard procedures for DNA removal and used for transcript-based analyses if needed. Each soil or rhizosphere fraction that was collected into a 2.0 ml bead tube containing PBS buffer was washed by brief mixing on a vortex mixer , then centrifuged (2500 x g) for 5 min at 4 °C and the supernatant was removed. Cell lysis and separation of nucleic acids was performed by the addition of 10 μl β-mercaptoethanol (Sigma), 0.5 ml phosphate buffer , and 0.5 ml phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8.0) (Sigma) to the cell pellet, followed by 2 min. of agitation at room temperature using a Disruptor Genie apparatus (Scientific Industries), then centrifuged . The supernatant was transferred to a clean 2 ml microfuge tube, avoiding volume at the phase interface. A volume of NaCl (5 mM stock) was added to obtain a final concentration of 0.7 M, mixed gently by inverting the tube several times, followed by addition of 0.1 volume of cetyltrimethyl ammonium bromide (CTAB) , then mixed together by inverting tube several times. An equal volume of chloroform-isoamyl alcohol (24:1) was added and mixed gently by inverting tubes several times before mixture was centrifuged  briefly enough to achieve phase separation. The supernatant was then transferred to a clean 2 ml microfuge tube and the chloroform-isoamyl alcohol clean-up step was repeated again, except the supernatant was transferred the second time into a clean 2 ml microfuge tube containing 1 μl glycogen (20 mg ml5’-AGA GTT TGA TCM TGG CTC AG-3’) was labeled at the 5’ end with 6-carboxyfluorescein (6-FAM) and when coupled to the reverse primer 1492 (5’-GGT TAC CTT GTT ACG ACT T-3’), amplifies the 16S rRNA genes from a broad diversity of bacteria. The primers were HPLC-purified and obtained from IDT . Stock concentrations (100 μM) of each primer were made by adding Invitrogen™ UltraPure™ DNase/RNase-Free Distilled Water  and subsequently diluted for use in PCR accordingly for amplification of the target gene. PCR reactions were performed in 50 μl volumes using the Takara ExTaq PCR kit (Clontech) and a MJ Research PTC-200 Gradient Thermal Cycler. The optimized reaction mixture was the following: 1X PCR buffer, 0.2 mM each deoxynucleoside triphosphate (dNTPs), 0.025U μL-1 TaKaRa Ex Taq DNA polymerase, 1.0 μM each forward and reverse primers, and ~1 ng DNA. T4 gene protein 32  was added to the reaction mixture to obtain a final concentration of 62.5 μg mL-1. The following thermocycling program was used: initial denaturation step at 94°C for 5 min, followed by 25 cycles of 94 °C for 90 sec, 55 °C 90 sec, and 72 °C for 90 sec, then a final extension at 72 °C for 10 min. PCR products were resolved by gel electrophoresis in 2.5% High Resolution Agarose (fragments < 1 Kb)  at 4 V cm-1 for 70 minutes or 1% Seakem LE Agarose (fragments > 1 Kb) (Lonza) at 4 V cm-1 for 50 minutes in 1X TBE buffer on a HU13 Midi horizontal gel unit . DNA ladders consisted of 10 μL of Quick-Load Purple 1kb DNA Ladder (0.1–10.0 kb)  or 5 ul of 1kb DNA ladder (Gold Biotech). The PCR amplified mixture was purified further to remove excess primers and salts using the QIAquick PCR Purification Kit (Qiagen) following manufacturer’s instructions except 30 ul of nucleic acids-free water was used to elute the DNA from the column and the final DNA suspension was stored at -20 °C until restriction enzyme digestion.The 16S rRNA genes in the DNA pools were amplified using primer set F27/1492R . The for®Purple 1 kb Plus DNA Ladder). Terminal fragment size analysis was performed using services available at the Roy J. Carver Biotechnology Center, University of Illinois, Urbana, IL.The amplified 16S rRNA gene products were digested using the restriction enzyme HaeIII, previously determined to be the most optimal for generating a wide range of terminal fragments (TRF) sizes generated following enzyme digestion of the amplicon pools typically resulting from local Illinois soils. Additional enzyme candidates for digestion include HhaI and HpaII and can be used instead of HaeIII. HaeIII digestion was performed for 12 h at 37 °C using the NEB restriction enzyme kit (New England Biolabs) according to manufacturer’s instructions. Digested fragments were visualized using 2.5% high resolution agarose gel electrophoresis along with reference DNA containing fragment sizes ≤ 1 kb , calibrated against size standards 50–1000 bp (ROX1000). Data was sorted according to size after removal of fragments <50 bp and >1000 bp fragments , and a threshold <80 relative fluorescence intensity was determined as background fluorescence signals based on analysis of water samples used as blank controls. The resulting dataset were unique patterns (i.e. T-RFLP profiles) comprised of TRFs that represented the bacterial 16S rRNA gene pool amplified from DNA recovered from each sample. The technical replicates of one sample used for internal assessment of extraction and amplification variability yielded identical T-RFLP patterns.a posteriori within each fixed factor or combination of factors  following PERMANOVA results. Principal coordinate analysis (PCO) based on Bray-Curtis dissimilarities was used to visualize the community assemblages. This analysis provides a pseudo F-value, based on 999 permutations, that quantifies the deviance from the null-hypothesis where large values of F correspond to large changes in community composition accordingly to the experimental factor (or combination of factors) defined for the analysis.To assess whether the type of plant species and their rhizosphere fractions correspond to statistically different microbial communities as measured by 16S rRNA gene-based T-RFLP profiles, permutational non-parametric MANOVA (PERMANOVA) was used to assess significant differences using the PERMANOVA+ package in PRIMER v.6 software . The PERMANOVA routine was performed using permutation of residuals under a reduced model and tests were based on a Jaccard (metric Bray-Curtis) community dissimilarity metric calculated from the relative abundance values of the T-RFLP fragments following square root transformation of the values. Statistical significance was set at p-value ≤ 0.05. Pair-wise tests were performed Amaranthus given the greenhouse growth conditions used in our experimental protocol. There was variability in the resulting plant height within each of the replicate sets established for each plant species, with overall shortest among A. retroflexus  followed by A. tuberculatus  and A. palmeri . Within the dioecious species, the A. palmeri female plants  on average were taller than the male plants , while the A. tuberculatus females  were on average similar in height to male plants . Most female plants had mature seeds, but lacking in one female plant replicate of A. tuberculatus and one replicate of A. retroflexus.The total time required to attain plant maturity starting from seed germination was ~78 d for all three species of A. palmeri and A. retroflexus with fewer unique fragments (n = 103–110) present in the rhizoplanes of these two plant species .The T-RFLP microbial community profile of each sample comprise unique size fragments (TRFs) that represent the most dominant taxa among the complex diversity of soil microbial populations typically present in varying abundances. The average number of TRFs obtained from the soil used for plant growth (SF) taken prior to the start of the experiment and considered the baseline source of microbial recruitment by the plant was n = 96, in contrast to an average n = 81 in plant-free soil controls taken at the end of the experiment. Similar numbers of TRFs ranging from n = 123–136 in the rhizosphere bulk soils (RB) and rhizosphere soils (R) were obtained from  species . The numAmaranthus plant species  . InteresA. tuberculatus, the communities within the rhizoplane (RP) fractions of A. palmeri and A. retroflexus demonstrated higher dispersion than their corresponding rhizosphere soil fractions (RB and R). The result suggests beta diversity does drive to some extent the differences between rhizosphere sample type.To analyze significant differences in the variability within the groups based on the replicate samples analyzed for each rhizosphere fraction, a distance-based test for homogeneity of multivariate dispersions measuring the deviation from the group centroid revealed differences between the type of rhizosphere compartment  for all three plant species . In contrast to Amaranthus that appear to be driven by the type of plant species and the distance in relative proximity to the plant root surface. To the best of our knowledge, the results present novel information that demonstrates a solid basis for continued detailed explorations regarding the general nature of the microbial communities found in association with the root surfaces and surrounding soil zones associated with weedy plants. Despite the close genetic relatedness of the three plant species chosen for study, the distinct differences in rhizosphere zones suggest important plant-microbe relationships specific to plant species that may be critical to the underlying mechanisms that drive microbial assemblages to form around the root and how these microbes may impact the success of these and other weed species. The molecular-based community profiling afforded by techniques such as T-RFLP analysis, while unable to provide taxonomic identification, allows a relatively rapid and inexpensive approach to compare a subset of members within the microbial community likely to be the responsive populations and present in the highest abundances. Importantly, such a methodological approach can be designed with high replication of specimens for maximum statistical power before investing in significantly more costly and time-consuming microbial analyses  that become potentially prohibitive with large number of samples to analyze.The present study provides a statistically robust qualitative analysis of microbial community differences in three species of Amaranthus species, predicated on the expectation that differences in phenotype and growth characteristics known among this plant genus might manifest rhizobiomes that also differ between the species. When all three species of Amaranthus were compared together, the results suggest that the microbiomes of A. palmeri and A. retroflexus appeared to be more similar to each other than A. tuberculatus with respect to their corresponding rhizosphere zones  in localized zones around the individual plant that can be mediated by such microbial processes like nitrogen fixation and ammonia oxidation. Other functions occurring within the rhizobiome may include roles of herbicidal detoxification [phnJ, is harbored in many known bacterial species and generally an activity used to gain P for cell metabolism. This enzymatic activity may also be a key to glyphosate resistance, a trait that is emerging with higher occurrences in Amaranthus species [Amaranthus species to gain a competitive advantage over crops [The identification of the specific plant-related factors that appear to drive the formation of the rhizobiome compartments remain speculative, however root exudates are likely candidates to influence microbial growth. Microbial responses to root exudates are well known, among them providing a source of nutrients and cofactors, along with various mechanisms of interactions that can be beneficial to both plant and microbes –24. Chemfication , 26, plafication , 28, andfication , 30. Celer crops . While tsensu stricto largely depends on several factors such as plant species, the type of root exudates, and the environmental factors that influence the mobility of these compounds [Amaranthus species were clearly distinct from those in the background- and plant-free control soils  suppresses redroot pigweed (A. retroflexus) by changing its root exudates when sensing the presence of pigweed [Besides the direct response to root exudates, indirect effects on microbial selection in the rhizosphere zone could be mediated by other biotic factors that can ultimately affect competitive interactions between plants. For example, buckwheat  already known to be present in soil environments, the rhizobiome recruitment by the plant may still be driven toward establishing recurring underlying microbial functional characteristics, but be associated with a different set of microbial taxa. Consequently, while the resulting rhizobiomes may significantly differ in taxonomic structure between Amaranthus species, recruitment of functionally similar communities may be a form of generalized strategy in genetically related plants. While this study reports the efforts on bacterial  communities, fungi are also important members of the microbiome with many well studied interactions known to occur between plants and fungi. For example, AMF fungi are well known for enhancing plant growth [Amaranthus rhizospheres did not reveal distinction in the fungal community structure according to plant species, however, it is possible that using a higher resolution method to obtain fungal taxonomic identities in the future may eventually reveal dominating fungal taxa in the rhizobiome not previously described in plant interactions.Finally, the objective of characterizing the rhizobiomes of three closely related t growth , 37. To Amaranthus species used in this study, and the recruitment of microbial populations does not appear to be largely stochastic in nature. This work provides a solid basis to support the continuing pursuit of identifying the microbial taxa and potential functional genes using technically more complex methodologies such as high throughput sequencing that may include amplicons generated from targeted ribosomal- and functional genes of interest or in metagenomic or other -omics analyses. Such approaches are both labor intensive and more costly, and employment of practical designs will be needed to necessarily reduce the number of specimen to analyze but still provide statistical robustness. This is particularly the case for future pursuit of gene expression studies, where RNA-based approaches can reveal the microbial activities that may be significant in interactions with the plant rhizosphere. More in-depth community characterization based on gene sequencing is currently underway with the same three Amaranthus species described here. From this study, we further advocate for the use of relatively simple and inexpensive approaches such as that afforded by methods such as T-RFLP profiling of targeted genes  that can be applied to large numbers of specimens for analyses and can provide highly useful preliminary assessments of community patterns for comparative studies under different plant growth conditions. The importance of soil microbial communities in the life strategies of weedy plants may be one key area leading to potential avenues that can be exploited in the pursuit of ecologically sustainable weed management practices in the future. Such burgeoning knowledge promises to contribute to more thorough understanding of the larger overall ecology of complex crop/weed/microbe/soil systems.We conclude that plant species-specific rhizosphere communities formed among the three S1 Table(DOCX)Click here for additional data file.S1 Figth and 75th percentiles indicated by the lower and upper box edges, respectively, and the line representing the median plotted within each box. Whiskers show the tails of the data distribution. Filled circles depict outlier values.Control samples were taken from plant-free experimental units. SF = SoyFACE soil was used as the plant growth medium and represents the baseline community. The box represents the central 50% of the data with the 25(TIF)Click here for additional data file.


In patients with non-valvular atrial fibrillation (NVAF) prescribed warfarin, the association between guideline defined international normalised ratio (INR) control and adverse outcomes in unknown. We aimed to (i) determine stroke and systemic embolism (SSE) and bleeding events in NVAF patients prescribed warfarin; and (ii) estimate the increased risk of these adverse events associated with poor INR control in this population.2DS2-VASc score was 3.5 (SD = 1.7), and the mean follow up was 4.3 years for both analyses. Mean TTR was 71.9%, with 34% of time spent in poor INR control according to NICE criteria.Individual-level population-scale linked patient data were used to investigate the association between INR control and both SSE and bleeding events using (i) the National Institute for Health and Care Excellence (NICE) criteria of poor INR control . A total of 35 891 patients were included for SSE and 35 035 for bleeding outcome analyses. Mean CHASSE and bleeding event rates (per 100 patient years) were 1.01 (95%CI 0.95–1.08) and 3.4 (95%CI 3.3–3.5), respectively, during adequate INR control, rising to 1.82 (95%CI 1.70–1.94) and 4.8 (95% CI 4.6–5.0) during poor INR control.P < 0.001] and bleeding  in Cox-multivariable models.Poor INR control was independently associated with increased risk of both SSE [HR = 1.69 (95%CI = 1.54–1.86), Guideline-defined poor INR control is associated with significantly higher SSE and bleeding event rates, independent of recognised risk factors for stroke or bleeding.  Major clinical guidelines now recommend that anticoagulation with direct oral anticoagulants (DOACs) should also be considered where appropriate.14Guidelines stress the importance of assessing INR control, achieving acceptable TTR, and re-evaluating therapy if adequate control cannot be achieved. The UK National Institute for Health and Care Excellence (NICE) defines poor anticoagulation as any of the following: (i) TTR <65%; (ii) two INR values >5 or one >8 within the past 6 months; and (iii) two INR values <1.5 within the past 6 months.15 However, the magnitude of the impact on major adverse outcomes of suboptimal control according to NICE clinical guideline criteria  has not been demonstrated. We were also interested to see if outcomes differed in those with evidence of poor control according to NICE criteria to those with inadequate control according to ESC and US criteria, which only consider the TTR.We have previously demonstrated in a large population that a considerable proportion of patients exhibited suboptimal INR control according to NICE and ESC guideline criteria.We aimed to (i) determine SSE and bleeding event rates in patients prescribed warfarin for NVAF, and (ii) estimate the incremental risk of these adverse events associated with poor INR control (defined using NICE and ESC/US guideline criteria), accounting for patient clinical and demographic characteristics.16–18A population-scale retrospective observational cohort study was conducted using individual-level linked anonymised routine electronic health record data sources for patients prescribed warfarin for NVAF in Wales, United Kingdom, between January 2006 and December 2017 using the Secure Anonymised Information Linkage (SAIL) Databank.Patients eligible for the study had a diagnosis of AF/atrial flutter recorded in their primary care record at any point before or during the study period (2006–2017) and were ≥18 years old at time of diagnosis. Patients were excluded if they had valvular AF  or venous thromboembolism (VTE) including deep vein thrombosis (DVT) and pulmonary embolism (PE) before, or within 6 months of inclusion, or were pregnant during the study period. Patients who were subsequently diagnosed with a VTE or valvular AF after 6 months from entry into the study were censored at the point of new diagnosis.The cohort was restricted to those prescribed warfarin and with ≥6 months of recurrent INR tests recorded in their primary care record during the study period .19 We also stipulated that there be: (i) at least four INR readings within each 6-month period; (ii) no gap >12 weeks between consecutive INR readings, and (iii) a gap of at least 3 months between the first and last INR reading within each 6-month window (2A–D); and (iv) at least one warfarin prescription in any 12-week window.15Individual TTR was calculated at each INR result within 6-month rolling windows using the modified Rosendaal method.Based on these criteria, an algorithm was developed to allow the temporal calculation of INR control, and assign patients to ‘unknown,’ ‘adequate,’ or ‘poor’ INR control status at each INR reading. Two established guideline criteria for poor INR control were assessed: Firstly, NICE criteria for poor INR control, one (or more) of the following (i) TTR <65%; (ii) two INR values higher than 5 within the prior 6 months; or a single INR value higher than 8; and (iii) two INR values less than 1.5 within the past 6 months. Secondly, ESC/US criteria for poor INR control, defined as periods of TTR <70% .Patients could move between adequate, poor, and unknown control status; assignment of patients to unknown status would result in temporary exclusion from analysis for any period during which there were insufficient INR results or no warfarin prescriptions available. Patients could re-enter the analysis when another six-month window became available with sufficient INR results and warfarin prescriptions for evaluation.An index date was assigned to each patient when all inclusion criteria were first met. The number of days a patient was in adequate and/or poor control was calculated to the end of 2017. Patients were censored at death or when an adverse event occurred (see below), including those adverse events occurring outside of periods of INR calculation or when lost to follow-up (end of primary care record).Tables S1A-D for diagnostic codes).Adverse events comprised (i) SSE and (ii) bleeding events  recorded in either the primary or secondary care records  prior to the index date for each patient were identified. Age and deprivation quintile22 were assigned at the index date. Heart failure, hypertension, vascular disease [defined as prior myocardial infarction (MI) or peripheral vascular disease (PVD) including peripheral artery disease and aortic plaque], prior stroke [including transient ischaemic attack (TIA)], diabetes, sex, and age were used to calculate the individual CHA2DS2-VASc score at the index date.20Demographic and clinical data  and SSE and bleeding outcomes, adjusting for relevant individual risk factors, including components of CHA2DS2-VASc score . Analyses were performed using IBM SPSS v26 and R version 3.5.3.SSE and bleeding event rates were calculated during periods of adequate or poor INR control using each guideline’s thresholds for INR control. Relationships between INR control, SSE and bleeding events were then calculated using NICE criteria. Since our data allowed estimation of times during which patients move between poor and adequate INR control status, we considered INR control as a time-dependent variable and estimated hazard ratios (HRs) representing the risk at any specific time point. Initial multivariable-models utilized Cox-regression to estimate the risk of SSE or bleed according to INR control status, adjusting for the baseline individual CHAt-tests for continuous variables.Comparisons were made between those included in the final cohorts for analysis and (i) those with NVAF prescribed warfarin but with inadequate or no INR test results for analysis, and (ii) those with insufficient INR tests recorded in the primary care dataset to classify INR control prior to either a SSE or bleed. Finally, within the final cohort, comparisons were made between those with and without deprivation quintile data available. Differences in these characteristics between groups were summarised using chi-squared tests for categorical variables and independent Figure 1) (see Table S2).Over 4 million patient records were identified in the SAIL Databank during the study period; 124324 patients had a diagnosis of AF and aged over 18 at diagnosis of which 10 633 had a diagnosis of DVT, PE, or valvular prior to or within 6months of the study. A total of 37638 were prescribed warfarin with ≥6 months INR data . The percentage of time spent with poor INR control using the NICE criteria was 34.0% and using ESC/US criteria was 40.9%, for both SSE and bleeding outcome analyses.A total of 35 891 patients had sufficient data to analyse associations between INR control and SSE, and 35 035 patients had sufficient data to be included in the bleeding analysis. Both cohorts had a mean follow up of 4.3 years, mean TTRs of 71.9%, and mean CHAOver the study period, a total of 2802 SSE events occurred in 2422 patients. During periods where there were sufficient data to allow calculation of INR control, 1868 SSE events occurred in 1837 patients. Of those with SSE events, 1650 were strokes and 218 were non-stroke systemic emboli.2DS2-VASc score, and higher prevalence of hypertension, prior ischemic stroke and PVD (Table 1) than those who did not. Females were less likely to suffer SSE than males.Patients experiencing SSE during the study period tended to be older, have higher CHAP < 0.001]; using the ESC/US criteria for poor control the HR for SSER was1.84 (95%CI 1.68–2.02).In univariable Cox analyses, the HR for SSE associated with poor INR control according to NICE criteria was 1.84 . Using the ESC/US criteria of poor INR control as a univariable the HR for bleeding was 1.45 (95%CI 1.38–1.54).In univariable Cox analyses, the HR for bleeding associated with poor control according to NICE criteria was 1.43 [(95% CI 1.35–1.51), Table 5) after adjustment for CHA2DS2-VASc score, and deprivation quintile. CHA2DS2-VASc score was also independently associated with bleeding events, after mutual adjustment but deprivation quintile was no longer associated.In the first multivariable models, poor INR (according to NICE criteria) was associated with both an increased risk of bleeding (Table 6). After mutual adjustment, increasing age was also associated bleeding events, as was diabetes, prior ischaemic stroke, and prior bleeding events, ischaemic heart disease, heart failure, and chronic kidney disease (stage 4+) was associated with bleeding events. Female sex was associated with fewer bleeds.Poor INR control according to NICE criteria was again associated with bleeding independently of other risk factors in the second multivariable model (Tables S3 & S4).Very similar relationships were seen between poor INR control according to ESC/US and bleeds in the multivariable models (see Table S6).The bleeding event rate was 3.9 (95%CI 3.8–4.0) in the overall population; 3.4  during periods of adequate INR control and rising to 4.8 (95% CI 4.6–5.0) during periods of poor INR control according to the NICE criteria. Very similar bleeding event rates were seen in those meeting ESC/US criteria for poor INR control  compared to the NICE criteria (34.0%), the relationships between SSE or bleeding event rates and poor INR control were very similar in magnitude with both guidelines.12 as well as those reported in randomised controlled studies where VKA therapy was included as a comparator against the DOACs (SSE range 1.50–2.2% and bleed range 3.1–3.4% in VKA arms).23–26The overall SSE and bleed rate in this study was 1.3 and 3.9 per 100 patient years, respectively. This was similar to rates recently reported in another real-world study of INR control in patients with AF,23–26 Differences in methods of calculating TTR, and the absence of reporting INR control assessed by very low or very high individual INRs, limits the comparisons that can be made between studies, and health care systems. Lastly, the comparison in bleeding event rate between studies is further limited by the lack of consistent reporting of bleeding events and severity.While the mean TTR in this study was 72% compared to 55–64% in randomized controlled trials (RCTs), the lower bleeding event rate observed in the RCTs may be explained by differences in definition of bleeding events, selective patient enrolment and enhanced observation of patients compared to real-world studies.This study evaluated the impact of multiple clinical and demographic factors as well as temporal INR control according to multiple criteria in one of the largest real-world studies of INR control in patients with NVAF. The use of a population-scale, individual-level rich linked anonymised data sources is a particular strength. The linked primary and secondary care data held by SAIL enable the investigation of a very large cohort of individuals longitudinally over a period of years and across multiple data sources, giving a much more complete picture of patient treatment, health, and clinical characteristics in a diverse and representative population than in previous studies.2DS2-VASc score was, as expected, associated with an increased risk of both SSE and bleeding, as were many individual characteristics commonly associated with stroke or bleeding. Increasing age (≥75 years) was associated with the highest risk of SSE and bleeding. Prior bleeding events were also strongly associated with further bleeding events and prior ischaemic stroke was associated with SSE, as demonstrated in previous studies.27–29Increasing stroke risk, assessed by the CHA30 Notably, INR control was worse in females than males, as we demonstrated previously in this cohort.15 It is therefore uncertain whether the sex imbalance in observed SSE outcomes could be diminished by improving INR control in women.We found that females had a lower likelihood of bleeds, but a slightly higher likelihood of SSE independently on INR control, in keeping with previous studies.Surprisingly, we observed no independent association between deprivation and INR control, SSE or bleeding in this study. The data for this study were obtained from routine data sources following patient interactions within the Welsh National Health Service, where prescriptions and INR monitoring are free to patients at the point of delivery, potentially mitigating important financial access barriers to healthcare, as seen in more economically-disadvantaged individuals or populations. This should be an important consideration when comparing the findings of our study to other healthcare systems.Table S7A & B). These patients had slightly lower prevalence of hypertension and a slightly higher prevalence of heart failure, but otherwise had very similar characteristics to the overall cohort. Importantly, all major comorbidities were well represented in the multivariable models and the inclusion of this small group would not be expected to have materially influenced the strong associations between poor INR control and adverse outcomes.Deprivation index data were missing in ∼1.9% of patients and were therefore excluded from the first multivariable models  are unlikely to have their individual INR data entered consistently in their primary care record. It was also not possible to identify periods of temporary discontinuation of warfarin for patients undergoing surgery which if recorded in the primary care record may have resulted in periods of INR control recorded as out of range.Table S8A & B). It cannot be determined why these patients had insufficient INR readings recorded in the respective period. However, these patients had a significantly higher prevalence of most SSE and bleeding risk factors, and it is likely that most of these patients were managed in secondary care, although it is also possible that they may not have been receiving warfarin nor having regular INR checks.We identified a number of patients prescribed warfarin, with at least 6-month follow up time but who had inadequate number of INR results during the study period who were therefore excluded from the analyses. A further group were excluded who had an had inadequate number of INR results in the 6 months prior to the event to allow calculation of INR control  was measured independently; pathology results, alcohol and illicit drug use are less robustly documented in the WLGP datasets, and aspirin and non-steroidal anti-inflammatory are frequently purchased without a prescription in the UK. Finally, the HASBLED score, unlike CHA9The temporal calculation of INR control allowed us to assign patients to adequate or poor INR control at each INR result based on the previous 6 months of INR data; periods where there were insufficient INR results were excluded from INR calculation but allowed patients to re-enter the analysis when there were sufficient INR results to recalculate INR control. While this conservative approach had the potential to exclude periods of INR calculation in patients who had planned extended periods between INR tests, fewer than 1.4% of INR tests had an interval of greater than 84 days. This approach provided greater surety that only periods of warfarin administration and monitoring were included in the calculation of INR control. Furthermore, the recalculation of INR control and assignment to adequate or poor INR control at each INR test allowed us to test the association between adverse events and INR control in the directly preceding period. Our approach provides a methodological improvement over previous studies that have reported the association between bleeding events and mean TTR, which may have been calculated over a period of years prior to an event.We found similar event rates in those with evidence of inadequate control by ESC criteria as those with inadequate control by NICE criteria at the population level. Whilst one could argue that the ESC guidelines could be adopted by the UK due to their greater simplicity, where clinical computer software can reliably identify those with ‘high’ or ‘low’ INR levels, this could still help guide individual patient management approaches and we would not advocate to change the UK guidance. However, our findings suggest that where those data are not readily available, following the ESC guidelines is acceptable in UK practice.This study was conducted prior to the COVID-19 pandemic when a greater proportion of patients were prescribed warfarin. Many healthcare providers have since moved patients to DOAC therapy which requires less intensive monitoring and patient contact. However, we are aware that across many healthcare systems large numbers of patients are still prescribed warfarin. The data in this study may provide valuable insight into selecting patients who are at the highest risk of bleeding with warfarin, who may require the greatest effort in improving INR and could be considered for alternative anticoagulation strategies where clinically appropriate.Periods of poor INR control, as well as increasing stroke risk and specific comorbidities for stroke and bleeding, were associated with a considerable increase in the risk of SSE and bleeding events in warfarin treated patients with NVAF. The potential to reduce these adverse events through improvement in INR control at a population level should be closely considered to help improve patient outcomes. For individuals, a detailed risk assessment, considering factors leading to poor INR control and comorbidities that increase SSE and bleeding risk, remains essential. However, it is clear that improved measures to optimise effectiveness of anticoagulation are likely to improve clinical outcomes.oead037_Supplementary_DataClick here for additional data file.


Denervation contributes to loss of force-generating capacity in aged skeletal muscles, but problems with quantification of denervated fibers mean the precise impact of denervation on muscle function remains unclear. This study therefore looked to develop a reliable assay for identifying denervated muscle fibers, and used this to explore the impact of denervation on age-related force-generation in mouse skeletal muscle. Thirteen young (6-month-old) and 10 old (24-months-old) C57Bl/6 J female mice were utilized. Anaesthetized mice were infused with the fluorescent deoxyglucose analog 2[N-amino]-2-deoxyglucose (2-NBDG) and the tibial nerve was repeatedly stimulated to label active skeletal muscle fibers by activity-dependent uptake of 2-NBDG. Data on muscle force generation were acquired as part of the stimulation routine. Labeled muscles were removed, snap frozen, sectioned, and slide mounted. Sections were imaged to show accumulation of 2-NBDG in activated fibers and lack of 2-NBDG accumulation in quiescent (denervated) fibers, then processed using immunohistochemistry to allow collection of data on fiber number and morphology. Soleus muscles from older mice had nine times as many denervated fibers as those from young mice . Older muscles developed significantly more passive force and less specific force, but denervation only partly accounted for age-related deficits in specific force. Further investigations are required to definitively identify contributors to the decrease in force generation that remain unaccounted for. Age-related skeletal muscle atrophy is accompanied by declines in muscle mass and function which threaten mobility and independence. The muscles of older individuals have been characterized as smaller , losses of force transmission, myosin isoform transformation, and changes in muscle structure  path for the delivery of force from neighboring fibers Sheard . They alWhilst several studies have documented fiber denervation  and 10 old (24-months-old) C57Bl/6 J female mice, equivalent to 18 and 72 years of human ageing amino]-2-deoxyglucose; Sapphire Bioscience, Australia) diluted in isotonic saline was infused. This procedure was to label active fibers with the fluorescent deoxyglucose analog 2-NBDG (see below).Prior to tissue collection, muscle force measurements were conducted on deeply anesthetized mice. The time of day and duration of all experiments were kept consistent. Anesthesia was achieved via an intraperitoneal injection of 30 mg.kgBoth legs were subsequently shaved and mice were placed in a prone position on a silicone elastomer  molded platform. A longitudinal incision was made in the skin along the posterior midline of the left leg and retracted by fixation to the underlying Sylgard with 38 mm insect pins. The calcaneal tendon was carefully detached with surgical scissors and the triceps surae was gently reflected rostrally. The soleus was identified and its lateral margins were freed from the gastrocnemius via blunt dissection. Its distal tendon was entirely freed from the calcaneal tendon and attached to a straight tungsten wire in turn attached to an isometric force transducer . The soleus nerve was identified and protected from stretching as the gastrocnemius was separated from the soleus. The portion of the calcaneal tendon attached to the gastrocnemius was pinned away to prevent interference with force measurements.A bipolar stimulating electrode was positioned under the posterior tibial nerve and exposed soft tissues were covered in mineral oil to prevent desiccation. Stimulator  settings were optimized to elicit the maximal force from a tetanic contraction. Force measurements were collected on Scope (v4.1.1) software via an ADInstruments PowerLab amplifier  running on an Apple iMac computer .Mice were given an intravenous bolus of 2-NBDG prior to stimulation, as outlined above. The presence of intravascular 2-NBDG was confirmed by popliteal artery fluorescence when excited under an epifluorescence-equipped Leica M3  surgical microscope. 2-NBDG is a fluorescent glucose analogue which is taken up by active cells via the GLUT2 and GLUT4 transporters  and of the active and passive tension at different muscle lengths (to generate length-tension curves)  and snap frozen in liquid nitrogen cooled L-isopentane. Mice were euthanized by sodium pentobarbitone overdose and cervical dislocation.Cryomatrix-embedded left soleus muscles were first bisected at an oblique angle, then mounted upright to a specimen disk and sectioned at a plane selected to avoid tendons and to ensure inclusion of all fiber profiles  overnight at 4 °C. The following morning, slides were washed in nine changes of Tris-buffered saline (TBS)  and re-incubated with an AlexaFluor 568 conjugated goat anti-rabbit IgG secondary antibody  for four hours at room temperature. Antibody concentrations and incubation times were optimized to work on fresh frozen mouse muscle tissue. Each staining batch also included a negative control in which the primary antibody was omitted from the immunodiluent. Once secondary antibody incubation was complete, slides were washed in another nine changes of TBS and cover-slipped using a 10:1 ratio of glycerol and TBS. The coverslip was sealed with clear nail polish. Sections were illuminated and photographed as described above.Digital micrographs were analyzed in a multistep manner that involved automated muscle fiber segmentation and manual refinement. Automated muscle fiber segmentation was done using a machine learning algorithm   in accordance with other publications . ACSA was calculated from fiber number and cross-sectional area derived from minimum feret diameter , using either one or two (one each for younger and older groups) curves. Analyses of force frequency curves were conducted in GraphPad Prism (version 6.01).0)  logistic function by least partial squares method. Akaike’s information criteria (AICc) were used to determine whether one (younger and older groups combined) or two curves (younger and older groups separated) modelled the data better. The difference in AICc between one and two curves was 45, indicating that two separate curves modelled the data better than one , indicating that it was right-shifted  and shallower . The muscles of older mice produced less active  and more passive force  at L0  Fig. E. Force one Fig. . The forPrior to force characterization mice were injected with an intravenous label 2-NBDG) which was subsequently taken into cells in proportion to their metabolic activity during stimulation. This technique was used to allow discrimination between recently activated and inactive fibers under the assumption that by nerve stimulation active muscle fibers should accumulate significant amounts of label while quiescent (denervated) fibers should not. This assumption was validated by a preliminary control experiment employing direct electrical stimulation of a soleus muscle’s posterior surface. When the muscle was sectioned, muscle fibers near the stimulation location at the posterior surface were strongly labeled   to direct muscle stimulation . When maximal force elicited from nerve stimulation was expressed as a percentage of that produced via muscle stimulation , we found that the presence of denervated fibers cost the young muscles 7% of their maximal force generating potential (NvM: 93 ± 4%), whilst denervated fibers in older muscles cost 18% of maximal force potential (NvM: 82 ± 9%). The functional consequence of denervation overall was more severe in older mice than younger (p < 0.001), Table . However2, p = 0.040)   (Table This investigation documented the contribution of muscle atrophy and denervation to age-related decline in muscle active tension using a novel technique to identify denervated muscle fibers. We did this by reconciling data on muscle function and histology from muscles exposed to a label (2-NBDG) which accumulated within recently activated muscle fibers. We found a 17% age-related deficit in muscle force . NaV1.5 is not normally expressed in adult skeletal muscles but its expression has been observed in the muscle fibers of older rats  on their sarcolemma . A version of this technique (using radioactive deoxyglucose) has previously been used to visualize the fibers of individual motor units  is fiber type transformation, though there is relatively little change in mouse soleus fiber type composition with age (Hill et al. 0 than younger mice, results that corroborate previous observations (Brown et al. 0.Muscles from older mice were found to produce less active tension and more passive tension at LThe application of a new method using activity dependent uptake of a fluorescent marker shows that muscle fibers with no functional nerve contact are present throughout adulthood in the mouse soleus, and that the proportion of such fibers increases with age. Old muscles generate less force than young muscles, but this deficit is only partly attributable to denervation. Fibers in old muscles are smaller than in young, and whilst this also contributes to the age-related loss of force there is a further remaining deficit that potentially arises as a consequence of age-related changes in sarcomeric proteins, fiber length, and connective tissue remodeling. The specific contributions of these and any other factors contributing to loss of force-generating capacity are yet to be determined.


Diastolic dysfunction is increasingly identified as a key, early onset subclinical condition characterizing cardiopathologies of rising prevalence, including diabetic heart disease and heart failure with preserved ejection fraction (HFpEF). Diastolic dysfunction characterization has important prognostic value in management of disease outcomes. Validated tools for in vivo monitoring of diastolic function in rodent models of diabetes are required for progress in pre-clinical cardiology studies. 2D speckle tracking echocardiography has emerged as a powerful tool for evaluating cardiac wall deformation throughout the cardiac cycle. The aim of this study was to examine the applicability of 2D speckle tracking echocardiography for comprehensive global and regional assessment of diastolic function in a pre-clinical murine model of cardio-metabolic disease. Type 2 diabetes (T2D) was induced in C57Bl/6 male mice using a high fat high sugar dietary intervention for 20 weeks. Significant impairment in left ventricle peak diastolic strain rate was evident in longitudinal, radial and circumferential planes in T2D mice. Peak diastolic velocity was similarly impaired in the longitudinal and radial planes. Regional analysis of longitudinal peak diastolic strain rate revealed that the anterior free left ventricular wall is particularly susceptible to T2D-induced diastolic dysfunction. These findings provide a significant advance on characterization of diastolic dysfunction in a pre-clinical mouse model of cardiopathology and offer a comprehensive suite of benchmark values for future pre-clinical cardiology studies. Currently, while there are new advances in treatments for patients with HFpEF, the benefits are largely seen in those with advanced disease4, thus a substantial research effort is underway for drug-discovery in pre-clinical models. There is an urgent need for validated tools for systematic evaluation of diastolic function in small rodent models of disease. Clinically important conventional echocardiography approaches to assess diastolic function clinically  have been applied to tracking diastolic function in small rodents6, but high heart rates (> 400 bpm) and small cardiac anatomy present unique challenges7. A high level of training and expertise is required to generate reliable outcomes. Speckle tracking echocardiography is a post-processing tool to track deformation of the myocardial wall throughout the cardiac cycle, and is increasingly used in both clinical11 and pre-clinical studies15. Global cardiac strain is the most commonly reported variable from speckle tracking studies, which is typically associated with characteristics of systolic function. Notably, speckle tracking-derived strain rate can be determined over the full length of the cardiac cycle and peak strain rate during the diastolic period may provide a robust additional measure to support the evaluation of diastolic function in small rodent models for pre-clinical studies7.Diastolic dysfunction is characterized by impaired heart relaxation due to increased myocardial wall stiffness. Identification of early diastolic dysfunction has important prognostic value for progression to heart failure. Heart failure with preserved ejection fraction (HFpEF), a condition characterized by diastolic dysfunction, is highly prevalent in diabetic patients and has worse prognosis than in non-diabetic patients16. The extent and speed of deformation of a specific area of the myocardium is recorded, in relation to the initial dimensions. A significant advantage of speckle tracking echocardiography is that cardiac function can be assessed in long and short axis viewing planes to obtain information on longitudinal, radial and circumferential strain for any given area of interest. Tracking regional changes in myocardial wall deformation has proven to be useful particularly in identifying ischemic regions in studies of myocardial infarction15. This approach may also be applicable to generating a more advanced understanding of regional cardiac dysfunction in chronic disease states.Speckle tracking echocardiography is based on tracking the motion of speckle patterns created by interference of ultrasound beams in the myocardium over time19. Regional differences in diastolic function have not been examined. An evaluation of speckle tracking echocardiography to monitor diastolic function in more clinically relevant models of diabetes is lacking. Thus the goals of this study were to validate speckle tracking echocardiography as a tool to assess diastolic myocardial wall deformation and evaluate regional differences in diastolic dysfunction in a dietary (high fat high sugar) mouse model of obesity and type 2 diabetes (T2D).Whilst speckle tracking echocardiography is becoming more widely used, particularly for reporting global strain measures, its utility in expanding the available tools for assessment of diastolic function in small rodent models has not been fully realized. Given the escalating prevalence of diabetes, and the predisposition for diastolic dysfunction (and HFpEF) in diabetic patients, diastolic strain rate may be an important measure for pre-clinical studies investigating novel drug targets for treatment of diabetic heart disease. The few studies available reporting diastolic strain rate in diabetic rodent models have mostly used genetic models of advanced diabetesTo develop a mouse model of T2D, mice were fed a high fat high sugar diet for 20 weeks. T2D mice exhibited significantly increased bodyweight from 2 weeks of dietary intervention Fig. A, impairTo evaluate cardiac dimensions and systolic function, we performed M-mode echocardiography and B-mode 2D speckle tracking strain imaging in control and T2D mice. Exemplar M-mode and B-mode echocardiography images are shown in Fig. A key advantage of speckle tracking echocardiography is the ability to measure longitudinal, radial and circumferential myocardial deformation throughout the cardiac cycle, and therefore distinct measures of systolic and diastolic ventricular kinetics can be acquired  was larger than the extent of increase in E/e’ detected in diabetic mice (22% increase). The use of high frequency echocardiography for measurement of diastolic strain rate and velocity provides a less angle-dependent method of assessing diastolic dysfunction in settings of fast heart rates in mice (> 400 bpm). The pulse-wave Doppler signal is influenced by alignment of the ultrasound beam with the flow of blood and is heart rate dependent. Further, E/e’ is measured near the mitral annulus and consequently does not capture the global relaxation properties of the heart21. The acquisition of high quality B-mode images for strain analysis is largely dependent on the resolution and frame rates of the echocardiography platform, and is less dependent on the operator than Doppler methods. Post-processing of B-mode images does however incorporate some element of operator subjectivity, and development of artificial intelligence tools for strain analysis has emerged in the clinical literature to overcome this limitation23.Although speckle tracking echocardiography was first introduced as a tool for in vivo assessment of myocardial strain in mice over 15 years ago24 although clinical studies suggest that severe diastolic dysfunction (Grade III) is characterized by a shortening of isovolumetric relaxation time.25 Mitral valve E-wave deceleration time was significantly increased in T2D mice, consistent with previous studies5. However, the nature of changes in both parameters are of relatively small magnitude (1–2 ms), and the underlying biological significance is difficult to assess. Deceleration time in particular has been noted for limited significance in the rodent setting of high physiological heart rates7. Further work needs to be undertaken to achieve consistency of measurement of these indices in rodents and to enhance the interpretation of these very small data shifts.In the present study, T2D mice exhibited a small but statistically significant decrease in isovolumetric relaxation time. Typically prolonged (or unchanged) isovolumetric relaxation time has been reported in settings of diastolic dysfunction,Early diastolic strain rate has been proposed as a measure of left ventricular relaxation, capturing the rate of ventricular wall deformation during early diastole, similar to the Doppler measure of tissue movement at the mitral annulus during the early filling phase (e’ wave). Surprisingly, e’ was unchanged in T2D mice in the present study, despite differences in diastolic strain rate. Although inter-observer variability of Doppler analysis (E/e’) was lower than diastolic strain rate (evidenced by higher Pearson correlation coefficient), the technical difficulty of acquiring tissue Doppler images in mice with small anatomy and high heart rates could be considered to be more limiting relative to acquisition of b-mode images for strain analysis. These findings suggest that, at least in this setting, diastolic strain rate is more sensitive to detection of left ventricular relaxation impairment than e’ wave measured by tissue Doppler imaging.13. In the present study, regional analysis revealed that impairment in longitudinal diastolic strain rate in diabetic mice was most prominent in the anterior free wall. Impairment in circumferential diastolic strain rate was most prominent in the inferior free wall and anterior septum. The underlying mechanism of regional diastolic impairment is not clear, but may relate to focal areas of fibrosis and/or localized thickening of the ventricular wall, and further investigation is required. These findings are similar to regional changes in myocardial deformation reported in adolescents with type 1 diabetes using cardiac MRI26. Significant impairment in diastolic strain rate was observed in the inferior septal and free wall regions26. This previous study calculated the diastolic relaxation fraction  and reported that adolescent T1D patients exhibit early diastolic left ventricular discoordination, despite otherwise normal cardiac function and left ventricular size. Thus assessment of regional myocardial wall deformation may provide an early indicator of diastolic dysfunction by identifying disparate regions of impairment. In the present study, regional changes in diastolic strain rate were concordant with differences detected in the global values. In future studies of more mild diastolic dysfunction, regional analysis of diastolic strain rate may be valuable to provide evidence of localized dysfunction which may not be detectable in the global output.In the pre-clinical literature, regional analysis of myocardial wall deformation has primarily been used in studies of myocardial infarction for identifying areas of dyssynchrony in the ventricular wall29. Global longitudinal strain quantifies the maximum change in length of the left ventricle during contraction , and thus is a measure of systolic function. Interestingly, in heart failure patients where ejection fraction is preserved (HFpEF) and systolic function is generally considered to be normal, global longitudinal strain is frequently reported to be decreased30. In addition, there are differences in methods of measuring ejection fraction in clinical and preclinical studies. In rodents, ejection fraction is typically measured using M-mode analysis in a short-axis view, while in humans, guidelines recommend the use of biplane measurements in long-axis views31. In humans therefore, the longitudinal function of the ventricle will contribute substantially to the measurement of EF, while in rodents, the EF is primarily measuring short axis contraction. As long axis shortening contributes approximately 75% of stroke volume in humans, compared to a contribution of approximately 25% by short axis shortening32, this is likely to make EF measurements in rodents difficult to directly translate to human studies. However, in both rodents and humans, global longitudinal strain is measured in the long-axis view. In the present study, measures of systolic function acquired from the short-axis imaging plane  were unchanged in diabetic mice. In contrast, global longitudinal strain was reduced, suggesting that longitudinal systolic dysfunction is present. These findings are consistent with other pre-clinical literature reporting that diabetic mice with normal ejection fraction exhibit reduced global longitudinal strain and diastolic dysfunction34. Clinically, global longitudinal strain has been incorporated into recently updated European Society of Cardiology guidelines for diagnosis of HFpEF35. In chronic settings of progressive cardiac dysfunction, such as diabetes, global longitudinal strain may provide a robust measure of early subclinical systolic dysfunction, with high predictive value, and improved translatability to clinical studies.While left ventricular ejection fraction has been the most widely used clinical measure of cardiac function for decades, global longitudinal strain is increasingly recognized as an important clinical indicator, with prognostic value in predicting adverse outcomes in chronic systolic heart failure0). Given that velocity is the rate of change of wall motion, it is therefore not dependent on the initial size of the ventricular wall. Thus, in a setting of diabetes where changes in the ventricular wall dimensions would be expected (as is the case in the present study), subtle differences in strain rate and velocity may be detectable. Additionally, there is some evidence from the clinical literature that strain rate may be more independent of acute changes in loading conditions (preload & afterload) than tissue velocities36, but further work is required to validate these findings in chronic settings.In the present study, both peak diastolic strain rate and peak diastolic velocity have been incorporated into the assessment of diastolic dysfunction in diabetic mice. In most cases, these variables show the same impairment, with the exception of unchanged circumferential peak diastolic velocity. By definition, strain rate is the derivative of strain, and is therefore presented as relative to the initial dimension  is notoriously difficult in small rodents with high heart rates. In the present study the early peak in mitral valve blood flow (E wave), mitral annulus tissue movement (e’) and diastolic strain rate was distinct from the atrial contraction-related peak in most cases. Some traces displayed partially fused early and late peaks, and some displayed only 1 peak in the diastolic period. Although quality images with distinct early peaks were available for every animal, replicate images where the early peak was not discernable were excluded from analysis and may have introduced an unavoidable level of bias to the dataset.38.In this study echo measurements were obtained at the end of the treatment period only, and no intermediate time point data were collected. An investigation involving repeated echo measurements during the development and establishment of diabetic heart disease would certainly be informative, especially in relation to refining the mechanistic interpretation of the parameters E and e’. The potential importance of molecular studies in distinguishing the components of myocardial stiffness and impaired relaxation in driving diastolic dysfunction has been recently highlighted.In conclusion, here we provide evidence to support the utility of 2D speckle tracking echocardiography to document global and regional diastolic dysfunction in a pre-clinical setting of murine cardiopathology. Peak diastolic strain rate and peak diastolic velocity constitute reliable indicators of diastolic dysfunction in diabetic mice, significantly correlated with traditional Doppler measures of impaired ventricular relaxation. Some evidence of longitudinal systolic impairment was detected by global longitudinal strain, despite preserved ejection fraction. The anterior free left ventricular wall appeared to be most vulnerable to longitudinal diastolic impairment. These findings provide a significant advance on characterization of diastolic dysfunction in a pre-clinical mouse model of diabetes and offer a comprehensive suite of benchmark values for future pre-clinical cardiology studies. Given the unmet clinical need for effective treatment options for HFpEF patients, implementing diastolic strain rate analysis as a robust tool for evaluating diastolic function in mouse models will support the ongoing drug-discovery effort in this field.www.arriveguidelines.org). Animals were randomly assigned to experimental groups and were group housed (4 mice per cage) in a temperature-controlled environment with 12-h light/dark cycles. Food and water was provided ad libitum.All animal experiments were approved by the University of Auckland Animal Ethics Committee and complied with the guidelines and regulations of the Code of Practice for the Care and Use of Animals for Scientific Purposes. This study is reported in accordance with the ARRIVE guidelines . After an initial 1 week transitional feeding period, animals were fed a high fat high sugar diet  or control reference diet  for 20 weeks duration. Fresh diet was provided twice a week. This is an extensively used model of diet-induced type 2 diabetes that is characterized by obesity, glucose intolerance, mild hyperglycemia and cardiac dysfunction.Glucose tolerance testing was performed after 11 weeks of dietary intervention following 6 h fasting. Baseline blood glucose levels were measured using an Accu-Check glucometer with a small blood sample from a needle prick to the tail vein. Glucose (1.5 g/kg body weight) was injected i.p. and blood glucose was measured at 5, 10, 30, 60, 90 and 120 min after the glucose injection.40, thus the 20 week timepoint allowed for sufficient time for diastolic dysfunction and other features of diabetic heart pathology to be fully established. Animals were anaesthetized with 3% isoflurane (with 1L/min O2 gas) and transthoracic echocardiography was performed using the VEVO LAZR-X 3100 with a MX400 probe (20–46 MHz), linear array transducer coupled with digital ultrasound system . The mice were secured to a warm platform containing ECG lead pads, and anesthesia was maintained via a nose-cone at 1.5–2.5% isoflurane (with 1L/min O2) throughout the procedure. All analyses were performed by an investigator blinded to study group allocation.Cardiac function was assessed following 20 weeks of dietary intervention. Our previous studies have confirmed that diastolic dysfunction (increased E/e’) is evident after 14 weeks of high fat high sugar diet feeding3 − LVIDd3, with correction factor 0.8. At least 3 consecutive cardiac cycles were sampled per cineloop image and 3 images per animal were analyzed and averaged.Left ventricular (LV) M-mode two-dimensional echocardiography was performed in a parasternal short axis view at the mid papillary level to measure LV wall and chamber dimensions. LV volumes were derived from dimensions using the Teichholz equation (end diastolic volume (EDV) = )) × LVIDd; end systolic volume (ESV) = (7/(2.4 + LVID at end-systole (LVIDs))) × LVIDs). Systolic function parameters: % ejection fraction ((EDV − ESV)/EDV) × 100), and % fractional shortening ((LVIDd − LVIDs)/(LVIDd) × 100). LV mass was calculated: 1.053 × Pulse wave Doppler and tissue Doppler imaging were acquired from the apical 4 chamber view to assess LV diastolic function parameters: velocity of mitral inflow during early passive filling (E) and during atrial contraction (A), and velocity of mitral annulus during early passive filling (e’) and during atrial contraction (a’), isovolumetric relaxation time (IVRT) and E-wave deceleration time. At least 3 cardiac cycles were sampled per cineloop image and 2–4 images per animal were analyzed and averaged. Wave forms from trans-mitral and tissue records were evaluated with temporal registration to validate waveform identification.Two-dimensional echocardiography B-mode loops were acquired from the left ventricular long axis  and short axis  views with a frame rate of 215 fps, and analyzed using Vevo strain software . Endocardial and epicardial borders were traced and checked throughout three cardiac cycles to ensure optimal tracking. The time at maximum relaxation (end-diastole) was used to determine the starting phase of the strain trace. Endocardial strain and displacement analyses were performed providing global and segmental  values. Endocardial strain rate and velocity parameters were derived from the strain–time and displacement–time relations respectively. The strain rate and velocity profiles for each image were examined to confirm that the early (passive filling) peak was identified by the software. In some cases, manual identification of the early peak was required, and images where the early peak was not discernable were excluded from the analysis. Average values were calculated from 2–3 independent images per animal.Data are presented as mean ± SEM and statistical analysis was performed using Graphpad Prism v7.0. Statistical outliers (> 2 standard deviations from the mean) were excluded to avoid data selection bias due to stochastic measurement of sampling anomalies . All data sets were tested for normal distribution using Shapiro–Wilk test. Differences between two groups were analyzed by Students unpaired t-test. For analysis of body weight, glucose tolerance and regional strain parameters, a repeated measures ANOVA with LSD post hoc tests was used. For correlation analyses, Pearson’s correlation coefficient was used. A p-value of < 0.05 was considered statistically significant.Supplementary Information.