We, therefore, explored whether subtraction imaging, comparing findings between two Met-PET scans with medical therapy-induced suppression of tumor activity in the intervening period, could increase confidence in adenoma localization. In addition, we assessed whether normalization to a reference region improved consistency of pituitary gland signal in healthy volunteers who underwent two Met-PET scans without medical suppression.Pituitary adenomas (PA) affect ~ 1:1200 of the population and can cause a wide range of symptoms due to hormone over-secretion, loss of normal pituitary gland function and/or compression of visual pathways, resulting in significantly impaired quality of life. Surgery is potentially curative if the location of the adenoma can be determined. However, standard structural  imaging, in the form of MRI, is unable to locate all tumors, especially microadenomas (< 1 cm diameter). In such cases, functional imaging [We found that the mean percentage differences in maximum pituitary uptake between two Met-PET scans in healthy volunteers were 2.4% for (SUVr) [cerebellum], 8.8% for SUVr [pons], 5.2% for SUVr [gray matter] and 23.1% for the SUVbw [no region]. Laterality, as measured by contrast–noise ratio (CNR), indicated the correct location of the adenoma in all three image types with mean CNR values of 6.2, 8.1 and 11.1 for SUVbw, SUVbwSub and SUVrSub [cerebellum], respectively. Subtraction imaging improved CNR in 60% and 100% of patients when using images generated from SUVbw [no region] and SUVr [cerebellum] scans compared to standard clinical SUVbw imaging.Met-PET scans should be normalized to the cerebellum to minimize the effects of physiological variation in pituitary gland uptake of 11C-methionine, especially when comparing serial imaging. Subtraction imaging following endocrine suppression of tumor function improved lateralization of PA when compared with single time point clinical Met-PET but, importantly, only if the images were normalized to the cerebellum prior to subtraction. Such trThe subtraction imaging techniques described above differ in their approach; subtraction imaging to detect sepsis uses standardized uptake value—body weight (SUVbw) images, whereas parathyroid and ictal subtraction imaging uses normalized images. Here, we have applied both techniques and assessed their effectiveness to aid pituitary tumor localization compared to standard Met-PET. To effectively normalize the images, an appropriate reference region must be selected. Therefore, we compared three commonly used reference regions from other nuclear medicine studies of the central nervous system Research Registry 2070). Data from healthy volunteers allowed the most appropriate reference region for normalization of the pituitary uptake to be determined. The patient group was used to explore which subtraction technique provided the clearest localization of the pituitary tumor (matched with findings at surgery). The cohort of patients with TSH-secreting pituitary adenomas included 2 men and 8 women, with a mean age of 58 years (range 27–75). In each case, the patient had confirmed hyperthyroxinemia (raised free thyroid hormone levels) with non-suppressed TSH and met clinical and laboratory criteria for a diagnosis of autonomous tumoral TSH secretion .The baseline Met-PET (PET1) was registered to the baseline MR (MR1) to create PET1 [Co-Reg].The on-suppression PET (PET2) was registered to the on-suppression MR (MR2 [Co-Reg]) resulting in PET2 [Co-Reg].The PET2 [Co-Reg] was resampled into a matrix of the same size and shape as PET1 [Co-Reg] to create PET2 [Resampled].All registrations were performed in 3D Slicer  version.The on-sStep 4 was required to enable voxel-wise manipulations to produce the subtraction images without the requirement for position and interpolation calculations while using the world coordinate system.Normalized PET images (SUVr) were created by dividing the voxel values of the PET image by the mean voxel value in a reference region Fig. . This prSubtraction images were created by subtracting the on-suppression Met-PET image (PET2[Resampled]) from the baseline Met-PET image (PET1[Co-Reg]) Fig. . This prTo quantitatively compare subtraction images using SUVbw images and SUVr images, ten patients who had confirmed lateralization of a microadenoma < 10 mm) were assessed using a semi-automated technique. This was undertaken by assessing lateralization using a modified contrast-to-noise ratio equation which selected the maximum signal from each side of the pituitary gland. The midline of the gland was defined on the FSPGR MR sequences as the point of insertion of the infundibulum  viewed in the coronal plane Fig. a. Therea0 mm wereThe mean CNR for both techniques was found, and the technique with the highest value was considered the most appropriate for highlighting the differences.To determine the reference region to use for normalization, the mean absolute percentage difference between the maximum signal in the pituitary gland at both time points was found using three reference regions  and using SUVbw without normalization. The mean absolute percentage differences were found to be 2.4% for SUVr [cerebellum], 8.8% for SUVr [pons], 5.2% for SUVr [gray matter] and 23.1% for the SUVbw [no region]. The percentage differences are shown as a box plot in Fig. Both subtraction imaging techniques and the standard clinical Met-PET scans were compared using CNR see Eq.  for ten The images for a representative subject are shown in Fig. We have shown that subtraction imaging, generated from normalized Met-PET scans performed before and after medical suppression of tumor function, permits accurate lateralization of pituitary tumors, with an improved CNR when compared with standard clinical imaging.The registration process was optimized for direct registration between the PET and MRI images Fig. , withoutThe process of creating subtraction images Fig. , step 4 The PET2 to PET1 registration was also used to resample the PET2 image into an identical matrix as PET1  method. Therefore, outlining these regions could be improved by automating the process using artificial intelligence (AI). There is a growing body of work that shows that AI and in particular deep-learning techniques (DL) have potential in automatically segmenting various areas of the body including the cerebellum .To assess which reference region would be used for normalization, we compared the maximum signal in the pituitary gland using two images from each of the healthy volunteers acquired at different time points. As well as the regions we also compared the differences seen in the original SUVbw images in case no reference region was needed. We found that the maximum signal from the SUVbw images could vary on average by more than 20% see Fig. . DiffereQuantifying the CNR between the left and right side of the pituitary gland Fig.  acted asThe CNR results indicate that normalized subtraction imaging (SUVrSub [cerebellum]) improved detectability of pituitary adenomas when directly compared to the original (SUVbw) images and non-normalized (SUVbwSub) images. In every case, the SUVrSub [cerebellum] image had the highest CNR out of the three image sets, but importantly the SUVbwSub images did not always generate a higher CNR over SUVbw images. In four out of the ten patients, the CNR was lower after subtraction which highlights the importance of normalization.This technique could also find application with other pituitary tumor subtypes where there is the possibility to suppress tumor function using medical therapy . Similarly, the basic principles underpinning our methodology are potentially applicable to other PET ligands and other tumors. In particular, PET ligands that target tumoral SSTR receptor expression in TSH-secreting pituitary adenomas may be considered  althoughIn conclusion, this study demonstrates that pituitary adenoma localization can be aided by normalized subtraction imaging. By highlighting areas of change between a baseline Met-PET image and registered suppressed Met-PET, this novel technique can allow improved localization of an adenoma when compared with conventional imaging modalities, especially when the cerebellum is used as a reference region. Implementation of this technique in clinical practice may be of particular added value when definitive intervention  is being considered, to guide targeted intervention and mitigate the risk of disrupting normal pituitary gland function.


These data suggest that some Ediacaran biotas were tolerant of at least intermittent anoxia, and thus had the capacity for a facultatively anaerobic lifestyle. Alternatively, these soft-bodied Ediacara organisms may have colonized the seafloor during brief oxygenation events not recorded by redox proxy data. Broad temporal correlations between carbon, sulfur, and uranium isotopes further highlight the dynamic redox landscape of Ediacaran-Cambrian evolutionary events.The Ediacaran biota were soft-bodied organisms, many with enigmatic phylogenetic placement and ecology, living in marine environments between 574 and 539 million years ago. Some studies hypothesize a metazoan affinity and aerobic metabolism for these taxa, whereas others propose a fundamentally separate taxonomic grouping and a reliance on chemoautotrophy. To distinguish between these hypotheses and test the redox-sensitivity of Ediacaran organisms, here we present a high-resolution local and global redox dataset from carbonates that contain in situ Ediacaran fossils from Siberia. Cerium anomalies are consistently >1, indicating that local environments, where a diverse Ediacaran assemblage is preserved in situ as nodules and carbonaceous compressions, were pervasively anoxic. Additionally, δ Geochemical data from sedimentary rocks in Siberia indicate that members of the soft-bodied Ediacara biota (the earliest macroscopic life on Earth) were tolerant of low-oxygen conditions, suggesting they had the capacity for anaerobic metabolisms. However, because of their unusual moldic preservation in sandstones (or as nodules or carbonaceous compressions in carbonates) and bizarre quilted morphologies, the metabolic requirements of these organisms remain controversial. Given the extreme surface area of the fronds, which preserve similar patterns at increasingly smaller scales3, recent interpretations of Rangeomorph feeding strategies range from osmotrophy (absorption of labile dissolved organic carbon from seawater), to chemosymbiosis (with autotrophic sulfur-oxidizing bacteria), to extracellular digestion within semi-isolated chambers (including a resident microbiome)10. Other hypotheses suggest an aerobic metabolism for the Rangeomorphs12, leading to the hypothesis that a rise in the oxidation state of seawater along continental shelves was the primary controlling factor in their evolution, and its fall to their extinction13. To the contrary, if the Rangeomorphs did not require oxygen for their metabolism, their evolution may have been independent of ocean redox conditions.Of the enigmatic Ediacaran biotas—the earliest macroscopic organisms on Earth that persisted for about 35 million years (ca. 574-539 Ma) in globally distributed marine environments—the Rangeomorphs, characterized by their modular organic-walled fronds and fractal level of organization, are considered by some to represent stem group eumetazoans16 and anoxic seafloor conditions18. Furthermore, data support the presence of redox gradients across facies on individual platforms, suggesting that the regional distribution of Ediacaran organisms was dependent on the oxidation state of overlying seawater23. Globally, most of the fossils of soft-bodied organisms are preserved as casts and molds in medium- to coarse-grained sandstones—which are not amenable to paleo-redox reconstruction—rather than in fine-grained shales, which do contain distributions of redox-sensitive elements that may be used to reconstruct environmental conditions24. In rare and fortuitous instances, Ediacaran biotas have been preserved as nodules and compressions in carbonates, for which proxies for both local and global redox conditions have been calibrated. Here, we present high-resolution time-series data for both local and global ocean redox in carbonates from arctic Siberia that also contain Rangeomorphs, along with a numerically-abundant assemblage of macrofossils consisting of Arboreomorphs (frondose organisms with holdfasts rooted in substrate), undifferentiated bulbous structures with tubules reaching into surrounding sediments, and large carbonaceous compressions.Distinguishing between these two hypotheses is critical to understanding the biological affinity of the Ediacaran biotas, and requires constraints on the redox conditions under which they lived. Emerging evidence suggests a highly dynamic oxidation state through Ediacaran space and time. Based on geochemical evidence, the geological period may be divided into discrete intervals with different proportions of oxic28  the Maastakh Formation, consisting of partially silicified peritidal dolostone with chert nodules, cross-bedding, and wave ripples; (2) the fossiliferous Khatyspyt Formation, consisting of thin- to medium-bedded bituminous limestone; (3) the Turkut Formation, comprising dolostone and limestone with several levels of collapse breccia filled with pyrobitumen that may be related to evaporite dissolution; (4) siliciclastics and volcanics of the Syhargalakh Formation; and (5) mixed siliciclastics and allodapic limestones of the Mattaia Formation the Maas29. Specifically, 87Sr/86Sr values for the Khatyspyt Formation are lower  of the N. sunnaginacus Zone and thereby the onset of the Cambrian explosion28.Detrital zircon U-Pb isotopic ages provide a maximum depositional age of 550 Ma for a sandstone near the top of the Maastakh Formation  appear near the base of the Syhargalakh Formation, the lowest occurrence of Treptichnius pedum (the ichnospecies associated with the Ediacaran-Cambrian boundary) and Rusophycus (a likely arthropod resting trace) are found at the top of the Syhargalakh Formation. Stratigraphically higher in the Mattaia Formation, more complex traces of organisms that burrowed deeply into the sediments, as well as transported SSFs of the lower Cambrian Fortunian Stage Purella antiqua and Cambrian Stage 2 Nochoroicyathus sunnaginicus assemblage zones, are found in siliciclastic and carbonate facies28.The Olenek Uplift is known for containing well-preserved nodules and carbonaceous compressions of Ediacaran organisms, including the first preserved appearance of 13C) are used to correlate Olenek Uplift strata with other terminal Ediacaran and basal Cambrian successions worldwide, in particular to coeval intervals in South China , while a second positive carbon isotope peak in the Gaojiashan Member associated with the first appearance of Cloudina in South China35 most likely equates to a similar magnitude excursion in the Khatyspyt Formation. The negative δ13C excursion seen across the Ediacaran-Cambrian boundary (BACE event37) beginning at the top of the Dengying Formation in South China  may begin in the upper Turkut Formation26, but the sedimentary rocks preserving the negative carbon isotope anomaly are regionally truncated at an unconformity surface below siliciclastics of the Syhargalakh Formation. Alternatively, the shift towards negative δ13C values in the upper Turkut Formation could correlate with the A4 anomaly, which is a negative δ13C excursion identified as older than the BACE event in Oman32. Higher in the succession, peak δ13C values of the Mattaia carbonate platform are potentially equivalent to those in the Zhujiaqiang Formation of South China, which are also biostratigraphically constrained to Cambrian Stage 2 based on the first appearance of N. sunnaginacus assemblage Zone fossils.Carbon isotopes  compositions of pyrite in acidified residues from Khatyspyt limestones may further provide a chemostratigraphic marker of terminal Ediacaran time. The stratigraphic data show a profound shift from −20 to +30‰ that has been interpreted as a change in redox conditions and/or sulfate availability on a regional or global scale20. Notably, the first appearance of soft-bodied Ediacara biotas in the Khatyspyt Formation and a positive δ13C excursion are associated with this prominent perturbation in seawater chemistry. A remarkably similar sulfur isotope anomaly is recorded in the Gaojiashan Member of the Dengying Formation, which is associated with the upper of the two positive carbon isotope excursions in the formation, and the first evidence for metazoan biomineralization in South China35. Examples of Ediacaran ‘superheavy pyrite’ in the aftermath of the Shuram Excursion are also known from Oman38, Namibia39, northwestern Canada40, and northwestern China41. Some Ediacaran ‘superheavy pyrite’ values have been attributed to anomalies in laboratory extraction methods42 or thermochemical sulfate reduction within hydrothermal fluids43. However, their coincidence specifically in post-Shuram successions worldwide supports the view that these events can be used as chemostratigraphic markers, and most-likely reflect significant changes in the redox landscape of the global ocean.The time-series trend in sulfur isotope (δ2/[Nd]) < 1 with element concentrations normalized to the post-Archean Australian shale)44. In contrast, Ce stays in solution under anoxic conditions and anoxic waters have little to no Ce anomaly, as evidenced by Ce/Ce* values that are close to or >1 below the chemocline in many modern anoxic basins45. Cerium oxidation is closely linked to the redox kinetics of manganese (Mn) such that the presence/absence of a Ce anomaly is specifically sensitive to the position of a sample with respect to the Mn redoxcline45. In the suboxic portion of the modern Black Sea, Mn is actively oxidized at O2 concentrations below analytical detection (<3 μM)46, which helps constrain the threshold needed to generate a negative Ce anomaly. Marine carbonates can preserve a reliable record of seawater REE patterns—as a substitution for Ca2+ in carbonate minerals47—if diagenetic alteration and detrital contamination are minimized48, with a recent study of modern Bahamian carbonates indicating that REE signals are particularly resistant to diagenesis49. Samples can be screened for detrital influence using yttrium to holmium (Y/Ho) ratios, with values >36 indicating an authigenic seawater signal48.Here we investigate local redox conditions in both limestones and dolostones of the Olenek Uplift succession using the magnitude of cerium (Ce) anomalies. Cerium is a redox-sensitive rare earth element (REE) where Ce(III) can be partially oxidized to Ce(IV) under oxic conditions. This causes a decrease in solubility, which results in Ce removal from seawater. Modern well-oxygenated seawater is thus depleted in Ce relative to other trivalent rare-earth elements (REEs), resulting in a pronounced negative Ce anomaly (Ce/Ce* = [Ce]/([Pr]2S in the water column) conditions, using uranium (U) isotopes. Uranium in nature is dominated by two long-lived isotopes, 235U and 238U, whose half-lives are 0.7 and 4.5 Ga, respectively. In nature, U exists in predominantly two oxidation states: soluble U(VI) and insoluble, particle-reactive U(IV). Soluble U(VI) forms unreactive, stable calcium-uranyl-bicarbonate complexes in seawater. It is well-mixed and isotopically homogenous in the modern ocean with an isotopic composition of −0.39‰50. Among various sinks for U in the ocean, the strongest isotopic fractionation is associated with removal of U to euxinic sediments, which preferentially sequester isotopically-heavy 238U, thereby leaving residual seawater more enriched in isotopically light 235U. Thus, an expansion of ocean euxinia will drive the δ238U composition of global seawater towards lower values. Recent data suggest that substantially less fractionation occurs under ferruginous (free Fe2+ in the water column) conditions51 and suboxic conditions52 than under euxinic conditions. Thus, U isotopes are increasingly considered a proxy specifically for marine euxinia53. Reference 238U values. The δ238U signal of seawater can faithfully be recorded in marine carbonate sediments under certain conditions, with modern Bahamian carbonates showing a small but consistent isotopic offset between seawater and sediment regardless of burial depth and mineralogy (Bahamas median = −0.15‰ vs. seawater = −0.39‰)55. Taken together, a shift towards lighter U isotopes in ancient carbonates can be interpreted as an expansion of global marine euxinia.We also estimate global seawater redox conditions, in large part related to the extent of euxinic  and global (U isotope) redox conditions in the same carbonate strata that preserve in situ Ediacaran fossils, thus allowing us to examine the redox context of the enigmatic biota. The occurrence of well-preserved Ediacaran fossils in carbonate environments allows us to directly link in situ oxidation state, global ocean redox, and fossil assemblages in the same strata, thus providing important constraints on the redox-dependence and metabolism of early macroscopic life on Earth.18O) compositions. Petrographic analysis  are in a reasonable range for well-preserved Proterozoic carbonates57 with the heaviest values in the Turkut Formation consistent with the presence of collapse breccias potentially associated with evaporative conditions. We find no relationship between carbon and oxygen isotope abundances in our sample set that would suggest diagenetic alteration  and U isotope  ratios and oxygen isotope δ1O composi49 to evaluate the fidelity of the samples. The majority of our samples show Y/Ho elevated above the detrital background (>36), resulting in a positive Y anomaly and a strongly hydrogenous (seawater) REE signal  or a shift in global sulfate availability. Regardless, our Ce anomaly data from Olenek Uplift carbonates strongly suggest deposition beneath an anoxic water column through the entirety of the succession.REE distributions in carbonates are currently recognized as a powerful tool to evaluate seawater patterns and redox conditions in ancient depositional basins. Given their low abundance, however, careful leaching of samples is required to avoid contamination from siliciclastic components. To this end, we used a step-wise extraction technique using dilute acids, and measured Y/Ho in our leachateskground >6, resultnal Fig. . No nega235U isotope. The median δ238U value for syn-depositional and post-depositional Bahamian carbonates deposited in the well-oxygenated modern oceans is −0.15‰55, such that lower values recorded in ancient carbonates likely indicate an expansion of global marine euxinia compared to today. In our sample set, the median δ238U value is −0.58‰ in the Maastakh Formation, −0.64‰ in the Khatyspyt Formation, −0.57‰ in the Turkut Formation, and −0.53‰ in the Mattaia Formation . The dataset as a whole has a median δ238U value of −0.58‰, which is significantly lower than modern carbonates 18 and South China (median = −0.87‰)59  are the lightest recorded thus far in the geological archive, including those measured in multiple sections across the Permo-Triassic boundary during which anoxia is thought to be a primary driver of the largest Phanerozoic extinction event65  is preferentially removed to sediments, leaving residual seawater U(VI) enriched in the light Nenoxites curvus, a rangeomorph Charnia masoni, an arboreomorph Khatyspytia grandis, macrophytes, and various holdfast structures34. The combination of in situ Ediacaran macrofossils and geochemical data suggesting a locally reducing water column is strong evidence that this Ediacaran assemblage was living under anoxic conditions. Whereas basal, sessile metazoans have a lower oxygen demand than large, motile animals67, all animals require some oxygen for their metabolism, with the only possible exception being meiofaunal loriciferans living permanently in the absence of molecular oxygen69, although the true oxygen demand of these organisms is still debated71. The absence of a negative Ce anomaly in Olenek basin waters suggests the lack of Mn-oxidation, which occurs in modern environments at sub-micromolar oxygen concentrations (3 μM or less)46. Bilaterians have been recorded in modern environments at O2 concentrations as low as ~1 μM, which ref. 2 conditions than are needed to generate a negative Ce anomaly. These exceedingly low O2 concentrations would severely limit the size and metabolic rate of metazoans, however, which is inconsistent with the macroscopic size of the Khatyspyt fossils . The sub-micromolar O2 living conditions of the Khatyspyt Ediacaran assemblage suggest instead that these organisms may not have metazoan affinities, unless they developed special adaptations to permanently anoxic conditions 68. Another intriguing possibility is that this Ediacaran assemblage lived in association with epibiotic or endosymbiotic bacteria10, by analogy with recent metazoans persisting at very low oxygen levels of only a few micromolar per kg76.Despite our carbonate-based determinations of pervasive positive Ce anomalies indicative of anoxic depositional conditions in the Olenek basin, the Khatyspyt Formation contains a diverse array of Ediacaran macrofossils preserved in situ  associated with the evolution and diversification of Ediacaran macroorganisms. Despite the spread of H34S-enriched pyrite (in which the sulfur isotopic composition matches or even exceeds that of carbonate-associated sulfate [CAS] or bedded sulfates) is seen in multiple Ediacaran successions worldwide, including in northwestern China84, South China36, Namibia42, northwestern Canada40, and Oman38. Various explanations for the ‘superheavy pyrite’ have come forth, but none of them have addressed the issue in the framework of prolonged late Ediacaran euxinia. As documented in the δ238U compositions of Ediacaran carbonates, the oxygenation state of the oceans oscillated between oxic and anoxic states. Following δ238U evidence for ocean ventilation (oxidation) during the Shuram negative δ13C excursion13, the subsequent terminal Ediacaran nadir of δ238U values is associated with expanded post-Shuram ocean euxinia and positive δ13C anomalies recorded in both South China and arctic Siberia. This coupling supports the canonical interpretation of δ13C fluctuations as resulting from enhanced burial and sequestration of organic matter in the global ocean86, which is promoted under conditions of expanded anoxia. This interval also reveals a profound stratigraphic step up (>40‰) in the δ34S signature of pyrite, with stratigraphic trends in multiple sections rising to values that meet and exceed those of seawater sulfate. Such a phenomenon requires removal of 32S from the oceans through the microbial production of H2S and its burial as insoluble pyrite in sediments when soluble iron is available. The mixing of globally expanded euxinic waters  with ferruginous waters that may have still dominated the deep Ediacaran oceans87 provides a biogeochemical mechanism for the starvation of sulfate in the ocean  after the Shuram Excursion and before the Ediacaran-Cambrian boundary. These observations contrast with δ238U and δ34S evidence for greater oxygenation with higher sulfate and alkalinity in seawater associated with the earlier Shuram Excursion89, as well as across the subsequent Ediacaran-Cambrian transition, when bedded sulfates are recognized worldwide59. This geochemical and lithological worldview further highlights the oscillatory redox landscape of the Ediacaran Period.Our data for both local and global redox conditions also shed light on the anomalous behavior of the terminal Ediacaran sulfur cycle. 90. This metal enrichment event was followed by a return to crustal values in the terminal Ediacaran Period, with another oxygenation event occurring at ~540 Ma. Together, widespread oceanic euxinia in the terminal Ediacaran Period can explain: (1) positive carbonate δ13C values promoted by enhanced organic carbon burial; (2) anomalously light carbonate δ238U values; (3) ‘superheavy pyrite’ δ34S values caused by seawater sulfate distillation; and (4) muted trace metal enrichments in euxinic shales caused by drawdown of the oceanic metal inventory. By contrast, oxygenation during the preceding Shuram Excursion and across the subsequent Ediacaran-Cambrian boundary was accompanied by negative carbonate δ13C values, near-modern carbonate δ238U values, 34S-depleted pyrites, and Phanerozoic-style trace metal enrichments.The emerging picture of Ediacaran redox trends is further consistent with a spike in redox-sensitive trace metal concentrations in euxinic shales at ~560 Ma, which was interpreted by ref. 38, ref. 238U evidence for a second euxinic pulse in Cambrian Stage 2. In addition, ref. 238U data. Our δ238U data from the biostratigraphically constrained Mattaia carbonate platform are consistent with the expansion of ocean euxinia at this time. In addition, our Ce/Ce* data further indicate locally anoxic water column conditions. SSFs preserved in the Mattaia Formation reveal evidence of transport and are not found in situ, possibly indicating that these animals were living in shallower shelf and shoreface environments  and then transported offshore. A shift back to a strongly euxinic ocean in Cambrian Stage 2 could have slowed the emergence of complex Cambrian life after its initial proliferation during the more oxygenated Fortunian Stage. Frequent redox oscillation has also been invoked as a driver of animal diversification beginning in Cambrian Stage 292. Alternatively, the persistence of euxinia in the global ocean—as indicated by light δ238U values—through the studied Olenek Uplift section could indicate that redox conditions were only a minimal factor in controlling the evolution of early complex life on Earth. These results are consistent with a compilation of shale iron speciation data that suggest only limited oxygenation across the Neoproterozoic-Phanerozoic transition93, with more permanent ocean oxygenation perhaps delayed until the Devonian Period98.After widespread terminal Ediacaran euxinia and potential re-oxygenation across the Ediacaran-Cambrian transition associated with the accumulation of thick sulfate deposits worldwide238U data are comparable to other coeval terminal Ediacaran sections worldwide, indicating that ocean euxinia was more widespread than at any other interval in Earth history examined to date using U isotopes. Evidence of local anoxia recorded in strata that preserve Ediacaran organisms in situ, combined with strongly euxinic terminal Ediacaran oceans as indicated by global U isotope data, strongly suggests that some members of the Ediacaran macrobiota were tolerant of low-oxygen conditions and might have been capable of a facultatively anaerobic lifestyle. One intriguing possibility is that Ediacaran organisms used chemolithoautotrophy  either directly or through symbionts, exploiting redox gradients at the sediment-water interface. Alternatively, these soft-bodied Ediacara organisms might have colonized the seafloor during brief oxygenation episodes not recorded by local redox proxy data. Ultimately, our combination of local and global redox data with Ediacaran fossils in the same succession offers an integrated perspective on the relationship between ocean redox and the evolution of early macroscopic life on Earth.In summary, this study combines proxy evidence for both local and global redox conditions with paleontological observations of a diverse assemblage of Ediacaran macroorganisms preserved in carbonate rocks of the Olenek Uplift of arctic Siberia. Our data indicate that: (1) a wide array of Ediacaran macroorganisms are preserved in environments that were often anoxic, and (2) the Ediacaran macrobiota persisted on multiple continental margins through a period of widespread ocean euxinia. Our δSamples were collected during a 2009–2010 field expedition to the Khorbusuonka and Olenek river areas of arctic Siberia. Hand samples were collected, cut, and bulk powders created in Russia before being transported to the United States. Samples were previously analyzed for a variety of geochemical proxies by refs. Thin sections were prepared by Vancouver Petrographics for a select group of samples and analyzed using a standard petrographic microscope in plane- and cross-polarized light in order to understand diagenesis. Diagenetic pathways were assessed by visual inspection of the degree of fabric retention, and petrographic characteristics were compared to geochemical data in order to interpret the diagenetic history of the succession.3, followed by 5 mL of concentrated HNO3, and finally, 20 additional mL of 1 M HNO3. Each sample was shaken and subject to vortex mixing to ensure all powder had reacted with acid. Samples were left to sit for 24 h at room temperature to allow for maximum carbonate dissolution. Digests were centrifuged at 3800 rpm for 9 min and the supernatant was decanted from the residue. 200 to 350 mL of the supernatant was removed, placed into 10 mL centrifuge tubes, diluted to ~200 ppm calcium (Ca) with 2% HNO3, and analyzed for a full suite of major and trace element concentrations on a Thermo iCAP™ quadrupole inductively coupled plasma mass spectrometer (Q-ICP-MS) at Arizona State University. Typical precision is reported based on repeated analysis of simultaneously run standards, and in this study, relative % s.d. was <6% for all reported elements. Total U concentrations were also analyzed using an Element-2 ICP-MS at the University of Maryland. Analytical precision was better than 3% based on duplicate analyses.50 mL centrifuge tubes were rinsed with 6 M HCl and Milli-Q water prior to adding powdered samples. Approximately 1.5 g of sample powder was first digested by 15 mL of 1 M HNO3 were dissolved with the appropriate amount of 2% HNO3. The insoluble residue was then separated by centrifugation and the solution decanted and disposed. The next 40% of moles of CaCO3 were then dissolved using the appropriate amount of 2% HNO3 and centrifuged again. The solution was pipetted, diluted to 10 mL with 2% HNO3, and shipped to Arizona State University for elemental analysis by Q-ICP-MS. For dolomite samples, a one step of digestion, which dissolves the first 20% of moles of CaMg(CO3)2 in the appropriate amount of 2% HNO3 was utilized. This multi-step digestion process follows the methods suggested by ref. 100 and the cerium anomaly (Ce/Ce*) was calculated using: Ce/Ce* = [Ce]SN/([Pr]SN2/[Nd]SN) where SN denotes a PAAS-normalized value48.Rare-earth elements were extracted using a multi-step leaching process. For calcite samples, the first 20% of moles of CaCO236U/233U double-spike (0.8 mL of spike per 500 ng U) was added to each sample before U isotope column chemistry101. The samples were digested sequentially using reverse aqua regia, H2O2, and concentrated HNO3. The spiked samples were evaporated to dryness and re-dissolved in 3 M HNO3. Uranium was separated twice from matrix elements by ion exchange chromatography using UTEVA resin. Separated U was dissolved in 2% HNO3 to a concentration of 40–50 ppb and analyzed on Thermo Scientific Neptune multi-collector ICP-MS instruments at Arizona State University and the University of Maryland  Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


N = 5 110) were followed up to 8 years after blood sampling. We included patients that attempted suicide during follow-up (N = 348) and matched bipolar disorder patients from the same cohort who did not attempt suicide during the study period (N = 348) and analyzed a total of 92 proteins with a neuro exploratory multiplex panel. Using a multivariate classification algorithm devised to minimize bias in variable selection, we identified a parsimonious set of proteins that best discriminated bipolar disorder patients with and without prospective suicide attempts. The algorithm selected 16 proteins for the minimal-optimal classification model, which outperformed 500 models with permuted outcome (p = 0.0004) but had low sensitivity (53%) and specificity (64%). The candidate proteins were then entered in separate logistic regression models to calculate protein-specific associations with prospective suicide attempts. In individual analyses, three of these proteins were significantly associated with prospective suicide attempt . Most of the candidate proteins are novel to suicide research.Suicide is a major cause of death worldwide. Several biological systems have been implicated in suicidal behavior but studies of candidate biomarkers have failed to produce clinically relevant biomarkers for suicide prediction. The objective of the present study was to identify novel candidate biomarkers for suicidal behavior. We used a nested case-control study design where a large cohort of patients with bipolar disorder ( More than 800,000 people die from suicide each year  and an eSeveral biological systems have been explored in relation to suicidal behavior –17, but future suicide attempt through an exploratory approach focusing on candidate systems rather than specific candidate markers. Using a neuro exploratory multiplex biomarker panel, we measured a total of 92 unique proteins involved in, e.g., neurogenesis, neural plasticity, immune system processes, and stress response. We employed a nested case-control study design where a large cohort of patients with bipolar disorder (N = 5 110) were followed up to 8 years after blood sampling. We included the patients that attempted suicide during follow-up  and matched bipolar disorder patients from the same cohort who did not attempt suicide during the study period .The aim of the present study was to identify novel potential biomarkers for Participants were enrolled in the Swedish Bipolar Collection (SWEBIC) study between 2009 and 2013 . PatientA total of 5 110 bipolar patients were enrolled in the SWEBIC study. Blood was donated at nearest lab or hospital and drawn in 9 ml EDTA tubes  that were sent by regular overnight mail to the Karolinska Institutet Biobank, Stockholm. Upon arrival, the samples were centrifuged for 15 min at 2000 x g, and blood plasma were separated in 0.5 ml aliquots and stored at −80 °C. Typically, the blood samples arrived at the biobank within 24 h of sample collection.Data on suicide attempts were obtained by linking data from the SWEBIC study to the Swedish National Patient Register, which has nationwide information on psychiatric inpatient care since 1973 and outpatient visits in specialist care since 2001 . Most diN = 52) or (ii) due to failed samples in the biomarker analysis (N = 2). In order to achieve follow-up period uniformity between cases and controls, we also excluded 56 persons whose attempted suicide occurred later than four years after blood sampling. Four persons were misclassified as cases due to mistaken ICD-10 codes and were moved to the control group. A total of 586 persons were included in the final analysis: 288 cases and 298 controls.Figure The study was approved by the Regional Ethical Review Board in Stockholm, Sweden, and all participants provided written and oral informed consent.Protein concentrations in plasma were quantified using the Olink® Neuro Exploratory panel provided by Olink Bioscience  . This muWe first identified a parsimonious set of proteins associated with prospective suicide attempt by removing uninformative proteins using the MUVR algorithm . This alProteins selected in the minimal-optimal PLS-DA model were then individually included in logistic regression models to calculate the odds ratios (ORs) of attempting suicide during follow-up for each protein, adjusted for sex, age, and lithium treatment. A majority of cases had also attempted suicide prior to blood sampling (168/288). To disentangle state from trait, we calculated a second set of ORs after excluding the 168 persons who had attempted suicide prior to blood sampling.N = 288); the number of bipolar disorder hospital admissions during the follow-up period (N = 502); and the severity of bipolar disorder as rated on the Clinical Global Impression scale within four years of blood sampling (N = 396). Finally, Welch’s t-tests were performed to compare mean NPX levels between cases and controls for each of the 77 included proteins.We calculated Spearman’s rank correlation coefficients to explore the association of each protein selected in the minimal-optimal PLS-DA model with: the number of days from blood sampling to the suicide attempt  method  was usedPatient characteristics are presented in Table candidate proteins. Box plots of NPX levels of the 16 candidate proteins are included in Supplementary Fig p = 0.0004) as seen in Fig. The variable selection algorithm selected 16 of the 77 included proteins for the minimal-optimal PLS-DA model, henceforth referred to as p < 0.05/16) associated with prospective suicide attempt after adjusting for sex, age, and lithium prescription  of the 16 candidate proteins were significantly . Of the 16 candidate proteins, three  were significantly associated with prospective suicide attempt in individual logistic regression analyses. When patients with previous suicide attempts were excluded, no associations survived correction for multiple comparisons and many proteins showed smaller effect sizes. There were no correlations between candidate proteins and the number of days that elapsed from blood sampling to suicide attempt. In addition, we found no significant correlation of candidate proteins with disease severity or the number of bipolar disorder hospital admissions.We conducted an exploratory biomarker study aimed at discovering novel candidate biomarkers to predict suicidal behavior. We sampled blood plasma from 5 110 patients with bipolar disorder—a high-risk condition for suicide—and followed them prospectively for four years in the Swedish National Patient Register. We compared baseline protein concentrations of 92 proteins from 288 patients with a prospective suicide attempt during follow-up (cases) with 298 patients from the same cohort who had never attempted suicide (controls). Using PLS-DA modelling within repeated double cross-validation, we identified a parsimonious set of 16 candidate proteins associated with prospective suicide attempts , but most candidate proteins are novel to mood disorders and suicide research.Uteroglobin (SCGB1A1), also known as clara cell protein, was the highest ranked protein in the multivariate model and showed the second-most decreased OR for prospective suicide attempt. Low SCGB1A1 concentrations have previously been observed in serum of patients with depression , 32, in Annexin A10 (ANXA10), ranked second in the multivariate model, is novel to psychiatric research. The annexins are a family of calcium-dependent phospholipid binding proteins implicated in pathologies such as cancer, cardiovascular disease, and inflammation . SeveralCentrin-2 (CETN2), ranked third in the multivariate model, is a calcium-binding component of the centrosome. It is required for duplication of centrioles during cell division  and alsoFK506-binding protein 5 (FKBP5), ranked fourth in the multivariate model, is a protein that might play a role in the dysregulation of hypothalamic–pituitary–adrenal (HPA) axis activity observed in affective disorders. It inhibits glucocorticoid receptor sensitivity  and has Fibroblast growth factor receptor 2 (FGFR2) showed the most increased OR for prospective suicide attempt. FGFR2 belongs to the fibroblast growth factor family, which is a widely researched system that has been associated with depression  as well Of the six candidate proteins associated with an increased risk for prospective suicide attempt, three are involved in immune system processes . In contrast, CD63 and CCL27, involved in immune cell migration, were associated with a decreased risk for prospective suicide attempt. Low CCL27 serum concentrations have previously been associated with higher anxiety scale scores , which iThe remaining candidate proteins  are involved in, e.g., cell cycle-related processes , cell migration (NAA10), chaperone activity (PFDN2), and metabolism (PTPN1 and ATP6V1F).A major strength of this study is the prospective design. More than 5000 blood samples were collected from patients who were then followed for four years to capture suicide attempts. An additional strength is that both cases and controls were diagnosed with bipolar disorder, which is important to disentangle biological mechanisms implicated in suicidal behavior from those related to mood disorders. A limitation is that psychiatric medications other than lithium were not adjusted for in the logistic regression analyses, due to the large number of missing data on prescription of antidepressants and antipsychotics. Further, blood samples were sent to the biobank by regular mail leading to a delay between sampling and centrifugation. While most assays are resilient to factors of pre-analytical processing, pre-centrifugation delay can heavily impact certain assays . HoweverWe explored a panel of 92 proteins in blood plasma from a large cohort of patients with bipolar disorder to discover novel potential biomarkers for future suicide attempts. We identified a set of 16 biomarker candidates involved in, e.g., responses to hormonal and steroid activity, immune system processes, cellular growth and metabolism. Most of our findings are novel to research on mood disorders and suicide and provide insight into the biological underpinnings of suicidal behavior. While observed effect sizes were generally small, these candidate proteins could feature in future biomarker studies evaluating the viability of combinations of candidate biomarkers as prediction models for suicide risk.Supplementary MaterialSupplementary Figure 1


Stress cardiovascular magnetic resonance (CMR) has been proposed as a non-invasive tool for detection of CMD. The aim of this study was to determine relationship between CMD and diastolic function in patients with HFpEF using a novel CMR technique.– Patients with obesity and HFpEF without epicardial coronary artery disease (CAD) underwent Doppler echocardiography to measure diastolic function, followed by vasodilator stress CMR, using a single bolus, dual sequence, quantitative myocardial perfusion mapping to measure myocardial blood flow (MBF) at rest and at peak hyperemia. With this, myocardial perfusion reserve (MPR), global stress endocardial-to-epicardial (endo:epi) perfusion ratio, and total ischemic burden  were calculated. Results are reported as median and interquartile range.– Nineteen subjects were enrolled . Median age was 64 [56–72] years. Global stress MBF was 2.43 ml/min/g [2.16–2.78] and global myocardial perfusion reserve (MPR) was 2.34 [2.07–2.88]. All had an abnormal subendocardial perfusion with an endo:epi of less than 1 (0.87 [0.81–0.90]). Regional myocardial hypoperfusion was detected in 14 (74%) patients with an IB of 6% [0-34.4]. Endo:epi ratio significantly correlated with IB  and measures of diastolic function .– Using a novel quantitative stress CMR myocardial perfusion mapping technique, women with obesity and HFpEF were found to have patterns of abnormal subendocardial perfusion which significantly correlated with measures of diastolic dysfunction. Heart failure with preserved ejection fraction (HFpEF) is a heterogenous clinical syndrome accounting for approximately one-half of the total HF patients , 2. Obes2) and with stable symptoms of heart failure (NYHA class II-III), preserved left ventricular EF (> 50%) and without obstructive CAD were prospectively recruited at Virginia Commonwealth University Health System. Patients underwent transthoracic echocardiogram to confirm diagnosis of HFpEF defined as left ventricular EF > 50%, and evidence of diastolic dysfunction as evident by at least two of the following criteria defined by the American Society of Echocardiography guideline . Angina was present in 10 (53%) of the patients. Medications at the time of enrollment are summarized in Table We enrolled 21 patients between July 2020 to December 2020 and March 2021 to November 2021, with interruptions in recruitment related to COVID-19 pandemic. Two patients were excluded after CMR: one due to new diagnosis of hypertrophic cardiomyopathy that became evident on CMR, and one due to severe perfusion abnormality suggestive of epicardial CAD and later confirmed by cardiac catheterization. Thus, a total of 19 patients met the enrollment criteria and were included in the analysis. All 19 (100%) patients were female and 8 (42%) were self-referred Black or African American. Age was 64 years [56–72] and BMI was 34 kg/mCMR characteristics of the patients are summarized in Table By quantitative perfusion analysis, segmental myocardial hypoperfusion was detected in 14 (74%) of the patients with a median ischemic burden of 6% [0-34.4]. In contrast, abnormal perfusion by visual assessment was detected in only 3 (16%) of the patients   and E/e’   all patients had an impaired stress endo:epi perfusion ratio (< 1.0); (2) the endo:epi ratio correlated with echocardiographic measures of diastolic dysfunction (E/e’ ratio and e’ velocity); and (3) the great majority of patients (74%) had evidence of regional hypoperfusion with median ischemic burden of 6%, also correlating with higher E/e’ ratio, a surrogate for elevated left ventricular filling pressures.CMD is common in patients with HFpEF, and a decrease in myocardial blood flow has been shown to be associated with higher incidence of mortality and adverse cardiovascular outcomes independent of traditional risk factors , 18. It Ischemia and myocardial injury have been thought to be the underpinning of hemodynamic derangement in patients with HFpEF, and CMD may promote myocyte injury, myocardial fibrosis, and stiffening in pathogenesis of HFpEF . MyocardIschemia caused by CMD may have a patchy distribution and may not be confined to a singular vascular bed . The uneHFpEF is a heterogenous clinical syndrome in which cardiovascular risk factors culminate in heart failure symptoms. In our study we have demonstrated that CMD is present in obese women with HFpEF and can be measured non-invasively using a novel CMR perfusion technique. It is difficult to determine to what extent the perfusion abnormalities are related to the underlying comorbidities; nonetheless, reduced myocardial perfusion is a marker of higher cardiovascular risk and associated with death and heart failure hospitalization . The relWe do recognize that there are limitations to our study. First, we recognize that the sample size is small and all patients were female, although not by design; while a female predominance in CMD and HFpEF is expected this is a potential limitation of this study. Nevertheless, the results of this pilot study may help develop future larger trials in the pathophysiology of heart disease in women, a condition that is too often neglected, and in a field of medicine where underrepresentation of women is often the prevailing problem. We also recognize that there was no invasive coronary assessment for CMD to validate our measures of MBF. Our study is, however, consistent with findings of others using quantitative non-invasive assessment of MBF. Additionally, adenosine is shown to detect non-endothelial-dependent coronary flow changes, and therefore we cannot completely rule out endothelial-dependent mechanism of abnormal coronary flow . FinallyIn conclusion, we have demonstrated that coronary microvascular dysfunction is common in women with obesity and HFpEF as demonstrated by abnormal stress endocardial-to-epicardial perfusion ratio using a novel fully automated quantitative perfusion mapping with cardiovascular magnetic resonance. The great majority of the patients also had evidence of regional hypoperfusion which correlated with abnormal transmural perfusion gradient. Abnormal sub-endocardial perfusion and regional hypoperfusion also correlated with measures of diastolic dysfunction.


Environmental enteric dysfunction (EED)—a chronic inflammatory condition of the intestine—is characterized by villus blunting, compromised intestinal barrier function and reduced nutrient absorption. Here we show that essential genotypic and phenotypic features of EED-associated intestinal injury can be reconstituted in a human intestine-on-a-chip lined by organoid-derived intestinal epithelial cells from patients with EED and cultured in nutrient-deficient medium lacking niacinamide and tryptophan. Exposure of the organ chip to such nutritional deficiencies resulted in congruent changes in six of the top ten upregulated genes that were comparable to changes seen in samples from patients with EED. Chips lined with healthy epithelium or with EED epithelium exposed to nutritional deficiencies resulted in severe villus blunting and barrier dysfunction, and in the impairment of fatty acid uptake and amino acid transport; and the chips with EED epithelium exhibited heightened secretion of inflammatory cytokines. The organ-chip model of EED‐associated intestinal injury may facilitate the analysis of the molecular, genetic and nutritional bases of the disease and the testing of candidate therapeutics for it. A human intestine-on-a-chip lined by intestinal epithelium derived from patients with environmental enteric dysfunction and cultured in nutrient-deficient medium recapitulates essential features of intestinal dysfunction associated with the disease. Previously described as ‘tropical enteropathy’ or ‘environmental enteropathy’, EED has gained renewed interest in recent years due to its devastating effect on millions of children in low- and middle-income countries. The intestine of EED patients commonly exhibits villous atrophy, nutrient malabsorption, barrier dysfunction and inflammation7. As there is currently no existing in vitro model of EED and only a limited number of animal models, mechanistic understanding of this disease is limited, which has hampered efforts to define biomarkers for diagnosis or develop therapeutics8. For example, deficiencies of micronutrients, such as zinc9 and vitamin A10, may contribute to EED pathophysiology as they are associated with abnormal lactulose:mannitol (L:M) ratios—a measure of intestinal permeability. However, attempts to treat EED-related stunting by nutritional interventions have been disappointing probably due to ongoing problems with nutrient absorption and inflammation in these patients8. Similarly, other dietary interventions, such as administering omega-3 long-chain polyunsaturated fatty acids11, optimizing amino acid profiles12, supplementing with multiple micronutrients13 or improving food digestibility through fermentation, hydrolysis or enzyme supplementation12, have also been tested with limited success.Environmental enteric dysfunction (EED) is a paediatric disorder characterized by chronic intestinal inflammation that is associated with malnutrition, stunted growth, cognitive impairment and attenuated response to oral vaccines14. Low serum tryptophan levels are also linked to stunting in children suffering from EED16. Tryptophan is both a building block for proteins and a precursor for niacin, melatonin and neurotransmitters (such as serotonin and tryptamine)17, and supplementation with amino acids including tryptophan was recently shown to improve symptoms in EED patients18. In animal studies, symptoms of tryptophan deficiency, including anorexia and impaired growth, may occur with intakes as little as 25% below the standard requirement, which translates to 2–2.5 mg kg−1 body weight for human infants 6–24 months old19. Recently, niacin (nicotinic acid) deficiency has also been implicated as a contributor to EED18 and other inflammatory intestinal conditions as administration of niacin was shown to ameliorate dextran sodium sulfate-induced colitis via prostaglandin D2‐mediated D prostanoid receptor 1 activation20. In addition, niacin serves as a precursor for coenzymes, such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which are essential for the normal function and survival of living cells. However, a mechanistic role for malnutrition in driving EED pathophysiology in humans remains to be demonstrated.Inadequate dietary intake not only leads to stunting but can also increase host vulnerability to environmental factors. For example, diets low in the essential amino acid tryptophan lead to decreased antimicrobial peptide secretion and increased susceptibility to chemical-induced intestinal inflammation in mice21. Thus, establishing an in vitro human EED model would help to elucidate disease pathophysiology and enable the development of new prevention and therapeutic measures. Here we describe how human organ-on-a-chip microfluidic culture technology that faithfully recapitulates the structure and function of many human organs, including the intestine31, can be leveraged to meet this challenge. We used human intestine chips lined with organoid-derived primary intestinal epithelium isolated from either healthy children or paediatric EED patients who were refractory to nutritional intervention. We have previously shown that these intestine chips support the formation of differentiated three-dimensional villus-like epithelial structures as well as the production of an overlying mucus layer, which require the presence of dynamic fluid flow34. Our studies comparing healthy versus EED intestine chips revealed that both nutritional deficiencies and genetic or epigenetic changes in the intestinal epithelium contribute to the clinically observed EED phenotype. Moreover, by comparing healthy and EED patient-derived intestine chips, we were able to study phenotypic responses to nutritional deficiencies, such as villus blunting and barrier dysfunction, which are known to be common to multiple intestinal pathologies (such as inflammatory bowel diseases and coeliac disease), and distinguish them from responses due to transcriptomic and cytokine signatures that are seen in EED.Studying a multifactorial disease such as EED raises substantial methodical and modelling challenges and, at present, there are only a few murine models and no human in vitro models that can be used to study this disease28 . Compared with healthy chips, EED chips showed differential expression of 287 genes  , neuregulin-4  and the intestinal stem cell marker SMOC2, whereas brush border peptidase MME, oxidative stress and inflammatory response controlling ectoenzyme VNN1, tight junction protein CLDN10 and secreted goblet cell protein CLCA1 were all downregulated  Fig. . EED chi38 versus samples from healthy control patients who were investigated for gastrointestinal symptoms but had normal endoscopic and histologic findings . When we compared gene profiles from EED chips versus healthy chips cultured in control medium , the differentially expressed genes had some overlap with the clinical EED signature, including most notably a shared downregulation of metallothioneins   5) Fig. .19. When we compared expression profiles from the healthy chips exposed to nutritional deficiency  versus the healthy control chips, we detected differential expression of 690 genes   and the inflammation-associated gene LCN2 , selected on the basis of past work implicating their role in EEDq < 0.05 and fold change ≥1.5; 522 upregulated, 447 downregulated)  and downregulation of not only metallothioneins but also metabolic and digestive genes . This congruence was most noticeable among the top 10 upregulated genes of the clinical EED signature, 6 of which (60%) were also upregulated when EED chips were exposed to nutritionally deficient medium  Fig. . Cultureed) Fig. . This waium Fig. .39. This analysis revealed several pathways that were significantly upregulated when EED chips were exposed to −N/−T media, including a chemokine pathway  and a pathway associated with amino acid starvation  , DNA damage , p53 methylation  and amino acid transporters . Many of these same genes were also upregulated in nutritionally deficient healthy −N/−T chips, including ATF4, TP53, AARS, YARS, MDM2, CCND2 and several amino acid transporters  Fig. . Within 45. In control medium, enterocyte markers were expressed at lower levels in EED chips compared with healthy chips  and were even more broadly downregulated in EED chips that were exposed to nutritional deficiency , but were differentially regulated when chips were exposed to nutritional deficiency. Healthy chips exposed to nutritional deficiency upregulated Paneth cell markers , while EED chips exposed to nutritional deficiency exhibited further Paneth cell marker downregulation . These findings are consistent with the histopathological finding of Paneth cell depletion in EED patient intestinal biopsies38.We also compared gene expression changes in our model with previously identified clusters of intestinal cell-type gene markersAs the transcriptomic analysis revealed downregulation in pathways involved in cell growth and intestinal barrier formation, we carried out differential interference contrast (DIC) and immunofluorescence microscopic analyses, which indeed confirmed that both healthy and EED intestine chips showed dramatically reduced growth of villus-like structures when cultured under nutrition deficient (−N/−T) conditions compared with healthy and EED control chips perfused with complete medium Fig. . QuantifMYO1A), which links actin to the overlying apical membrane and whose absence results in irregularities of microvilli packing and length46; protocadherin-24 (CDHR2) that forms links between adjacent microvilli and is the target of enterohemorrhagic Escherichia coli-mediated brush border damage47; and mucin-like protocadherin (CDHR5) that forms heterophilic complexes with CDHR2  values, which were measured by calculating the transfer of Cascade Blue fluorescent tracer (596 Da) from the epithelial lumen in the top channel to the underlying parallel channel below. Both healthy and EED control chips exhibited a tight barrier under baseline conditions (within the 10−7Papp range) and displayed small but statistically significant reductions in barrier function when exposed to nutritional deficiency, as indicated by 8.9- and 2.5-fold increases in Papp for the healthy and EED chips, respectively , microsomal triglyceride transfer protein (MTTP), apolipoprotein B (ApoB) and apolipoprotein C-III (ApoC3) were all lower in nutritionally deficient epithelium  member 2 (SLC2A2) and solute carrier family 2 (facilitated glucose/fructose transporter) member 5 (SLC2A5). Other nutrient transporters (such as amino acid absorption and metabolizing factors) that were found to be significantly downregulated in EED chips in response to −N/−T deficiency (EED −N/−T vs EED control), include SLC36A1, which encodes the proton-coupled amino acid transporter 1, ANPEP membrane enzyme alanyl aminopeptidase that is responsible for peptide digestion at the brush border, retinol binding protein 2 (RBP2), cytochrome P450, family 27, subfamily A, and polypeptide 1 (CYP27A1) , conditionally essential amino acids  and non-essential amino acids .We then assessed differences in absorption of nutrients between the healthy and EED intestine chips grown in control medium by performing untargeted metabolomic analysis by liquid chromatography tandem mass spectrometry (LC–MS/MS) to analyse epithelial uptake of nutrients and transfer of these molecules from the lumen of the intestinal epithelium in the top channel to the underlying basal channel Fig. . We deteN-acetylglutamine, homoarginine, indolelactate, glutamine, alanine, betaine, imidazole lactate, 3-methylhistidine, 1-methylhistidine), nucleotides , xenobiotic metabolism (guaiacol sulfate) or lipids (3-hydroxy-3-methylglutarate). Interestingly, we also identified 4 metabolites unique to the EED chips cultured in control medium, including products of purine nucleotide metabolism (xanthine), vitamin B6 metabolism , citric acid cycle/energy metabolism  and fatty acid metabolism (acetylcarnitine (C2))  in the perfusion medium. Of these metabolites, 17 were unique to the healthy control chips and included products of pathways related to metabolism of amino acids ) Fig. .PEPT1 transporter, and not due to passive inter- or intracellular diffusion, as this response could be completely prevented by adding Gly-Gly dipeptide (1 mM), which is a specific inhibitor of this transporter54 , by the epithelial cells in the top channel, and its transfer to the lower channel using LC–MS/MS. This analysis revealed reduced uptake and transport of Gly-Sar in nutritionally deficient healthy intestine chips compared with the same chips cultured in control medium  and regenerating islet-derived protein 3 alpha (REG3A), were upregulated when EED or healthy chips were exposed to nutritional deficiency  in the epithelial lumen of healthy −N/−T intestine chips compared with the same chips cultured in control medium , chemokines (such as CXCL5), metallothioneins , membrane ion transport (such as SLC4A7 and SLC23A3), antimicrobial defence  and mucosal protection 55. Interestingly, a duodenal proteomic profile that correlated with dysbiosis in a nutritionally refractory stunted Bangladeshi patient cohort also showed overlap with genes related to brush border structure, mucosal function and inflammation 56. Additional organ-on-a-chip studies will need to be carried out with cells from a larger number of EED patients to rule out any potential biases due to small sample size and to determine their generalizability to other EED patient subpopulations.Importantly, although the number of EED donors used in this study was limited, the results obtained with EED intestine chips were consistent between donors and compared favourably with genetic data from clinical studies with much larger numbers of EED patients carried out in Pakistan (SEEM study), as well as in similar clinical studies in Zambia and Bangladesh. The Zambian study compared stunted paediatric patients who were unresponsive to nutritional intervention, to paediatric patients or adults with severe acute malnutrition, and EED relevant changes were seen in NADPH oxidases  because of their reported effect on intestinal development and function, and the suggested correlation between their deficiencies and EED development58. Indeed, we were able to show in this study that these phenotypes can manifest because of nutritional deficiencies regardless of whether the intestinal epithelium was derived from healthy or EED patients. This implies that villus blunting and barrier dysfunction observed in EED may be a response to environmental conditions, rather than an inherent genetic or developmental feature of this disease. This finding has high clinical relevance because the intestinal epithelium undergoes continuous shedding and renewal every 3–5 d; hence, the chronic negative effect of nutritional deficiencies might explain a fundamental aspect of the malabsorption and poor response to oral vaccination seen in EED patients. The ability to dissect and assess the factors leading to villus blunting in EED  in vitro is a unique capability enabled by intestine chips that support the formation of three-dimensional villus-like structures as well as mucus production and quantification of transepithelial transport, absorption and secretion.Villus blunting and barrier dysfunction are hallmarks not only of EED, but also of other intestinal pathological conditions, such as inflammatory bowel diseases, coeliac, diarrhoea and small-intestine bacterial overgrowth51. Our transcriptomic analysis was sensitive enough to detect several genes, including ApoB, that were downregulated in nutritionally deficient epithelium. We confirmed that the expression of this molecule is decreased, using immunofluorescence microscopy, and that fatty acid absorption is impaired in both EED −N/−T and healthy −N/−T chips (compared with their respective controls), using a fatty acid uptake assay.Fat is a macronutrient responsible for 30–40% of total caloric intake in children and 20–35% in adults, and fatty acid composition has a direct effect on health and development, including inflammatory status and cognitive developmentGiven that dietary protein is an important macronutrient directly linked to linear growth and the results from our transcriptome-wide analysis revealed downregulation of amino acid transporters in EED chips, we also conducted an untargeted metabolomic analysis that identified several amino acids that are significantly reduced in the EED control chips compared with healthy control chips. More interestingly, we identified metabolites that were secreted by the intestinal epithelium into the lower channel. Thus, the intestine chip may be used as a nutritional and metabolic screening tool where uptake, utilization and secretion of specific metabolites by and through the intestinal epithelium can be followed in high resolution and quantified over time. Moreover, future analysis of molecules released into the lower flow channel could lead to identification of biomarkers of disease severity and/or progression that might be detectable in blood.59. A more effective approach might be to first administer a diet composition that preferentially promotes intestinal recovery with formation of new villi and restores increased intestinal absorptive area before moving to supplementation required for catch-up growth.While reduction of intestinal absorptive surface area due to villus blunting caused by nutritional deficiency could impair nutrient uptake by the intestine, exposure to nutritional deficiency also directly suppressed expression of multiple genes related to nutrient absorption, specifically in chips lined by cells from EED patients. Thus, these results suggest that nutritional deficiency has a two-fold effect in these patients, which would probably manifest in a greater degree of intestinal dysfunction and a more severe EED phenotype. In our study, we also found that EED intestine chips exposed to nutritional deficiencies produced greater amounts of inflammatory cytokines compared with healthy chips grown under the same −N/−T conditions. This is a critical detail as inflamed intestine has higher caloric demands for basic maintenance and renewal, which could result in a negative caloric balance unable to support catch-up growth. Furthermore, chronic inflammation may negatively affect the efficacy of oral vaccines in the EED intestine. As such, it is possible that the lack of catch-up growth in EED children receiving nutritional intervention is due to current interventions only supplying nutrients aimed at replenishing the deficiency in tissues responsible for linear growth 38. Thus, this in vitro EED model may be useful for gaining further insight into the pathophysiology of this disease as well as for development of potent therapeutics. The intestine chip could also find uses in personalized medicine and nutrition by leveraging clinical biopsies, potentially allowing for personalized (patient-specific) digestion, absorption and allergic reactions to be assessed for different nutrients.The ability of the intestine chips to allow the study of the effects of environmental factors (such as cell source and nutrient levels) individually or in combination in a controlled manner and to explore multiple clinically relevant outcomes, such as cell and tissue morphology, barrier function, nutrient metabolism and absorption, inflammatory status and transcriptome modifications, enabled us to distinguish between manifestations more common to multiple intestinal diseases versus responses that are unique to EED. For example, we identified the CD36 gene, which has several functions  relevant to EED as well as to intestinal cancer and other intestinal diseases, to be significantly downregulated under nutritional deficiencies in both healthy and EED intestine chips. Remarkably, our findings relating to EED are directly in line with recently published clinical data that explored unique signatures of EED-affected children compared with healthy controls and children with coeliac disease60. A total of 3 healthy and 2 EED donors were used to generate the data in this study  were activated using ER1/2 (Emulate) and UV exposure for 20 min. Chips were then coated with 200 μg ml−1 collagen I (BD Corning) and 100 μg ml−1 Matrigel (BD Corning) in serum-free DMEM-F12 (Gibco) for 2 h at 37 °C. After washing, organoids were broken into smaller fragments using enzymatic activity  and seeded in the luminal upper channel of the chips. They were then allowed to adhere for 24 h before introduction of flow and mechanical deformation as previously described28. For the −N/−T treatment, niacinamide and tryptophan were removed from the basal medium  used to prepare the expansion culture medium  and no additional niacinamide was added28. After 16–18 d in culture with continuous flow (60 µl h−1) and mechanical deformation , the medium was changed to differentiation medium  in the luminal top channel and expansion culture medium in the lower channel for 4 additional days.Organoids from healthy donors or EED patients were generated from biopsy samples collected during exploratory gastroscopy following a procedure previously describedn = 3 biological replicates) and one EED donor  and were reflective of a recent clinical EED transcriptomic signature derived from a larger population . Subsequent validation studies were carried out using 1–3 healthy donors  and 1–2 EED donors  per experiment. RNA samples were processed using the Genechip WT PLUS reagent kit and hybridized to Affymetrix Human Clariom D arrays. Robust multichip average (RMA) was used to generate normalized expression intensity data for subsequent analysis . Differential expression analysis was performed with limma61 R package for each comparison pair; gene expression values were averaged for each condition. Template matching was used to extract genes that are differentially expressed between these conditions. Differential gene expression heat map analysis was performed using Euclidean distance and McQuitty’s linkage within the R package heatmaply39. To normalize values across experiments, columns were scaled to generate a Z-score. Pathway analysis was performed using the natural language-processing algorithm COmprehensive Multi-omics Platform for Biological InterpretatiOn (COMPBIO) to generate a holistic contextual map of the core biological themes associated with gene expression changes. Enriched concepts associated with differentially expressed genes were compiled from PubMed abstracts using contextual language-processing. Themes were scored using a complex function that incorporates an empirical P value and a standard score similar to a Z-score to give a final value representing fold enrichment over a random clustering as follows: 3–9, weak relationship; 10–99, modest relationship; 100–999, strong relationship; 1,000+, very strong relationship. A theme map was generated where themes are represented as nodes and interconnections between themes are represented as edges, with the thickness of an edge relating to the degree of interconnection.Initial microarray experiments were carried out using one healthy donor  at 3–6 months of age were selected and given nutritional intervention if they continued to be malnourished at 9 months of age. Those EED patients who were unresponsive to nutritional intervention underwent esophagogastroduodenoscopy and histological evaluation as part of the clinical workup. SEEM samples were sequenced using RNA-seq technology as previously detailedA12380) at room temperature for 60 min and washed with PBS; nuclei were co-stained with Hoechst 33342 . Microscopy was performed with a laser scanning confocal microscope (Leica SP5 X MP DMI-6000 or Zeiss TIRF/LSM 710).Immunofluorescence microscopic imaging was carried out using the following steps: the apical and basal channels of the chips were gently washed with PBS and fixed with 4% paraformaldehyde  in PBS for 30 min, then washed twice with PBS and kept at 4 °C. The fixed samples were sectioned to 150–250 µm sections using a vibratome (Leica), and then permeabilized and blocked with 0.1% Triton X-100 solution and 10% donkey serum in PBS for 30 min at room temperature. Then primary antibody  was added (1:100 in 1.5% BSA/PBS solution) and the samples were incubated overnight at 4 °C, followed by multiple PBS washes. Cells were then incubated with secondary fluorescent antibody  and phalloidin  was introduced to the luminal channel (at 60 ml h−1). After flowing overnight to saturate the microfluidic channels, outflows were discarded and collection for measurements were conducted for ~24 h. The fluorescence intensities (390 nm/420 nm) of the top and bottom channel effluents were measured using a multimode plate reader (BioTek NEO). The apical-to-basolateral flux of the paracellular marker was calculated using the following equation: Papp = (dQ/dt)/AdC, where dQ/dt (g s−1) is molecular flux, A (cm2) is the total area of diffusion and dC (mg ml−1) is the average gradient.To assess paracellular permeability, 50 μg ml(WGA)–Alexa Fluor 488 conjugate  for live cell imaging, as described previously26 with some modifications. Briefly, WGA solution  was allowed to flow through the epithelium channel for 30 min and then washed with continuous flow of HBSS for 30 min. Intestine chips were then cut sideways parallel to the length of the channel and imaged with an epifluorescence microscope (Zeiss Axio Observer Z1) with ×5 objective.Mucus was visualized using a wheat germ agglutinin Chips were starved for 1 h by replacing the luminal and lower channel media with HBSS. Then, fluorescently labelled dodecanoic acid combined with a quencher  were added according to the manufacturer instructions  to the luminal upper channel of the intestine chips. The entire length of the channels was then imaged with an epifluorescence microscope  with ×5 objective at 5, 10, 30 and 60 min.The medium in the lower channel of the chips was changed to HBSS and allowed to flow for 30 min to clear any residues. Then collection of outflows was conducted over 5 h. Samples were frozen immediately after collection (−80 °C) and submitted for LC–MS/MS analysis. The Metabolon global metabolomics platform was used to measure biochemicals in cell and media samples. Samples were prepared using the automated MicroLab STAR system (Hamilton). Several recovery standards were added before the first step in the extraction process for quality control purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000), followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/Ultra Performance Liquid Chromatography (UPLC)–MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC–MS/MS with negative ion mode ESI, one for analysis by Hydrophilic interaction (HILIC)/UPLC–MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap (Zymark) to remove the organic solvent. Raw data were extracted, peak-identified and QC processed using Metabolon’s hardware and software.61 R package (v3.32.10) to fit a linear model to the data. log2 fold change, P value and adjusted P value were calculated for each comparison using an unmoderated Student’s t-test and the false discovery rate (FDR) method for multiple testing correction62. Adjusted P values are shown in volcano plots. To assess transporter mediated uptake and transfer from the luminal upper channel to the lower channel, glycyl-sarcosine  alone or in combination with the specific PEPT1 transporter inhibitor Gly-Gly dipeptide  was added to the luminal medium and their abundance in the lower channel outflow was assessed.Beginning with the OrigScale values from Metabolon, which are normalized in terms of raw area counts, we calculated total ion count in the outflow to visualize sample-wise variance. Next, we normalized each sample by its total ion count and rescaled each metabolite’s value by dividing each metabolite by its root mean square. We visualized metabolite abundance using a heat map. Statistical analysis and heat map generation was performed using R . We performed differential expression analysis by using the limmat-test or 2-way analysis of variance (ANOVA) was performed to determine statistical significance, as indicated in the figure legends .Each intestine chip was used as a biological repeat for one terminal assay. Either a Student’s Further information on research design is available in the Supplementary InformationSupplementary figures.Reporting SummaryPeer Review FileSupplementary Table 1Comparison of gene-expression data between healthy and EED, control or −N/−T chips.Supplementary Table 2Upregulated and downregulated genes categorized according to themes.Supplementary Table 3Gene expression of different cell-type markers.Supplementary Table 4Summary of the donor data.Supplementary Table 5Source data for Supplementary Fig. 9.