Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract.Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla ( The human microbiota is an integral part of human physiology, influencing human development, host immunity, and nutrition  tract, especially the stomach, is a notoriously depauperate environment in terms of microbial composition and diversity, as compared to the oral cavity and the lower gastrointestinal tract. The stomach is a particularly unique and challenging locale from which to isolate and examine microbial constituents because of increased acidity and mucus production, which generally discourages microbial colonization. A number of methods are described in the literature with varied results in diversity, species selection and abundance  were further examined by WGS.t-test (p > 0.05). Although the total bacterial contents are different in six samples  when extracted using homogenization vs. lysis method, no specific extraction protocol provided consistently higher yields. This suggests both methods have comparable efficiency in extracting bacterial DNA from human tissue , Firmicutes , and Bacteroidetes (Gram negative) using each method , although this difference was not consistently in favor of one method or the other  showed a significantly higher abundance of Bacteroidetes using the homogenization method which was also in contrast to the lysis method that yielded higher abundance in gastric cancer samples . Notably, despite these differences in bacterial abundances, there was no trend as to which extraction method would routinely provide the greatest bacterial yield. For example, C233 tumor appears to have significantly greater total bacterial content with the homogenization extraction method  .Overall, no specific method consistently provides higher yields for either of above phyla/genus, and we found near equal quantitation between two methods of most clades across all three types of cancers. Of the 68 samples tested across four different primer sets, 53 samples show no significant difference in quantitation  of the full DNA content of the biopsy. Read counts and mapping rates to hg19 (95.773% ± 0.309) are comparable among all samples Table . The remp < 0.001)   Table . Since tOverall, Gram-negative and Gram-positive bacteria were represented by both extraction methods with similar efficiency, with some subtle differences Figure . For exaActinobacter, Firmicutes, and Bacteroidetes) as well as Fusobacterium relative to total 16S rDNA quantitation. Based on our qPCR results, no single method consistently generated higher readouts for any single clade across all samples. Our results therefore indicate that either extraction method, homogenization or prolonged lysis, can be successfully used to detect bacteria without introducing a significant bias when examining bacteria by qPCR.Study of the gut microbiome is of increasing importance with numerous studies identifying the role of microbiota in diseases such as cancer. Various protocols have been employed by the scientific community to extract bacterial DNA from human biopsies, which are then analyzed using next generation sequencing methods. In order to ensure that such an analysis of the gut microbiome is not biased due to the method of extraction, we analyzed and compared two widely used DNA extraction protocols, namely the mechanical disruption (homogenization) vs. the enzymatic lysis method. Furthermore, to account for the vast diversity of the human microbiome, it is important that such an analysis is performed on human biopsies, as opposed to preclinical samples. In order to realistically gauge the influence of a particular extraction method on the complexity of the human gut microbiome, we assessed 5 normal and 12 tumor samples across three different cancer types including gastric, esophageal, and colon cancer. Based on qPCR quantification, no single method consistently generated higher total bacterial content. There were a total of six samples that showed significant differences in the overall bacterial content between the homogenization and the lysis method, as assessed by absolute abundance of 16S rDNA using qPCR quantitation. However, this difference was not in favor of one particular extraction protocol. Moreover, it is possible that these differences may be intrinsic, since different regions of the same biopsy were used to extract DNA using the two protocols. To account for these intrinsic differences, we assessed abundance of three different phyla , Firmicutes  and Bacteroidetes phyla (gram-negative organisms) relative to total 16S rDNA quantiation. We found very similar yields from Actinobacter as well as from other phyla, irrespective of differences in their cell wall composition. Together, our data suggest that both extraction methods can be successfully employed in assessing the overall microbiome composition and content in human biopsies without skewing results due to the choice of extraction method.Gram-positive bacteria contain a single layer of peptidoglycan in their cell wall whereas gram-negative organisms contain an additional layer of lipopolysaccharide referred to as the outer membrane. In order to determine, if gram-negative vs. positive bacteria show differential susceptibilities toward lysis or homogenization methods, owing to their different cell wall composition, we compared bacterial yields using qPCR quantitation of 16S rDNA from https://cancergenome.nih.gov/). In our previous study, we successfully retrieved the microbiome information from TCGA samples with our computational pipeline . All participants provided written informed consent for use of their tissue samples in accordance with the declaration of Helsinki prior to study enrollment. Patients were enrolled in one of two studies—Weill Cornell Medical College Gastric Cancer and H. pylori Research Database and Tissue Repository (IRB 1203012274), and the NYPH-Weill Cornell Digestive Disease Registry (IRB 0908010582).A total of five colorectal tumor and matching normal pairs were obtained from the colorectal cancer biobank supported by the Translational Research Program at WCMC Pathology and Laboratory Medicine. For each sample, 3–4 frozen cores of 1.5 mm diameter each were obtained. One core for each of the samples was used for DNA extraction by either the homogenization or the lysis method. The DNA was subsequently used for qPCR and WGS studies.H. pylori Research Database, a registry and tissue repository to examine the natural history of H. pylori infection in patients with and without gastric cancer. Gastric biopsy samples were sourced from fundus or proximal body sections of the stomach from patients without gastric carcinoma. Biopsies were obtained using the Bard Precisor EXL® coated disposable biopsy forceps  and were immediately placed into individual sterile cryovials on dry ice and flash frozen while still in the endoscopic suite. The samples were then transferred to liquid nitrogen for prolonged storage.Three gastric mucosal biopsy pairs were obtained from the Weill Cornell Medicine Gastric Cancer Center and Esophageal cancer and pre-cancer tissue biopsies were sent to us by our collaborators at University of Kansas School of Medicine, Kansas City, KS. These biopsy samples were obtained from the esophagus at about 2–3 cm above gastroesophageal junction. Tissues were first stored in RNA later solution overnight at 4°C prior to freezing and were shipped to us on dry ice. The DNA from the biopsies was then extracted either using the homogenization method or the enzymatic lysis method as described.Non-paired biopsies from the same tissue were equally cut prior to gDNA isolation to enable extraction using the two extraction methods illustrated below.Frozen biopsy samples (~3–5 mg) were placed in a 2 ml sterile Eppendorf tube containing 350 μl of RLT lysis buffer (Qiagen®). Samples were then homogenized individually on ice for 20–30 s using a pre-sterilized homogenizer (Pro250® Pro Scientific) until tissue was uniformly disrupted. gDNA was subsequently isolated according to manufacturer's instructions from Qiagen Allprep Micro DNA/RNA kit . Briefly, the homogenized lysate were loaded on Allprep DNA mini spin column (Qiagen®) and was centrifuged briefly at 10,000 x g in a table top centrifuge. This step allows binding of DNA to the column. After subsequent washing steps with buffers AW1 (Qiagen®) and AW2 (Qiagen®), DNA is eluted using the elution buffer provided in the kit. Homogenizer was cleaned and sterilized before and after each individual sample with separate 15 ml falcon tubes containing 10% bleach, 70% EtOH, and RNAse/DNAse free sterile water.Frozen biopsy tissue (~3–5 mg) was placed in a 1.5 ml centrifuge tube and 180 μl of lysozyme (Gold Biotechnology Cat no: L-040-10) (20 mg/ml stock concentration prepared in TE) was added. Lysozyme was pre-warmed to 37°C prior to its addition to the sample. Samples were then incubated at 37°C for 1 h. Proteinase K  was added after the incubation. And sample was incubated at 56°C on a heat block and periodically vortexed until complete lysis of tissue was observed (4–6 h). Samples were then incubated in 200 μl Buffer AL from QIAamp DNA Mini kit (Qiagen) for 30 min at 70°C before continuing extraction using QIAamp DNA mini kit (Qiagen) according to manufacturer's instructions  and a Qubit™ 2.0 Fluorometer .To validate that the two kits used in the above two mentioned protocols themselves did not contribute to any variation, we re-extracted 1 μg of DNA from one of the gastric cancer samples using Allprep DNA/RNA micro kit as well as QIAamp DNA mini kit. This re-purified DNA from the two kits (5 ng each) was then subjected to qPCR using the 16S rDNA gene primer set to check for bacterial abundance. Furthermore, to prove that the columns itself were not a source of any contamination, we performed parallel purifications using water and subjected the eluate to qPCR against the 16S rDNA gene primer set Figure .Fusobacterium sp. abundances, genomic DNA was initially analyzed via qPCR. Reactions were carried out on a StepOne® 48-well thermocycler (Applied Biosystems). Primers used were FAM-labeled Genesig® 16S Eubacteria quantification primer/probe set (Proprietary sequence-PrimerDesign®) to determine total bacterial composition and a custom FAM-MGB Fusobacterium primer/probe (Applied biosystems) for Fusobacterium quantitation: Forward: 5′–AAGCGCGTCTAGGTGGTTATGT-3′; Reverse: 5′-TGTAGTTCCGCTTACCTCTCCAG-3′; Probe:FAM CAACGCAATACAGAGTTGAGCCCTGCATT  and Real-time PCR reactions were performed in triplicate on MicroAmp® fast-optical 48 well (0.1 ml) reaction plates (Applied Biosystems). Each reaction contained 1–2 μl template DNA, 0.5ul of 20X ABI primer/probe assay, 5 μl Taqman® Gene Expression Mastermix (Applied Biosystems), and RNase/DNase free water to a total volume of 10 μlCycling conditions included an initial denaturation at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.Escherichia coli gDNA (ATCC®) for 16S total quantitation and Fusobacterium nucleatum gDNA (ATCC® Strain VPI-4355) for Fusobacterium sp. quantitation.Absolute quantification was performed using a standard reference curve and controls prepared using known concentrations Genomic DNA (200–500 ng) was submitted from each sample to the Weill Cornell Medicine Epigenomics Core for library preparation and subsequent WGS using an Illumina TruSeq DNA-seq® DNA sample preparation kit and the Illumina HiSeq 2500® platform. Each sample was sequenced on a single flow cell lane as 50-bp paired-end reads at roughly 10x coverage to Human genome. Homopolymers, adapters and distribution of base quality of raw sequences from each sample were investigated using FastQC (version 0.10.1). In order to estimate the potential contaminations from experiment, two control samples containing only RNase/DNase free water went through two extraction procedures along with biopsies samples, and were similarly sequenced as the clinical samples. There were no bacteria detected by either qPCR or WGS in those two control samples.1) Filtering human DNA: All reads from WGS data were aligned to corresponding Human DNA databases with four different aligners  step by step. Any reads mapped to Human DNA databases were removed and the remaining reads were used as the input for bacteria identification.2) Mapping to bacterial genomes: 1,421 non-redundant bacterial genomes were collected from NCBI. Bowtie2 was used as the aligner to map the read to each bacterial genome, and each read was labeled as either uniquely mapped, unmapped or ambiguous (multiple genomic mapping).3) Genome coverage evaluation: For any bacteria with more than ten uniquely mapped reads we compute a genomic coverage measure to further remove false identifications.4) Calculating the relative abundance: Based on the identified bacteria from step 3, we applied a Bayesian statistical framework to assign all mapped reads to the most probable bacterial source genome ] for each experiment and each sample individually, and then performed a one-tailed one-sample MS, DB, and CZ conceived and designed project. PT and SP extracted DNA and performed the experiments. PS and SV collected clinical samples. CZ and DB performed the bioinformatic analyses. MS, PT, CZ, and DB interpreted the results. CZ, PT, SP, DB, and MS wrote the manuscript. All authors contributed to final revisions of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we report on our discovery of the Merensky Reef in the Bushveld Complex that occurs on subvertical to overturned margins of depressions in a temporary chamber floor. Such relationships preclude crystal settling and demonstrate that the reef crystallized in situ. This finding indicates that platinum deposits can grow directly at the chamber floor, with immiscible sulfide droplets sequestering ore-forming noble metals from strongly convecting silicate magmas. Our model also provides evidence for the paradigm that argues for magma chambers being masses of nearly crystal-free melt, which gradually loses heat and crystallizes from the margins inward.Most of the world’s economically-viable platinum deposits occur as ‘reefs’ in layered intrusions – thin layers of silicate rocks that contain sulphides enriched in noble metals. There are two contrasting magmatic hypotheses for their formation. The first suggests accumulation through gravity-induced settling of crystals onto the magma chamber floor. The alternative argues for  The Merensky-type platinum deposits occur as thin, continuous layers of sulphide- and chromite-bearing silicate rocks anomalously enriched in the platinum group elements , thus are referred to as ‘platinum reefs’. The physical and chemical processes responsible for their origin remain highly controversial, with two opposing magmatic models. One school of thought holds that they are produced by gravity-induced settling of crystals through the magma body onto the chamber floor16, with potential subsequent sorting of minerals during late-stage slumping of the cumulate pile18. The competing hypothesis states that they are formed by in situ growth of all minerals, including chromite and sulphides, directly on the chamber floor, accompanied by minor re-deposition of the minerals in association with convection in the magma chamber27. The resolution of this dilemma remains critical for our understanding of the formation of magmatic mineral deposits as well as for critical evaluation of the competing paradigms for magma chamber evolution52.Merensky-type platinum deposits in mafic-ultramafic layered intrusions Fig.  – named 30 which partly explains why the MR, together with two other principal ore deposits of the complex (UG2 chromitite and the Platreef), provide ~75% of the globally exploited platinum, ~35% of the world’s Pd and >80% of its Rh2  in mafic-ultramafic complexes  with four acid digests and nickel sulphide collection fire assay for PGE. Each ICP-MS analysis was accompanied by control standards GTS-2a, AMIS0167, and AMIS0013 and selected samples were reanalyzed to check anomalous results. For all elements the relative standard deviations were less than 10%. A complete list of analyses from the overhanging MR is available online within the electronic Supplementary Table One representative exposure of an overhanging MR package from a pothole in the Lonmin Platinum Karee mine, Western Bushveld was selected for detailed sampling. Bulk chemical analyses were obtained from five vertical and horizontal sections across the MR package in this exposure Fig. . RectangMicrobeam scanning X-ray fluorescence analysis was conducted on a polished rock slab using the Bruker Tornado™ desktop scanner equipped with a rhodium target X-ray tube operating at 50 kV and 500 nA without filters and an XFlash® silicon drift X-ray detector. Analyses were quantified using the Bruker ESPRIT software, which provides standardless semi-quantitative analyses for elements heavier than Na, with estimated detection limits for the given operating conditions and dwell times of approximately 1000–2000 ppm for first-row transition metals. Maps were created using a 25–40 μm spot size on a 40 μm raster with dwell times of 10–20 ms per pixel. Maps are represented as un-quantified background-corrected peak height data for Kα peaks for each element.Supplementary InformationSupplementary Dataset


Aethionema arabicum (Brassicaceae) is a dimorphic annual species that is hypothesized to survive stressful conditions during colonization due to adaptive plasticity in life-phase  and fruit morph (dehiscent [DEH] vs indehiscent fruits [IND]). We tested for adaptive plasticity in life-phase and fruit morphs along laboratory environmental stress gradients found in the natural habitats of Ae. arabicum. We considered optimal environmental conditions (750–2000 m above sea level) to be those that resulted in the following fitness parameters: higher biomass and a higher total number of fruits compared to stressful habitats. We found evidence of plasticity in life-phase and fruit-morph along a stressful environmental gradient. High hydrothermal stress proportionally increased the number of dehiscent morphs and non-dormant seeds germinating in autumn. This offsets natural phenology towards dry and cold winter , yielding fewer fruits that dehisce in the next generation. We conclude that the plastic responses of Ae. arabicum to natural stress gradients constitute a strategy of long-term adaptive benefits and favouring potential pathways of colonisation of the optimal habitat.Plasticity in plant dispersal traits can maximise the ability of a plant species to survive in stressful environments during colonization.  While animal migration to find suitable habitats is distinctly visible, for sessile plants, the strategy is cryptic and mostly involves adaptive structural/functional modifications suitable for such colonization. Morpho-physiologically diverse fruits and seeds (heteromorphic diaspores) and phenotypic plasticity are thought to influence greatly population establishment and local adaptation5. Moreover, plasticity plays an important role during environmental change or colonization and is likely to favour plastic individuals to more successfully survive and reproduce than the less plastic individuals7. However, empirical evidence is scarce on whether plants can tune their life strategies to colonize optimal habitat9.Spatial distribution of species diversity is vital to our understanding of why species assembly within the same area tends to be distinct from assemblages anywhere else11. This diversity allows alternative strategies of dispersal, germination, dormancy, and seedling competitive ability13. Both spatial dispersal and dormancy are considered part of ‘bet-hedging’ strategies in unpredictable environments, where offspring are distributed over space or time to optimize their opportunity for successful reproduction irrespective of the prevailing conditions. This strategy maximizes the geometric mean fitness across generations, which is very sensitive to extreme environmental conditions14, as on mountain slopes.The production of distinct heteromorphic diaspores is considered as an outcome of constant fine-tuning of different tactics for successful dispersal and appropriate germination timing15 considered two apparently independent environmental variables, stress and disturbance, to propose that inhabitants within continuously unproductive, disturbed conditions  exhibit a stress-tolerant strategy16. Organisms living at the boundaries (edges) of their ecological niches may experience environmental conditions that deviate from the optimal conditions for normal growth and development18. The induction of phenotypic plasticity via environmental cues are well documented , however, a thorough understanding is lacking about the plastic adaptation to changing environment at different altitudinal ranges and calls for more integrative eco-physiological studies.Early eco-physiological perspectives for classification of plant species21 and biotic13 stress experienced by the mother plant at different ontogenetic stages. As in the heteromorphic Diptychocarpus strictus (Brassicaceae), both severe water stress and simulated herbivory by defoliation22 led to the production of a greater proportion of indehiscent fruits. The diverse morphology of heteromorphic species, living in arid and semi-arid, stressful habitats24, provides an excellent opportunity for the diaspores to escape uncertain fate in time and space from the complex environmental gradient in the mountainous terrain.From an adaptive viewpoint, fruit and seed heteromorphism might pose a predicament, because not all diaspores are optimally adapted to the prevalent environmental conditions, which risk the long-term reproductive success of the offspring. Whereas an ecological perspective highlights the advantages of diverse fruit morphs considering their development is often plastically synchronized to abioticAethionema arabicum (L.) Andrz. ex DC. is an annual member of the Aethionemeae and occurs mainly in the East Mediterranean and Irano-Turanian region25, the hypothetical centre of origin for Brassicaceae27. The species constantly experiences unpredictable climatic conditions in the semi-arid steppe , along elevations of the mountain slopes and screes  and seeds  with complete lack of intermediate morph30. While the morph with low dispersal ability remain close to mother plants and germinate more quickly , the morph with high dispersal ability germinate later  due to their dormancy31. Such a combination has also been observed in other species and interpreted as a way to overcome high sibling competition in local populations32. The dimorphism displayed dynamic flexibility in the production of reproductive parts33 and in the proportion of two fruit morphs (DEH: IND) produced on the same plant in response to temperature29, branch removal34, mechanical damage, and herbivory35. This distinct dimorphism of Ae. arabicum presents a fascinating model system with which the optimal plastic response of parental generation is observed that allows the plants to modulate their dispersal and germination ability in response to environmental cues thereby gaining strategic advantage potentially via bet-hedging.ees Fig.  with occAe. arabicum regulate plasticity in life phases  and fruit morphs (DEH and IND) which promotes colonisation at the optimal habitat. In this study, we used an integrative empirical and meta-analytical approach i) to estimate the plant fitness and plasticity of life phases and fruit morphs in response to the natural equivalent of optimal (as control) and sub-optimal (as stress) abiotic and biotic conditions, and ii) to interpret the consequence of stress-mediated plasticity upon the life-strategies of dimorphic Ae. arabicum and possible pathways of migration towards its optimal habitat.Based on the above premise, we hypothesized that the environmental gradients along the habitats of Ae. arabicum collection sites in 10 countries on the East Mediterranean/Irano-Turanian mountain slopes highlighted the unpredictable and stressful natural habitats of the species  at the lower edge of the optimal habitat (0–750 masl). On the other hand, the spring temperature at the upper edge of the optimal habitat (2000–3000 masl) is sub-optimal , while during vegetative and reproductive growth the summer temperature is 20 ± 2 °C. However, the summer temperature is higher than optimal  confirmed the abundance of Ae. arabicum at the optimal habitat. While the basic geophysical parameters driving plant adaptation along the mountain slope is often studied in tandem with regional parameters38, the surface topology may confound such parameters and differs strongly from standard meteorological data39. Therefore, we considered all interacting factors equally during our study and analysed their overall effect on phenotypic plasticity.The congregation of ees Figs , S1, defAe. arabicum on the stony mountain slopes and screes  with low precipitation, which physiologically delimits the occurrence of cohabitants due to drought and nutrient stress41. Therefore, we tested the relevant intra-specific competitive stress gradient on plasticity.The observed occurrence of ees Fig.  was consees Fig. . Althougees Fig. , an expeAe. arabicum provided ideal fitness to the plants. Total biomass and the number of fruits produced in a lifetime per plant were significantly higher in control conditions (Ctrl.) than in hydrothermal (HT1-5), nutritional (N1-2) and competitive (C1-3) stress conditions  produced at control optimal habitat conditions is also corroborated by field-collected data  of the fruit morphs (DEH: IND = 1.9: 1) from the optimal and edge habitats. Approximately 5089 DEH fruits  were collected from optimal habitat compared to 2669 IND fruits in recent field visits in Çankırı district, Inner Anatolian mountain slopes (750–2000 masl).Hydrothermal stress to the parental generation significantly changed the proportion of vegetative and reproductive parts upon maturity, thus producing a varied number of fruits in their lifetime with a proportional change of DEH vs IND fruits per plant Fig. . Similar42. We found no significant proportional changes in the dry mass of vegetative and reproductive parts or the number of fruits produced in control and stressed conditions for nutrient and intra-specific competition  comparable to the one observed in field studies in 2017 at the optimal habitat (DEH: IND = 1.9:1) and validated our hypothesis on plasticity in life strategies for Ae arabicum  cannot be ruled out for both dimorphic diaspores, but it is especially important for the abscission of the IND diaspores31. Moreover, very rare cases of long-distance transport are possible by adherence to animal vectors (exo-zoochory) as was shown for other Brassicaceae taxa with intercontinental dispersal49. Likelihood of transport by attaching to the substrates that constantly move downhill in screes at East Mediterranean and Irano-Turanian mountains41 adds up to the fate of DEH seeds dispersed at uphill elevations to move to the optimal habitat. Indehiscent (IND) fruits developed under similar low-stress conditions remain dormant in the seed bank to germinate later in the spring, follow natural phenology, and maintain the natural fruit ratio at optimal habitat (DEH: IND = 1.7:1).Seeds of dehiscent (DEH) fruits developed under low hydrothermal stress or produced in the field at optimal habitat germinated immediately in late summer or early autumn possibly near the mother’s site by anchoring to the substrate with mucilage31 can be upgraded to a high potential distance by considering the real field conditions  with occasional strong wind gusts further enhances the opportunity of IND fruits to be dispersed by anemochory over hilly terrain via their aerodynamically winged pericarp Fig. . While tons Fig.  and the 31. However, the IND fruits developed under stressed conditions should remain in the seed bank to germinate in the spring to experience again high hydrothermal stress and produce an altered fruit ratio (3:1). Nevertheless, the winged IND fruits can potentially fly upwards with the help of high wind speed . Further extensive field experiments on the specific dispersal mode, direction, and proportion of both fruit morphs will elucidate the spatial-temporal dynamics of the species along the mountain slope. Our study indicates that complementary exploration by integrating field observations, ecological modelling, and experimental validation with simulated natural gradients is suitable to test the long-standing hypothesis of adaptive benefits of plasticity in dimorphic species in a tractable way.Aethionema arabicum is an annual herb widely distributed in the arid and semi-arid environments of East Mediterranean/Irano-Turanian countries , and digitized records in GBIF50. An exhaustive compilation of accessions was prepared, which included all possible synonyms of Ae. arabicum, from archived documents in Herbaria at Botanical Institute, Azerbaijan (BAK), Department of Plant Taxonomy and Geography, Armenia (ERE), and Georgian National Museum, Georgia (TGM). The accuracy of elevation for the recorded coordinates was validated in www.geoplaner.com. Field data on distribution pattern, fruit morph proportion and natural germination times in the mountainous habitat in Turkey  were collected from April to October 2017.ies Fig. . The geoAe. arabicum distributed over 0–3000 m above sea level (masl) were accumulated from the Worldclim database (http://worldclim.org/version2). We used the high spatial resolution GeoTiff images (30′′ grid = ~1 km2) to extract the average monthly climate data for minimum, mean, and maximum temperature (°C), rainfall (mm) and wind speed (m s−1) for 1970–2000 in R (ver. 3.4.3) using the packages raster, sp, rgdal, foreach, ggmap, rJava and xlsx51.The climate data for the natural habitats of 54 (https://soilgrids.org). Data for soil coarse fragment percentage (v/v), pH in H2O, cation exchange capacity (cmol + kg−1), and soil organic carbon content (g kg−1) at 0.05 m depth from the surface were extracted from GeoTiff images (resolution 1 km2) in R.All available soil parameters for 238 accessions were collected from global soil grid database29. After-ripened seeds were stratified on sterilized rooting-media agar plates  for four days at 4 °C in darkness followed by incubation in a growth chamber at 14 °C with 16 h daylight (155 µm s−1 m−2). After 15–20 days, the germinated seedlings were transferred to 0.5 l pots filled with soil consisting of Einheitserde (Einheitserdewerke Gebr. Patzer GmbH & Co. KG): autoclaved sand: perlite (7:2:1). The plants started flowering one month after germination and produced seeds after another two months.Mature plants were grown from seeds originally collected from Turkey in 2007 and provided by Eric SchranzAe. arabicum and used as control parameters, along with parameters extracted from previous hydrothermal modelling of seed germination and growth29. All control experiments (Ctrl.) were conducted in long-day light condition (16 h light) and optimal temperature cycle , corresponding to the natural phenology  and ca. 35 ml of added fertilizer  every week.Environmental conditions of the optimal habitat with high species distribution (750–2000 masl) were considered optimal for the growth of ogy Fig.  of the sHydrothermal stress gradients on the life phase and fruit morph plasticity were simulated in laboratory by providing 600 ml  and 400 ml  water, respectively to batches of 15 plants per week for each treatment. High thermal stress was induced at four-leaf rosette stage by exposing batches of 15 plants to higher temperature . To test the natural equivalent of hydrothermal stress on plasticity, two more binary combinations of stress gradients  were examined on groups of 15 plants.As suggested by the altitudinal gradient of soil cation exchange, pH and organic carbon content, equivalent nutrient stresses were tested by supplying less  or no additional nutrient  contrary to the control nutrient supply (0.2% v/v).The observed altitudinal gradient of soil coarse fragment size, which potentially regulates the habitability of plants in a particular habitat, were extrapolated to test the intra-species competitive stress. Instead of only one plant per 0.5 l pot in control experiments (no stress), three , five  or seven plants  were grown together with identical growth conditions. The target plant was surrounded by competitor plants during growth Fig. .After 3 months of stress treatments, aboveground vegetative parts (stem and leaves) and reproductive parts  from each of the treated and control plants were collected individually in paper bags for drying in an oven at 80 °C for 48 h and weighed for dry mass. Roots were excluded from the measurement of vegetative structure in all cases due to the technical difficulties of washing off soil/sand particles without significantly damaging the root system. The number of DEH and IND fruits was counted for each control and treated plant.Ae. arabicum along the elevation, the habitat range was further categorized into lower (0–750 masl) and upper edge (2000–3000 masl) of the optimal habitat (750–2000 masl).The average monthly data for climatic parameters were aggregated for the four seasons as follows: winter , spring , summer , autumn . All climatic and edaphic factor data were subjected to Locally Weighted Least Squares Regression (Loess) in R with the respective elevation at geo-locations of accessions with a 2D polar contour analysis to determine the optimal habitat features. Depending on the Gaussian distribution of Analysis of variance (ANOVA) was performed to compare the effect between control and stress treatment conditions on the dry mass of vegetative- (Veg.) and reproductive- (Rep.) plant parts and proportion of dehiscent (DEH) to indehiscent (IND) fruits, followed by post-hoc mean grouping with SPSS (version 24). For all non-parametric and proportional data, Kruskal-Wallis test (H) was performed to compare the effect of the individual or combined stress levels on life phase and fruit morph plasticity. A significant difference of means from the control was determined by Dunn’s pairwise tests for H and adjusted using the Bonferroni correction in SigmaPlot 13.0.To determine the effective magnitude of the stress gradient to the plasticity and interactive correspondence between the life phase and fruit morph changes we performed multivariate canonical correlation analysis in R package ‘cca’ and ‘vegan’.Supplemental information


IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies.Genomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis.Exome sequencing and data analysis revealed a novel homozygous missense variant  within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant  residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein.IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia.This study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in The online version of this article (10.1186/s12881-018-0709-3) contains supplementary material, which is available to authorized users. Aminoacyl-tRNA synthetases (ARSs) are evolutionarily conserved enzymes that catalyze amino acid attachment to their cognate tRNA. This catalytic process, termed tRNA charging, is a prerequisite for the translation of genetic sequences into polypeptide chains . Two disIle  population data. Artifact-prone gene families  were further excluded and the analysis focused on missense, stopgain/stoploss, startgain/startloss, splicing and indel variants. Variant prioritization was aided by the tools FATHMM  and c.2450G > A; p. [(Arg817His)] variants in exons 4 and 20, respectively, and exhibited delayed motor development, as well as infantile spasms and abnormal brain MRI diagnostic imaging leading to a diagnosis of Leigh syndrome. One of the two siblings had cataracts and the other sibling had a neonatal hearing screening result requiring follow-up. Both female children were in their first decade of life at the time of publication.Whilst this manuscript was in peer review, two additional publications described IARS2  in exon 4 and c.2575 T > C; p.(Phe859Leu) in exon 21; family 10: c.2446C > T; p.(Arg816*) in exon 20 and c.2575 T > C; p.(Phe859Leu) in exon 21). Both probands shared the Phe859Leu exchange, which affects the anticodon-binding domain and can be reasoned to exhibit only mild/moderate effects. One of the reasons for this hypothesis is that the amino acid exchange conserves hydrophobicity. Another being that the Gly203Arg that is located in the aminoacyl-tRNA synthetase domain and the Arg816* null allele can reasonably be predicted as causing more severe effects on the protein. If the Phe859Leu exchange were to cause a severe protein change, we would expect a severe or even lethal phenotype in the presence of the null allele. Thus, the allelic protein product with the Phe859Leu exchange should still maintain functionality.The second publication which characterised by far the mildest clinical presentation characterized to date described two probands from China with sporadic pediatric cataract . Both prIARS2 variants, expanding the clinical and mutational spectrum. Patient 1 also presented with type II esophageal achalasia, as well as growth hormone deficiency and central adrenal insufficiency, which could result from dysfunction of the hypothalamic-pituitary axis. The unusual combination of findings suggest that other endocrine disorders in patients with an IARS2-associated mitochondriopathy could likewise be possible and should be excluded. We propose that these additional phenotypes expand the syndromic constellation of symptoms in adults. Early recognition of IARS2-related pathophysiology has potentially important implications in clinical management, seeing that undetected adrenal insufficiency could lead to a life threatening crisis.In conclusion, we describe two additional independent families with a total of three affected individuals displaying clinical features overlapping with CAGSSS and novel Additional file 1:Sequence alignment used for homology modelling of IARS2 protein. (DOCX 16 kb)Additional file 2:Comprehensive clinical summary of patients with pathogenic variants in IARS2. (DOCX 24 kb)


Embryoid bodies (EBs) are three dimensional aggregates of pluripotent stem cells primarily used to investigate morphogenesis and cell toxicity, are also attractive tools in regenerative medicine. While embryonic stem cells (ESCs) and induced pluripotent cells (IPSCs) have been shown to form EBs in mouse, primate and humans, EB formation have not been previously demonstrated in mesenchymal stem cells (MSCs). Here we show that rat MSCs form EBs; which express regulatory T cell (Treg) marker Foxp3 and CC chemokine CCL1 receptor CCR8. We show a novel method for formation of EBs from MSCs under stress and demonstrate that the induction of FoxP3+ CCR8+ EBs is dependent upon CCL1 gradients which mediate cell proliferation, migration and invasion of mTregs. The identification of EBs and novel FoxP3+ CCR8+ regulatory T cells (mTregs) for selective conversion and isolation of bone marrow derived MSCs offers novel avenues for research, diagnosis and treatment. Drug based immunosuppressive regimens are effective in various disorders. However, their long term use causes malignancies, toxicity and infections. Stem cell therapy is an option which is rigorously being undertaken by various laboratories and clinical practices. Three types of stem cells which include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) are currently being tested in stem cell based therapies. Apart from ethical issues in using ESCs, the propensity for malignancy is the biggest drawback in using iPSCs and ESCs. MSC or regulatory T cell (Treg) based therapy offer alternative immunosuppressive treatments with lesser side effects. In 1970, Friedenstein et al. . These ain vivo. Further studies are needed to establish how and wherein these cells have to be administered and how they may function to safely modulate host immune response in vivo in clinical transplant setting.The success in MSCs transplantation is related to their immunosuppressive properties. The mechanism by which MSCs trigger their immune—suppression is by inhibiting CD4+ CD8+ T cells and preferential Treg differentiation . Tregs aAs MSCs are known to support wound healing and repair by immune suppression, we postulated that MSCs may divert a subpopulation of MSCs towards regulatory T cell (Treg) fate. Here we show that mouse and rat mesenchymal cells form spontaneous FoxP3+ CCR8+ EBs under stress which has immunosuppressive properties. In addition, the aggregation and induction of FoxP3+ CCR8+ EBs is dependent upon the CC chemokine CCL1 and its receptor CCR8CCL1 /CCR8 interactions. We have previously shown that CCL1/ CCR8 may play a role in vascular pathobiology [2 tissue culture flask and incubated at 37°C with 5% CO2.MSCs were isolated from rat bone marrow as previously described  with som2 tissue culture flask and incubated at 37°C with 5% CO2.For the isolation of mice, FVB/N strain (2months young) was used. These mice are characterized by vigorous reproductive performance and consistently large litters mice The same procedure was followed as described for rat MSCs, however, the final pellet was suspended in Dulbecco Minimum medium (DMEM)  containing 10% horse serum and 0.5% Penicillin /streptomycin/fungizone (P/S/F) in 25 cmHuman umbilical vein endothelial cells (ECs) and human aortic smooth muscle cells (SMCs) were obtained from American Type Culture Collection  and grown according to the manufacturer's instructions. While ECs were cultured in Dulbecco modified Eagle medium (DMEM) as described , SMCs we+ T lymphocytes cells were counted before further analysis and/or cell culture.Using aseptic conditions spleen was removed from young (8 week) and old (17 week) FVB/N mice and placed in a plastic dish with sterile PBS. Next the spleen was teased apart with forceps and passed through a stainless steel mesh to obtain a single cell suspension. This suspension was washed with sterile PBS. Red blood cells were removed using mouse Erythrocyte Lysing Kit  following package instructions. The cell pellet was disrupted briefly by “racking” the tube and re-suspending the cells in erythrocyte lysis solution. The cell suspension was vortexed, incubated for 5–10 minutes at room temperature, and the cells were washed with Wash Buffer obtained from the lysing kit. Splenocytes were next suspended in buffer or medium, further enriched for CD4Embryoid bodies (EBs)/mTregs were formed spontaneously in confluent rat derived MSCs when serum was removed. EBs/mTregs also formed when rMSCs were treated with injured SMC/ECs. The cells detached and started aggregating within 24–48 of deprivation. They range from 200 μm to 600 in size. These cells were further characterized for the presence of Treg markers . Once confirmed for Treg characteristics the Ebs/mTregs were used for subsequent studies.Recombinant , goat polyclonal antibody against CCL1, as well as IgG controls were obtained from R&D systems . Poxvirus MC148 was obtained from Abcam . Anti-CCR8 was generated as described (10). Anti-CCR8 was conjugated using the APC Conjugation Kit (ab201807) which purchased from Abcam. Antibodies against MSC markers PDGFRα (clone αR1) and Purified Mouse Anti-Rat Nestin Clone Rat 401 (RUO) was purchased from BD Biosciences. One Step Staining Human Treg Flow Kit (FOXP3 Alexa Fluor 488/CD25 PE/CD4 PerCP) was purchased from Biolegend. Secondary antibodies Alexa Fluor 633 goat anti–rabbit IgG, Alexa Fluor 633 goat anti–mouse IgG, and Alexa Fluor 633 goat anti–rat IgG were obtained from Molecular Probes.mMSCs/rMSCs were grown into four well TC slides  until they reached more than 70% confluency. Serum deprived MSCs developed EBs/mTregs under overnight serum deprivation. When cells reached 90–100% confluency spontaneous EBs were formed. To study the modulation by CCL1/CCR8 MSCs were treated with either recombinant CCL1, antibody against CCL1 (>CCL1) overnight at 37° C with 5% CO2. For Treg specific staining of EBs/mTregs conjugated Alexa Fluor 488/CD25(red) PE/CD4 (yellow) PerCP FoxP3 (green), and CCR8 (APC red) were used. The EBs/mTregs were treated with conjugated antibodies for 30 minutes, washed 3 times and grown on culture slides for 24–48 hours and observed under Nikon T 90 microscope. Images were taken at 10x, 20x and 40x magnification.OpenUrl.Essen Bioscience). Wound Confluence is a report of the confluence of cells within the wound region, given as the percentage of the wound region area occupied by cells. Relative Wound Density(RWD) relies on measuring the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at every time point. The metric is self-normalizing for changes in cell density which may occur outside the wound due to cell proliferation and/or pharmacological effects. Importantly, the RWD metric is robust across multiple cell types as it does not rely on finding cell boundaries.Human SMCs and ECs were plated separately in a 96-well format and grown overnight. Upon reaching confluency cells were subjected to automated scratch assay using Incucyte plate scratcher. The plates were subsequently was imaged using an Zoom  every 2 h for 30 hours. Cells were imaged under phase contrast as well as fluorescence. Analysis was performed using Incucyte’s Imaging software. The IncuCyte ZOOM Scratch Wound assay utilizes the WoundMaker-IncuCyte ZOOM-ImageLock Plate system to analyze both 2D-migration and 3D-invasion in label-free, live cells  were isolated and expanded. These cells demonstrated multipotency. They may remain adherent  and/or aWe have observed that non adherent rMSCs have a capacity to form spheres or embryoid body (EB) like structures as those reported for embryonic stem cells in the absence of serum . This haEndothelial cells (ECs) were grown overnight on 96 well plates. They were then subjected to automated scratch assay and immediately co- cultured with rMSCs  or mTregVascular smooth muscle cells (VSMCs) were grown on 6 well plates overnight. They were then subjected to automated scratch assay and immediately co- cultured with rMSCs  or mTregS2 DataIn order to see the role of CCL1/CCR8 in the EB formation mMSCs were either treated overnight with anti CCL1 (>CCL1)  or left Since mTregS /EBs express CCR8 we tested ligand specificity of this receptor and co-localization with FoxP3. It was found that EBs/mTregs derived from rMSCs express CCR8 , red andWe have developed a method to induce and expand EBs from stress induced MSCs, isolated a novel subtype of regulatory T cells with immunosuppressive properties and have identified the chemokine CCL1 and its receptor CCR8 as modulators of this function. When rat MSCs were exposed to stress  or inflammatory microenvironment, they formed colonies or EBs which express Treg specific markers CD25  and FoxP3 (transcription factor forkhead box P3). Specifically, MSC derived Tregs or mTregs are obtained from serum deprived rat MSCs (rMSCs), injured smooth muscle cells (SMC) + rMSC and injured endothelial cells (EC) + rMSCs co-cultures. Adherent non-colony forming MSCs do not express these markers. These MSC derived EBs show self-renewal capacity and are multipotent as observed by their ability to differentiate into chondrocytes, adipocytes and neurons which has relevance in designing both MSCs and Treg based therapies in human diseases.. Thus CCL1/CCR8 may play an important role in migration; homing and proliferation of MSC derived cells and mTregs/EBs.Our findings that non adherent MSCs have a capacity to form spheres or EBs as those reported for embryonic stem cells opens up novel avenues for research and therapy. While ESCs and IPSCs are known to make EBs, this has not been previously described for MSCs. In addition, the identification of CC chemokine CCL1 receptor CCR8 on EB subsets offers an explanation for the mechanism of recruitment and homing of these cells to the site of injury. CC chemokine CCL1 is the known ligand for CCR8. CCR8 is involved in the regulation of the Th1-Th2 response and is known to be expressed in different subsets of Tregs. We have found CCR8 to be a functional receptor on rMSCs as its specific ligand CCL1 increases proliferation of non-adherent cells after migration across a chemotactic chamber in vitro immune suppression of the patient's Treg cells seems to be the basis for these diseases. This has led to an increasing interest in the possibility of using Tregs in immunotherapy to treat or prevent chronic infections, autoimmune diseases, allergies and transplantation-related complications, such as graft rejection or graft-versus-host disease (GvHD) [Treg defects have been discovered in patients with multiple sclerosis (MS), type I diabetes (T1D), psoriasis, myasthenia gravis (MG) and other autoimmune diseases . Similare (GvHD) . Treg the (GvHD) –18. WhilIn this study we show that mTregs exhibit distinct immunomodulatory and remodeling properties when co-cultured with injured SMCs or Huvecs. This was observed SMCs) / Huvecs were subjected to scratch assay and co- cultured with either MSCs or mTregs. Both SMCs and Huvecs quickly recovered after injury in the presence of MSCs; as observed by the relative wound confluency (RWC). However, this was accompanied by the invasion or relative wound density (RWD) of cells leading to increased cell proliferation in the presence of MSCs and aberrant wound repair. When SMC/Huvecs were co-cultured with mTregs, the RWC remained intact; however, the RWD was inhibited showing that mTregs modulate immunosuppression. The mTreg specific EBs when re–introduced into the inflammatory microenvironment have inhibitory capacity. For example, when non-adherent FOXP3+ mTregs are added to injured SMC or endothelial cells, significant decrease in cell migration, invasion (relative wound density or RWD) and proliferation (relative wound confluency or RWC) is observed. On the other hand, when MSC is added alone, there is complete wound closure and higher RWC and RWD, suggesting that the conversion of MSC to mTreg is an important factor in wound healing and remodeling.versus damaged or diseased tissues must be understood in order to design future clinical applications. The restoration of a functional niche will be essential to safeguard durable repair and ensure continual replacement of mature cells lost to physiological turnover or subsequent stress or damage. Our novel findings expand upon current therapies that rely upon MSCs and/or Tregs by employing MSC specific EBs to produce niche specific mTregs for various downstream applications.A stem-cell niche is a spatial structure in which stem cells are housed and maintained by allowing self-renewal in the absence of differentiation. The crosstalk between MSCs and neighboring cells (SMCs/ECs) activate cytokines/ chemokines in the microenvironment facilitating the formation of EBs and mTregs which mediate immunomodulation and vascular remodeling. mTregs influence the phenotypic conversion of interacting cells in response to inflammatory microenvironment. Therefore, the interactions between MSCs with their nearby microenvironment, i.e. the stem cell niche, in healthy There are certain limitations to this study as target antigens and mTreg subtypes remain to be elucidated. Identification of antigen specific mTreg subtypes will lead to development of methods for designing specific therapeutic regimens and targeting specific cell and tissues. Drug interaction, combination therapy with mTregs needs to be elucidated which may identify novel pathways and insights into stem cell biology result in beneficial outcomes. Therefore, MSC derived EBs/mTregs needs further investigation. Studies in our lab are underway to characterize MSC derived EBs and in the identification of mTreg subtypes/target antigens.In conclusion, it is proposed that MSCs have the capacity to form EBs and generate mTregs that have immune—modulatory and regenerative capabilities. As mTregs subtypes are generated in response to specific microenvironment, they are antigen/tissue specific and therefore capable of more precise targeting as opposed to direct MSC or peripherally obtained Treg treatments. This targeted approach has the potential for development of adoptive transfer methodologies which has more chances for success. Our method for the conversion of MSCs to EBs and mTregs in an inflammatory microenvironment offers a unique process for targeted stem cell therapy. To the best of our knowledge, MSC derived Tregs (mTregs) and their functionality have not been previously described. MSC derived immune-modulators will provide an alternate option to current therapies and is expected to have fewer side effects and thus a higher potential for therapeutic use. In addition, MSC derived EBs may serve as a replacement for embryonic stem cell derived EBs for tissue engineering with fewer ethical concerns. Thus, this method has far reaching applications in research, diagnosis and treatment.S1 Data(ZIP)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.


High-resolution respirometry was established in the myocardium to measure oxidative capacity ex vivo. The mitochondrial oxidative capacity was 90% higher in ventricular compared to atrial tissues  of explanted hearts. Respiration rates were comparable in ventricular samples of heart failure patients obtained during open heart surgery by standard tissue preparation or ex vivo endomyocardial biopsy , and the mitochondrial oxidative capacity in samples from these patients remained stable for 8 h when stored in either of two common preservation buffers. The oxidative capacity was 44% lower in heart failure than in transplant recipients  and correlated positively with heart function . High-resolution respirometry of ventricular tissue is feasible in transcatheter biopsies, facilitating clinical studies on myocardial mitochondrial function in patients not undergoing heart surgery.The lifetime risk of developing heart failure is approximately 20%, and survival rates remain poor. Myocardial mitochondrial function has been suggested to play a pivotal role in heart failure pathophysiology. Human studies on ex vivo mitochondrial function have mostly been limited to atrial tissue obtained during open heart surgery and have provided contradictory results. This study aimed at measuring myocardial mitochondrial function in transcatheter ventricular endomyocardial biopsies and assessing the relationship between oxidative capacity and heart function. We enrolled 40 heart failure patients undergoing ventricular assist device surgery or heart transplantation  was significantly higher than in the upper heart chambers (atria). Moreover, they found that mitochondrial oxidative capacity was reduced by 44% in heart muscle biopsies from patients with heart failure compared with biopsies from heart transplant recipients with normal ventricular function. Stimulating the respiration rate of mitochondria in ventricular heart cells could be a promising strategy for improving cardiac function. Due to the aging of the population and improved survival from acute cardiac diseases, HF prevalence is increasing steadily, with a 20% lifetime risk for the development of HF2, which has a poor prognosis. The pathophysiology of HF is complex, but growing evidence from both preclinical and clinical studies points to impaired mitochondrial function in HF4. Thus, mitochondria appear to be a promising therapeutic target to improve cardiac function5.Heart failure (HF) is a rising medical and societal burden8. Polarographic measurement of ex vivo mitochondrial oxygen consumption in the presence of specific substrates has been widely accepted as the gold-standard method for analyzing mitochondrial oxidative capacity and efficiency in human tissue samples, i.e., skeletal muscle and the liver13. Thus far, assessment of ex vivo cardiac mitochondrial respiration has been limited to samples acquired during open heart surgery, which precludes follow-up measures during clinical trials and restricts patient groups. Of note, the mitochondrial oxidative capacity of left ventricular (LV) tissue obtained during open heart surgery was decreased by 40% in terminal HF patients compared to patients with an LV ejection fraction >45%14.Current in vivo noninvasive measurement of heart energy metabolism relies on the relative concentrations of phosphorous metabolites and is restricted to selected clinical centers15. However, EMB has not yet been employed to assess mitochondrial function.Human transcatheter endomyocardial biopsies (EMBs) of the interventricular septum (IVS) are routinely applied for the management of cardiovascular diseases according to the latest scientific statement of the American Heart Association, the American College of Cardiology, and the European Society of Cardiology17, referred to as the “control group”, while analyses of ventricle tissue were mostly applied to patients with end-stage HF following heart transplantation or left ventricular assist device (LVAD) surgery18. Meanwhile, accumulated evidence suggests that the atrial mitochondrial oxidative capacity does not reflect that of ventricular tissue; thus, comparisons of different patient groups must address corresponding heart areas18. Moreover, the use of mitochondrial preservation medium19 or standard tissue perfusion solution17, as well as the storage time until mitochondrial respiration analysis, might also affect respiration rates. Taken together, standardized procedures applicable to clinical trials in larger cohorts are warranted.A number of studies analyzed atrial tissue in patients undergoing valve replacement or coronary artery bypass surgeryTo this end, we analyzed mitochondrial respiration in right atrial, LV, and IVS tissue specimens from heart explants. We then established high-resolution respirometry (HRR) in human transcatheter EMB of the IVS. We tested for the impact of the time lag between tissue acquisition and the start of the experiment and the effect of different preservation media on respiration measurements. Finally, we tested the clinical applicability in a cohort of heart transplant recipients with normal LV function and in patients with terminal HF and related mitochondrial respiration rates to heart function.This registered clinical trial protocol was approved by the ethics board of the university hospital of the Heinrich-Heine University Düsseldorf . All procedures conformed to the World Medical Association Declaration of Helsinki. Before inclusion, all participants gave written informed consent.n = 40), tissue specimens were obtained surgically during LVAD implantation (n = 28) or heart explantation (n = 12). In stable heart transplant recipients with a normal LV function , tissue specimens were obtained during routine surveillance EMB  or ice-cold CUSTODIOL® perfusion solution containing [in mM] 15 sodium chloride, 9.0 potassium chloride, 1 potassium hydrogen 2-ketoglutarate, 4.0 magnesium chloride, 18.0 histidine HCl, 180.0 histidine, 2.0 tryptophan, 30.0 mannitol, and 0.015 calcium chloride in sterile water. Samples were transferred to the laboratory in ice-cold buffer within a few minutes. Part of the ventricular tissue was directly prepared for HRR. The remaining tissue was kept on ice in BIOPS or CUSTODIOL® for 3 or 8 h to assess the impact of storage buffer and time on respirometry. To assess the impact of sample size and tissue acquisition technique on mitochondrial respiration, we harvested EMBs ex vivo using an EMB bioptome from tissue samples of eight patients obtained during LVAD surgery and compared the mitochondrial respiration in these EMBs to that in standard preparations of tissue specimens of the same patients using scissors and forceps.During LVAD surgery, the part of the cardiac apex that corresponds to the LV was excised  for 30 min, and then washed in respiration medium 22. Thereafter, tissue specimens were blotted on filter paper, weighed, and transferred to oxygraph chambers containing 2.3 ml MiRO5. Respiration was measured at 37 °C at oxygen concentrations between 120 and 450 µM in duplicate and analyzed using Datlab software . Oxygen fluxes represent basal or substrate-saturated respiration before adding ADP, ADP-stimulated coupled respiration with octanoyl-carnitine, glutamate, and succinate (state III), and maximal uncoupled respiration after adding the uncoupling factor fccp (state u)23. The oxidative capacity of the mitochondrial oxidative phosphorylation system (OXPHOS capacity) was derived from state III respiration after addition of octanoyl-carnitine, glutamate, and succinate. Mitochondrial leak respiration (state 4o) was induced through inhibition of the enzyme ATP synthase by adding oligomycin12. The respiratory control ratio (state III/state 4o) and the leak control ratio (state 4o/state u) were calculated as surrogate markers of mitochondrial coupling efficiency12. The integrity of the outer mitochondrial membrane was analyzed via exogenous stimulation of state III respiration by cytochrome c24. A stimulation of oxygen flux less than 10% after cytochrome c addition is considered to indicate a preserved outer mitochondrial membrane24.Myocardial mitochondrial respiration in permeabilized myocardial muscle fibers and isolated mitochondria was analyzed ex vivo using HRR following procedures previously established and/or developed for skeletal muscle and liver tissue 26. In short, CSA was measured spectrophotometrically in lysed cells at a 412 nm wavelength after addition of 10 mM dithionitrobenzoic acid, 30 mM acetyl-CoA, and 10 mM oxaloacetic acid. The results have been normalized to the protein content measured colorimetrically via bicinchoninic acid assay.The mitochondrial content was assessed from the citrate synthase activity (CSA) as described previously26. An aliquot of tissue sample prepared for the HRR measurements was transferred to oxygraph chambers containing 2.3 ml MiR5 with 10 mM of the extrinsic fluorophore Amplex red  and horseradish peroxidase 27. Catalyzed by horseradish peroxidase, Amplex red, and hydrogen peroxide produce the red fluorescent compound resorufin (excitation wavelength 563 nm and emission 587 nm). The change of the emitted fluorescence intensity during stepwise addition of the mitochondrial substrates malate and succinate is directly proportional to the production of hydrogen peroxide28. Calibrations were performed with a standard hydrogen peroxide addition.Fluoroscopic measurements of mitochondrial hydrogen peroxide release were performed using an Oxygraph-2k combined with the Fluorescence-Sensor Green of the O2k-Fluo LED2-Module for hydrogen peroxide 9. Oxygraph measurements of isolated mitochondria were performed following the same protocol as for permeabilized fibers, but in a different respiration buffer  at saturating oxygen levels.Mitochondria were isolated by differential centrifugation as described previously29. The cardiac index was calculated using a modified Fick method during right heart catheterization at the time of EMB or at the time of evaluation for heart transplantation and/or LVAD implantation30.The LV ejection fraction was analyzed using the modified biplane Simpson’s rule according to current European Society of Cardiology HF guidelines with a Philips HD15 ultrasound system 9.Relative quantification of myocardial messenger ribonucleic acid (mRNA) expression of genes related to mitochondrial respiratory chain complex I (NDUFS1) and an important mitochondrial transport enzyme, carnitine palmitoyltransferase 1B (CPT1B), was assessed with real-time PCR  and the comparative threshold cycle method (DDCt) with 18S ribosomal RNA as the reference gene, as described previously, in tissue specimens of a small subset of HF patients undergoing LVAD surgery and heart transplant recipients undergoing EMBP-values ≤0.05 were considered significant. Data were analyzed using the paired or unpaired Student's t-test, two-way analysis of variance (ANOVA), and the Sidak post hoc test. Datasets with no Gaussian distribution were analyzed using the Mann–Whitney U test. To test for a linear association between two variables, we used Pearson or Spearman correlation models and partial correlation, depending on whether data were normally distributed.Statistical analyses were performed using Prism 7  and SPSS . Data are expressed as the means ± standard deviation or means ± standard error of the mean. The distribution of age, sex, and body mass index was comparable in HF patients and heart transplant (HTX) recipients Table . Table 1p < 0.01). After addition of the citrate cycle-derived substrates glutamate and succinate, mitochondrial respiration in ventricular samples was approximately 90% higher    Fig. . The unc01) Fig. . CSA was01) Fig. . The res01) Fig. .Fig. 2Cor = 0.9988, p < 0.0001) .There was a strong correlation between oxygen fluxes measured in EMBs harvested using a bioptome from ventricular apex tissue samples obtained during LVAD surgery of HF patients and fluxes measured after standard tissue preparation for the same tissues (01) Fig. . In tiss® preservation buffer. These measurements displayed stable oxygen flux rates for each timepoint and buffer in mitochondrial state III respiration on solely octanoyl-carnitine and the citrate cycle-derived substrates glutamate and succinate plus octanoyl-carnitine, with no effect on uncoupled respiration or the respiratory control ratio   higher and, after addition of the citrate cycle-derived substrates glutamate and succinate, 38%  higher compared to LV tissue specimens of HF patients obtained during open heart surgery  .Based on the above-mentioned experiments, the mitochondrial respiration in LV and IVS tissue specimens is comparable. In EMBs endoscopically harvested from the IVS of heart transplant recipients with a normal LV function, state III mitochondrial respiration on the medium-chain fatty acid octanoyl-carnitine was 44%  Fig. , even afp < 0.0001) and CPT1B  mRNA was significantly reduced in LV tissue specimens of HF patients undergoing LVAD surgery compared to IVS EMBs of HTX patients with a normal LV function 17 or did not focus on differences in mitochondrial respiration between IVS and LV tissue specimens.18 To the best of our knowledge, this is the first study analyzing regional differences in mitochondrial respiration for a significant number of cases (n = 9–11). Increased ventricular OXPHOS capacity compared to atrial tissues might relate to higher mitochondrial content, as suggested by CSA measurements, but calculation of the respiratory control ratio and leak control ratio as surrogate markers of mitochondrial coupling efficiency12 suggests qualitative differences in mitochondrial respiration between ventricular and atrial tissues.Analysis of state III myocardial respiration in LV, IVS, and right atrial tissue specimens obtained from explant hearts of HF patients revealed increased mitochondrial oxygen flux in ventricular compared to atrial tissue specimens. The ventricular OXPHOS capacity in our study was two-fold higher compared to atrial tissues, in line with previous results showing a factor of 2.3 between atrial and ventricular tissues in explant hearts of patients suffering from ischemic or dilated cardiomyopathyc addition resulted in no relevant increase in O2 flux in all measurements, indicating outer mitochondrial membrane integrity33. In contrast to earlier studies, where fiber bundles from 4–6 mg22 to 10–40 mg17 were obtained during heart transplantation18, LVAD, or heart valve surgery34, EMBs taken by a bioptome in our study had a tissue weight of at most 0.5–2 mg. These results suggest that the mechanical stress exerted by a bioptome and differences in tissue weight do not impact oxygen flux measurements compared to standard tissue preparation. To the best of our knowledge, this is the first study analyzing gold-standard myocardial mitochondrial oxidative phosphorylation in bioptome-harvested ventricular EMBs from humans. This facilitates the possibility of including samples from patients in mitochondrial respiration analysis that would not be accessible by a surgical approach. These patients comprise all patients with a clinical indication for EMB. According to the latest scientific statements of the American Heart Association, the American College of Cardiology, the European Society of Cardiology and the International Society for Heart and Lung Transplantation, these include especially patients with new onset of HF with hemodynamic compromise, HF patients with an unexplained restrictive cardiomyopathy, and heart transplant recipients undergoing routine surveillance EMB to test for heart transplant rejection35. Especially in heart transplant recipients, scheduled follow-up EMB facilitates the possibility of longitudinal analysis of myocardial mitochondrial respiration under various clinical influences. Consequently, a great number of patients with various pathologies are becoming accessible for HRR analysis, providing the opportunity for a deeper understanding of myocardial mitochondrial energy metabolism.We analyzed mitochondrial respiration in myocardial samples excised by a scalpel compared to biopsies harvested from the same sample using an endomyocardial bioptome. Both tissue acquisition techniques revealed equal oxygen flux rates. Cytochrome ®, independent of cold storage time up to 8 h. This corresponds to findings in surgically assessed human heart samples showing no changes in respiratory parameters during the first 9 h of tissue preservation before membrane defects occurred17. Cardioplegic solutions and BIOPS have previously been used as a storage buffer in studies analyzing myocardial mitochondrial function36, but to the best of our knowledge, there are no studies comparing mitochondrial respiration after storage in different solutions. We compared the mitochondrial preservation abilities of BIOPS with CUSTODIOL®, an organ preservation solution frequently used for heart transplantation in Europe38. While respiratory parameters were similar, the mitochondrial peroxide release increased after 8 h of CUSTODIOL® storage compared to BIOPS at all time points. This comparison demonstrates the comparability of different study protocols and buffers used in previous studies and suggests the preferential usage of BIOPS for tissue preservation, especially if a delay in measurement initiation is expected and ROS formation is analyzed.To analyze the impact of the cold storage time and preservation buffer on heart energy metabolism, we assessed respiration and ROS production in tissue specimens obtained during LVAD surgery. The mitochondrial state III respiration on fatty acids and citrate cycle-derived substrates, maximal uncoupled respiration, and coupling efficiency were comparable for the two most widely used preservation media, BIOPS and CUSTODIOL16, LVAD vs. valvular surgery34, and failing explant vs. donor hearts18, we not only detected differences in mitochondrial energy metabolism between HF and control patients but also, for the first time, were able to show a significant correlation of mitochondrial respiration and CI measurement, highlighting a direct and close link of energy metabolism to cardiac function. This correlation over a large number of patients remained significant, even after correction for HbA1c serum concentrations, underlining the profound connection of mitochondrial respiration and CI, considering that there is some evidence for impaired mitochondrial respiration in myocardial tissue specimens of patients suffering from type 2 diabetes mellitus39.The mitochondrial OXPHOS capacity in terminal HF patients was reduced 44% compared to HTX recipients with a normal LV function. In contrast to previous studies comparing mitochondrial respiration in surgically obtained tissue specimens of patients with coronary vs. valvular heart disease14. In contrast, analysis of myocardial mitochondrial respiration in isolated mitochondria from LV tissue specimens revealed no differences in OXPHOS capacity between terminal HF and control patients36, possibly indicating that the intrinsic mitochondrial capacity is not affected.Our results support previous data showing 40% diminished OXPHOS capacity in LV tissue specimens of patients undergoing LVAD surgery compared to LV tissue specimens of control individuals with good LV function undergoing cardiac valve surgery40. Additionally, nonfailing explant hearts from patients undergoing combined heart-lung transplantation following severe pulmonary hypertension experience pressure overload and thus do not correspond to healthy hearts. Donor hearts that were not eligible for transplantation or could not be allocated also have limitations for use as controls, as these organs have a long cold ischemia time that affects transplant integrity41. Therefore, defining an adequate control group is one of the main limitations in analyzing human heart tissue. Here, we established HRR for the analysis of mitochondrial respiration in endoscopically harvested EMBs. Analyzing EMBs enables us to obtain tissue specimens independent of cardiac surgery and increase case numbers for a better understanding of mitochondrial respiration in HF pathophysiology.A major challenge of studies analyzing human myocardial mitochondrial respiration in terminal HF is the comparison of pathologic findings with an appropriate control group. It is well known that pressure and volume overload impact myocardial oxygen consumption and energy metabolism; thus, patients with valvular heart surgery might not serve as an adequate control group for patients suffering from HF42. Information on mitochondrial biogenesis in human HF conflicts with the murine results. The amount of mRNA for subunits of the mitochondrial electron transfer chain is normal in explanted failing hearts compared with donor hearts43. Our results support an HF-associated decline in the expression of genes associated with mitochondrial respiration. Further studies focusing on gene expression are needed to address this issue.In a murine model of induced HF, decreased expression of genes related to mitochondrial biogenesis, e.g., PGC-1α, has been shownAccording to previous studies in the field, defining a control group is challenging. We compared energy metabolism in terminal HF patients with HTX recipients. These patients were characterized by a normal cardiac index and no signs of allograft rejection but received an immunosuppressive therapy whose impact on mitochondrial OXPHOS capacity is unknown. However, our correlation of cardiac index measurements with myocardial OXPHOS capacity showed robust results over all patients, as well as within the HF or post-HTX group.In this study, we established HRR as a gold-standard method for the analysis of mitochondrial respiration in human EMB. We detected local differences in mitochondrial respiration, with markedly increased oxidative capacity in ventricular compared to atrial tissue specimens. HRR measurements generated robust results even after prolonged cold storage. Application of this technique in a clinical setting showed a 44% reduced myocardial OXPHOS capacity in HF compared to HTX patients, with a positive correlation of mitochondrial oxygen respiration to cardiac function. This technique will facilitate evaluation of mitochondrial energy metabolism in various patient cohorts, helping to decipher the pathophysiological impact of a disturbed myocardial energy metabolism on HF.Supplementary Table 1Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4


The World Health Organization’s (WHO) guidelines for cancer pain management were intentionally made simple in order to be widely implemented by all physicians treating cancer patients. Referrals to pain specialists are advised if pain does not improve within a short time. The present study examined whether or not a reasonable use of the WHO guideline was made by non-pain specialists prior to referral of patients with cancer-related pain to a pain clinic.Cancer patients referred to a pain specialist completed several questionnaires including demographics, medical history, and cancer-related pain; the short-form McGill Pain Questionnaire (SF-MPQ); and the Short Form Health Survey SF-12. Data from referral letters and medical records were obtained. Treatments recommended by pain specialists were recorded and categorized as “unjustified” if they were within the WHO ladder framework, or “justified” if they included additional treatments.Seventy-three patients  aged 55 years  participated in the study. Their pain lasted for a mean of 6 (1–192) months. Mean pain intensity scores on a 0–10 numerical rating scale were 7 (2–10) at rest and 8 (3–10) upon movement. Most patients complied with their referring physician’s recommendations and consumed opioids. Adverse events were frequent. No significant correlation was found between the WHO analgesic medication step used and mean pain levels reported. There were 63 patient referrals (85%) categorized as “unjustified,” whereas only 11 patients (15%) required “justified” interventions.These findings imply that analgesic treatment within the WHO framework was not reasonably utilized by non-pain specialists before referring patients to pain clinics. The treatment of cancer-related pain is a primary goal for healthcare professionals. Pain affects two-thirds of patients with advanced cancer, reaching intensities of moderate-to-severe in more than half of them.5A principal concept of the WHO guideline, the “three steps analgesic ladder,” refers to the pharmacological management of cancer-related pain . It emph8Regardless of the growing understanding of cancer pain mechanisms and treatment, and despite increasing efforts to disseminate the WHO guideline worldwide, only little progress has been made during the past three decades in terms of reducing the prevalence and severity of cancer-related pain.10International as well as several national guidelines on cancer-related pain have been published and disseminated in Israel.The study was approved by the Ethics Committee at Rambam Health Care Campus in Haifa, Israel .Both hospitalized patients and out-patients with cancer who were referred to a pain specialist for cancer-related pain management were recruited for the study. Inclusion criteria for the study consisted of being aged 18 and above; having a referral for cancer-related pain or cancer treatment-related pain; having the ability to understand the study aims; and giving written informed consent to participate in the study. Patients were excluded if their pain diagnosis was not related to cancer or cancer treatment, as determined by a pain specialist.Patients were asked to complete a questionnaire upon arrival to their consultation with a pain specialist at the Institute of Pain Medicine at Rambam Health Care Campus. The questionnaire consisted of questions regarding their demographic data; medical and cancer history; intake of pain medications during the previous 24 hours; medication-induced adverse effects; minimal, mean, and maximal pain intensity at rest and movement during the previous 24 hours ; and the number of pain exacerbation episodes during the previous 24 hours. Data were also obtained on the recommended treatments noted in their physicians’ referral letters and in the medical records of hospitalized patients. Study participants also completed the short-form McGill Pain Questionnaire (SF-MPQ)Pain specialists were informed that their patients’ data would be collected for the study. However, to avoid treatment selection bias, the specialists were not informed of the study’s aim.The normality of data distribution of each variable was examined with the Shapiro–Wilk normality test. Data are presented as median (range) and/or mean± standard deviation (SD) for demographic variables, cancer parameters, pain intensities, SF-MPQ and SF-12 scores, and adverse effects.The pain specialists’ consultations were retrospectively analyzed and categorized as “unjustified” or “justified” based on their treatment recommendation. Referrals to pain specialists were categorized “unjustified” if the specialists’ treatment recommendations remained within the WHO ladder framework and included one or more of the following four recommendations: (1) changing drug dose or rotating to an alternative drug within the same analgesic ladder step; (2) adding a rescue medication; (3) moving to the next step of the WHO analgesic ladder; or (4) adding an adjuvant treatment. Referrals were categorized “justified” when specialists’ recommendations fell outside of the WHO ladder in the following cases: (1) an invasive procedure; (2) administration of a systemic drug other than an opioid ; (3) adding medical cannabis, which can only be carried out in Israel by a pain or palliative care specialist or by an oncologist; or (4) referral to radiation therapy, radio-isotope therapy, etc.Complete data were available for 73 patients . Their median age was 55 years .Cancer type was heterogeneous, with breast cancer being the most common type (18%), followed by lung (14%) and colon (11%) cancer. Additional cancer types included pancreatic cancer, female reproductive cancers, and others .Fifty-two patients (68%) had metastatic spread of their cancer to the following body regions: bones (26%), lungs (13%), liver (12%), other sites in the abdominal cavity (12%), lymph nodes (8%), and additional sites (10%). Notably, some patients (13%) had more than one metastatic site. The median cancer duration was 24 months .Pain duration was 6 months . Pain intensity at rest (NPS) was 7  and at movement 8 . Fifty-one patients (72%) experienced pain exacerbation episodes. The number of episodes was 2 . Although a majority of patients had fewer than 5 such episodes, four patients had 20, and one reported 30 pain exacerbation episodes.The most frequent pain location involved the extremities in 36 patients (47%) and included focal extremity pain, pain radiation from another site, or in the form of distal neuropathy. The second most common sites involved the chest and the abdomen in 32 (42%), followed by upper and lower back in 30 (39%), pelvis in 19 (25%), and head and neck in 8 (10%). Most patients had more than one painful site.Pain type was specified by a pain specialist as nociceptive in 52 patients (71%), neuropathic in 20 patients (27%), and visceral in 18 (25%). Hence, about one-quarter of the patients had a mixed type of pain. Pain was attributed to the cancer itself in 53 patients (73%) and to cancer treatment in 20 patients (27%).Seventy-one patients (97%) completed the SF-MPQ, and all patients completed the SF-12 questionnaire. The median MPQ score was 25 . The median SF-12 physical component score was 23 , and the median mental component score was 35 .At the referral evaluation, 56 patients (77%) were treated with opioids. Strong opioids alone were used by 51 patients, weak opioids were used by two, and a mixture of a strong and a weak opioid by three patients.Of those using opioids, long-acting oral or transdermal preparations were used by 43 patients (77%), immediate-release opioids by 41 (73%), and rapid-onset opioids (ROOs) by two patients (3%) only. An additional 13 patients were treated by parenteral opioids, typically intravenous morphine infusions with or without rescue boluses. Non-steroidal anti-inflammatory drugs (NSAIDs) or cox-2 inhibitors (Coxibs) were taken by 12 patients (16%), simple analgesics by 32 (44%), and adjuvant drugs by 20 (27%). Herbal cannabis was used by five patients (7%). Almost all patients (88%) took the analgesic medications as recommended by their referring physician. In contrast, four patients took analgesics not prescribed by their caregiver, on their own initiative. An additional five patients were only partially compliant with their physician’s recommendations and avoided using specific analgesics or took them at a lower dosage than was recommended.The most frequently reported adverse effects in our study are summarized in According to the administered medications, 69 patients (94%) were retrospectively categorized into one of the three WHO analgesic ladder steps. Three patients received only herbal cannabis, and one used only adjuvant drugs and therefore did not match any of the three formal steps. Fifty-four of the patients (74%) were compatible with step 3 of the ladder; three patients (4%) with step 2; and 12 patients (16%) with step 1. However, the mean pain intensity at rest and movement of the patients in all three WHO categories was 6 or above. This is also true for the four patients who received only adjuvant drugs or medical cannabis .r=0.31, P=0.807) or at movement .Spearman’s coefficient correlations were used to test relationships between the WHO analgesic step medications used and the mean pain intensities. No significant correlations could be demonstrated either at rest  were diagnosed with neuropathic pain by a pain specialist. Of them, only 10 patients received adjuvant treatments in an attempt to treat the neuropathic pain.All patients were referred to pain specialist consultation by their physicians: 49 (67%) by hospital physicians; 24 (33%) by community physicians; 17 (23%) by internists; 15 (21%) by family practitioners; 10 (14%) by gynecologists; nine (12%) by oncologists; and six (8%) by surgeons. The remaining 16 (22%) patients were referred by other physicians from various medical fields.For 62 (85%) patients, recommendations remained within the WHO ladder as follows: 24 patients  were advised to change opioid analgesic dose; in four patients (5%) an adjuvant medication was added; 10 patients (14%) were advanced to the next WHO analgesic step; and eight patients (11%) were given a rescue medication. Sixteen patients (22%) required two simultaneous changes within the ladder (i.e. both an increasing opioid dose and the addition of a rescue medication).n=5) or in combination with other changes in the medical regimen within the WHO ladder. One patient was advised to undergo a nerve block, and another received a prescription for methylphenidate to manage opioid-induced excessive daytime sleepiness. As mentioned earlier, a recommendation for cannabis use in Israel can only be completed by a specialist in pain, palliative medicine, or oncology. Since none of the patients for whom medical cannabis was recommended were referred by an oncologist, their referrals were considered “justified.” Similarly, methylphenidate is not registered in Israel for reducing opioid-induced sedation, and its administration requires a special recommendation from a pain specialist.The remaining 11 patients (15%) required an intervention not included in the WHO ladder: in nine patients (11%) medical cannabis was prescribed either alone  were advised to receive a treatment that is not part of the WHO analgesic ladder. Most of these patients (six patients) were referred from a community clinic by their family practitioner. The rest were referred from the hospital by an internist (three patients), an oncologist (one patient), or an occupational physician (one patient). Conversely, the majority of referrals (85%) were categorized as “unjustified.”Although cancer pain represents a complex medical condition, the WHO analgesic ladder has a simple and logical structure, allowing widespread implementation by all physicians caring for cancer patients. Validation studies have shown that up to 73% of cancer-related pain can be reduced when the WHO guideline is properly implemented.Congruent with the literature,The main finding in the present study is that, contrary to our hypothesis, 85% of patients were advised by a pain specialist a treatment within the framework of the WHO three-step ladder. This implies that the pharmacological management of cancer-related pain provided by the WHO guideline was not reasonably utilized by non-pain specialists who care for such patients. A second important finding is that patients exhibited high compliance with the analgesic treatment prescribed by their referring physicians. Close to 90% of the patients reported analgesic consumption that was consistent with the analgesic regimen documented in their referral letter or in their hospital medical record. This is despite the high percentage of reported adverse effects by the patients in our study. These two findings imply that patients’ concerns about treatment side-effects are not the main reason for the observed suboptimal treatment in the patients. Rather, the main problem seems to be an incomplete implementation of the WHO guideline by non-pain specialist physicians. As shown in the literature,20Several additional factors point to significant gaps in the implementation of the WHO guideline for the management of cancer-related pain. First, 16 patients 22%) did not receive opioids although their reported mean pain was moderate to severe (with an NPS of 6 or greater). These patients were treated by either simple analgesics, an adjuvant drug, or medical cannabis. Notably, in recent years medical cannabis has emerged as a new player in the cancer-related pain treatment armamentarium and can be prescribed for this indication in at least some countries, although evidence for its effectiveness is still anecdotal at best.% did not25It is important to refer patients to pain specialist consultations if pain is not improving within a short time or if patients are experiencing intolerable side-effects of analgesia. However, given the poor availability of pain clinics, attempts to optimize analgesic treatment based on the WHO guideline are expected to be made prior to such referrals. Although all referred patients in the present study were treated prior to their referral, such treatment was not successful, and 85% of the patients were provided treatment by their pain specialist that is within the WHO ladder framework. We have not investigated the reasons for this poor adherence to the WHO guideline. Several providers’ barriers have been identified in the past, such as poor pain assessment, insufficient knowledge about pain treatments, and prioritizing cancer treatment over pain treatment.20However, patient-related barriers such as fears or unwillingness to take pain medicationOur findings and those of othersOne possible limitation of our study is the lack of a follow-up session for evaluating the outcome of the pain specialists’ recommendations. Seemingly, this could have resulted in a more comprehensive clinical picture. However, this was clearly beyond the scope of this study, which focused primarily on referrals to pain clinic consultations rather than on the outcome of these consultations.The pharmacological management of cancer-related pain provided by the WHO guideline was not reasonably utilized by non-pain specialists in our study. This is despite the high compliance of patients with the analgesic treatment prescribed by their referring physicians. Future studies should aim at a better understanding of cancer-related pain management barriers and improving pain management guideline adherence.