Fundamental knowledge gaps exist in characterizing the biogeochemical mechanisms that transform microbe-mineral interfaces at submicron scales, particularly in complex field systems. Our objective was to develop methods targeting the nanoscale by using high-resolution microscopy to assess biological and geochemical drivers of weathering in natural settings. Basalt, granite, and quartz (53–250 µm) were deployed in surface soils (10 cm) of three ecosystems  for one year. We successfully developed a reference grid method to analyze individual grains using: (1) helium ion microscopy to capture micron to sub-nanometer imagery of mineral-organic interactions; and (2) scanning electron microscopy to quantify elemental distribution on the same surfaces via element mapping and point analyses. We detected locations of biomechanical weathering, secondary mineral precipitation, biofilm formation, and grain coatings across the three contrasting climates. To our knowledge, this is the first time these coupled microscopy techniques were applied in the earth and ecosystem sciences to assess microbe-mineral interfaces and  Microorganisms represent the bulk of subsurface terrestrial biomass, colonize almost all terrestrial environments, and are an important component in the biogeochemical cycling of carbon, nitrogen, and other life-supporting elements4. Microorganisms also actively and selectively interact with mineral surfaces by engaging in element capture and transfer processes from ecosystem to molecular levels. This is best demonstrated by the mycorrhiza “underground highway” that facilitates the transfer of carbon and nutrients within and between plants through a common hyphal network6. The feedback loops that plants and their rhizosphere microbiome develop with bedrock minerals emerged in the early Phanerozoic  during the colonization of terrestrial habitats by plants9, and continues to evolve in the present. However, knowledge gaps remain in characterizing biological-mineral interfaces at fundamental scales of interactions that dictate how the abiotic and biotic components of ecosystems contribute to elemental cycles and ecosystem function11. The study of biogeochemical interactions in soil can be aided by novel technologies that can enhance our ability to assess mineral surfaces and associated organisms at micro- to nanoscale resolutions. We demonstrate potential applications of Helium Ion Microscopy (HIM) as an emerging tool in the earth and ecological sciences in combination with Scanning Electron Microscopy (SEM)-Energy Dispersive X-ray spectra (EDX) using, as an example, samples from a field study.The weathering of Ca- and Mg-silicate rocks stabilizes global climate over geologic timescales by consuming carbon dioxide  transform minerals at submicron scales.Helium ion microscopy is a relatively new technology mostly used in the material and biomedical sciences that is well-suited for research in the natural sciences18. Microscopy studies have examined biomechanical and biochemical weathering mechanisms that: weaken mineral structures through the fungal-driven oxidation of Fe(II) in biotite19, induce secondary mineral formation and biomineralization20, enhance microbial growth around nutrient-rich zones21, incongruently leach major and trace elements22, and exert control of K nutrient uptake and related clay mineral modifications under changing land-use regimes23. Atomic force microscopy has been used to detect the simultaneous occurrence of indirect and direct biochemical weathering, where it was observed that abundant small etching pits formed on mineral surfaces exposed to siderophores (molecular Fe chelators) released to solution by microbes, whereas fewer yet larger “biopits” formed on surfaces colonized directly by bacteria24. Fungal biofilms imaged with SEM were shown to enhance biochemical weathering through the release of organic acids at the hypha-mineral interface and transformed grains via shrink-swell processes25. Backscattered electron images captured by SEM were utilized to characterize polysaccharide biofilms produced by microbes that also affect weathering by promoting mineral dissolution or serving as a protective coating that slows weathering27. Biochemical processes may have a larger effect on weathering than biomechanical transformations18 as observed in saturated liquid cultures where fungi accelerated biotite dissolution by acidifying the bulk solution at greater rates than direct fungal-mineral interactions28, but this may not be the case in unsaturated conditions found in field settings. High resolution imaging and elemental mapping presents an opportunity to target biological weathering processes occurring at micron to submicron scales30. Coupling the superior imaging capabilities of HIM with established methods for measuring elemental distribution using SEM can help resolve complex weathering processes found in natural soil environments.Unresolved questions pertaining to mineral transformation mechanisms involve assessing relative controls of biochemical versus biomechanical weathering, indirect or direct microbe-mineral interactions, and whether microbes enhance or slow mineral dissolution31 or to deployed mineral substrates that vary in nutrient content35. In-soil mesh bags have also been used to estimate fungal biomass in the field36, to explore the role of mycorrhizal fungi as soil carbon sinks37, and to examine how forest soil nutrient status impacts soil mycorrhizal foraging activities38. Mesh bags filled with granular rock substrates also showed how mycorrhizal fungi employ “biosensing” mechanisms to preferentially colonize and weather basalt compared to granite and quartz under both laboratory conditions39 and in forest systems40.Bacteria and fungi intricately interact with silicate minerals to promote weathering; however, it is not completely understood how surface morphology and elemental distribution in mineral grains  change under interactions with abiotic and biotic factors operating at various scales. In-soil mesh bags represent a successful approach to assess the response of soil fungal communities to contrasting soil nutrient patchesOur objective was to employ HIM and SEM to detect potential drivers and evidence of mineral weathering at the micron-submicron scale using in-soil mesh bags filled with granular substrates that were deployed in natural field settings. We hypothesized that a reference locator grid would be effective to identify and analyze individual grains by applying: (1) surface sensitive helium ion microscopy to capture details at micron to sub-micrometer scales and to image mineral-organic matter interaction hotspots on grain surfaces; and (2) scanning electron microscopy to quantify elemental distribution at the same locations using electron backscattering for chemical point analyses and elemental maps. We used a field experiment to demonstrate the effectiveness of our microscopy approach for assessing incipient weathering of grains exposed to the natural environment. Briefly, nylon mesh bags filled with granular granite, basalt, and quartz (53–250 µm) were retrieved from surface soils (0–10 cm) of contrasting climates  and landscape positions  after one year of deployment. The substrates were sealed into coarse (35 µm mesh size) and fine (0.5 µm mesh size) nylon mesh bags designed to include and exclude direct fungal hyphae contact with mineral surfaces. Both mesh sizes allow for solute, nanophase, and bacterial interactions with mineral grains. To our knowledge, this is the first time HIM has been used in conjunction with SEM to characterize complex field samples in the earth and ecosystem sciences, and we provide a benchmark method to assess microbe-mineral interfaces and biological inputs to incipient weathering in field and lab experiments at micro- to nanometer resolutions.The high-resolution imaging of grain surfaces and microbe-mineral interfaces revealed the intricacies of such features in unprecedented detail. We first examined untreated granular substrate controls and distinguished grain microtopography and nanocrystal edges characteristic of quartz substrate Fig. , the lamHelium ion microscopy further revealed interactions among fungal hyphae networks with the granular granite, basalt, and quartz in the coarse mesh bags (35 µm mesh size) in all ecosystems, i.e., semiarid Catalina desert scrub, subhumid Catalina mixed conifer forest, and humid Calhoun mixed hardwood-pine forest. For instance, basalt-fungal complexes in the Catalina desert ecosystem displayed morphological variability across resolutions Fig. : micron 43 and biomedical research45. However, HIM is often overlooked as a visualization tool to resolve complex environmental samples where various abiotic and biotic actors are at play, despite its elevated sensitivity and higher resolution capabilities compared to classical electron microscopy techniques.Our results demonstrate that HIM is well-suited for the non-quantitative imaging of microbe-mineral interfaces that are common to the environmental and ecosystem sciences. Specifically, we present potential evidence for incipient microbial-mineral interactions, biological specimens in the weathering environment with contrasting morphological characteristics, and weathering features on grains at different resolutions. High resolution imaging with HIM has been utilized in the nanosciences) biomechanical rock disruption manifested by fungal-enhanced expansion of mineral sheets ets Fig.  and 5, tets Fig. , and incets Fig. . Biomechnts Figs . For insnts Figs  that gronts Figs  or in bents Figs . Fungal nts Figs  and dispnts Figs .Figure 3+). These compounds originate from the organisms themselves or as by-products from the microbial decomposition of organic matter. Organic acids oxidize to produce carbon dioxide that results in greater concentrations of carbonic acid in solution47. The resultant higher pCO2 levels in soil solutions are made even higher due to plant and microbial respiration, which then creates more acidic, enhanced weathering conditions. Thus indirectly, microbe-mineral interactions enhance aggregate stabilization in soil, which allows for more effective, undisturbed weathering activity by bacteria and fungi48. Biochemically-induced changes to mineral surfaces following these processes involve pitting, etching, and other microtopographic changes to grain surfaces, mineral displacement and/or replacement by the formation of secondary nano-minerals, or the complete dissolution of mineral grains49.Biochemical weathering, often concurring with its mechanical counterpart, is mediated by microorganisms through mechanisms that include: the release of compounds that promote weathering, such as organic acids, chelators, and reactive species  and the microbial taxa present in a particular weathering environment50.We present potential evidence for contrasting mechanisms of biomechanical weathering versus biochemical weathering including interactions along mineral basal surfaces, side edges, and between mineral sheets. Our results and prior research suggest that distinct fungal species use varied strategies to mine nutrients that may place them in different ecosystem niches or that fungi exhibit contrasting responses to varying environmental conditions. For example, laboratory studies identified unique weathering signatures for individual fungal species grown under sterile conditions with phlogopite mica, where each species exhibited specific growth patterns  and biochemical weathering signatures when interacting with the minerals52  during mineral weathering23. Biominerals formed by free-living and symbiotic fungi  include aggregated iron hydroxides, calcium and magnesium oxalates, among others57. We observed fungal hyphae adhered to an augite mineral in a basalt sample from the subhumid site  that contained <250 nm aggregates embedded into a hyphal strand  produced during the fungal weathering of Icelandic basalt60. Our findings provide support for biomineralization in a field setting, where fungi produced secondary minerals utilizing cations present in pore solution or from incipient basalt weathering.Biominerals of comparable size (~1 µm) and morphology were documented during fungal diagenesis of carbonates, where Ca oxalate biominerals precipitated onto dolomite surfacesudy Fig. , Cu, Cd,We developed a method to overcome the limitation of HIM for quantifying elemental distribution and tested our technique by consecutively imaging the same microbial-mineral interface with both HIM and SEM Fig. . A granu43Ca nuclear magnetic resonance were combined with HIM to enhance the molecular and microscopic assessment of well-known organic matter reference materials 61. Helium ion microscopy was also coupled with atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy to characterize membrane nanodomains in mammalian cells, where HIM helped identify specimens with 1.5 nm resolution44. Our findings provide examples of microbe-mineral interfaces identified using HIM in field samples that would be well-suited for integration with other quantitative analyses. We envision combining our SEM-HIM technique with focused ion beam sectioning and transmission electron microscopy to quantify the geochemical composition of biomineral formation  from the mineral structure during the initial stages of weathering. Conversely, frayed edge weathering sites also represent nutrient refugia where the reduction of ferric octahedral clay minerals under low pore water oxygen concentrations provide sites that fix biologically relevant interlayer cations 23. Our coupled SEM-HIM method demonstrates a pathway to capture HIM micrographs of microbe-mineral interfaces at sub-nanometer resolution in combination with measures of elemental distribution by SEM. Our approach also shows strong potential for integration with additional microscopy and spectroscopy techniques.Helium ion microscopy has been successfully integrated with other technologies to enhance the strength of its superior imaging capabilities with additional quantitative measurements. X-ray diffraction and ion Fig. , coatingion Fig. , and to ion Fig.  and 3. FMicrobes support terrestrial ecosystems by extracting and transferring rock-derived nutrients through direct and indirect weathering mechanisms. Our study is the first to illustrate how helium ion microscopy, in combination with scanning electron microscopy, can be successfully applied in the earth and ecosystem sciences to image the complex nanoscale environment of microbe-rock interfaces that are keystone in understanding incipient biogeochemical cycles. We demonstrate the effectiveness of a bimodal microscopy approach to assess how microbes interact with basalt, granite, and quartz deployed in semiarid, subhumid, and humid climates for one year. The method we employed identified the intricate morphology of exposed mineral surfaces prior to and following exposure to biological or chemical effects in the field. We visualized individual mineral grains and associated mineral-organic matter interactions at the micro- to nanoscale using helium ion microscopy. We then quantified the elemental distribution at the same referenced interface using point analyses and elemental maps produced by scanning electron microscopy. The results enhance our understanding of incipient mineral weathering in complex field systems by characterizing the biomechanical expansion of mineral sheets by soil fungi in all three climates at sub-nanometer resolutions. Rock substrates deployed in the humid mixed hardwood-pine environment also present evidence for putative biomineral formation and the development of coatings on mineral surfaces that represent early sites of biochemical transformation. Coupling the superior imaging capabilities of HIM with the ability to investigate elemental composition by SEM helped address how microbes interact with mineral surfaces in natural settings by assessing nanoscale features in combination with measures of geochemistry on mineral surfaces.We addressed our research objective of assessing potential contributors to mineral weathering in field systems by utilizing in-soil mesh bags filled with unreacted basalt, granite, and quartz sand deployed in three climates for one year. Samples were buried in semiarid and subhumid landscapes in the Catalina Critical Zone Observatory (CZO) and humid environments in the Calhoun CZO. All samples were processed at Oregon State University and analyses were conducted with the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory.63. Climate end members include desert scrub (1100 m.a.s.l.), characterized by Saguaro (Carnegeia gigantea), Acacia (Acacia spp.), Agave (Agave spp.), and Ocotillo (Fouquieria splendens); and conifer forest (2400 m.a.s.l.) including Douglas fir, Ponderosa Pine, and white fir. The Catalina CZO spans intrusive Precambrian to Tertiary parent rock65 with granitic intrusions and granitic soils that present a relatively narrow range in mineral composition and grain size67. Desert scrub soils formed on the Catalina granitic pluton (Oligocene–Miocene) and the mixed conifer soils on the Wilderness granite suite . The most common soil types in the Catalina CZO include Entisols  in the desert scrub landscape as well as Mollisols and Entisols  in the mixed conifer forest67. The Calhoun CZO (South Carolina) represents the wet, humid end member of the considered climate gradient, receiving 127 cm of precipitation a year with an average annual temperature of 16 °C. Hardwood tree species comprise Shortleaf Pine (Pinus echinata), Northern Red Oak (Quercus rubra), and Sweetgum (Liquidambar styraciflua). The Calhoun sites are located within the Holcomb’s Branch watershed of the USFS Calhoun Experimental Forest where soils formed on gneiss bedrock  in the region of the Whitmire Complex or metadiorite in the Wildcat Complex comprised of plutons and sub-volcanics68. The most common soil orders classified for the watershed include Alfisols  and Ultisols 69. Herein, we reference three field areas by location and vegetation type: Catalina desert scrub, Catalina mixed conifer forest, and Calhoun mixed hardwood-pine forest.Sites extend from water-limited systems in the southwest US to wet, humid forests in the southeast. The Catalina CZO (Arizona) encompasses a semiarid to subhumid environmental gradient that spans significant range in average temperature (24–10 °C) and precipitation (25–85 cm) with well-defined vegetative communitiesi) are reachable by fungal hyphae, referred to as “fungal-accessible” mesh bags (35 µm) and (ii) those that exclude fungi, or “fungal-excluding” mesh bags (0.5 µm). Our approach was adapted from established in-soil mesh bag methods that found the 35 µm mesh size excluded plant root growth into the bags while permitting the penetration of fungal hyphae71. We selected grain sizes that would enhance fungal foraging in the field based on work that showed ectomycorrhizal fungi displayed greater foraging activity in finer grain treatments (53–90 µm) compared to coarser granular substrates (500–1000 µm)39.The in-soil mesh bag approach comprised granular granite, basalt, and quartz (53–250 μm) heat sealed into nylon mesh bags that  that was attached to an aluminum stub with double-sided carbon tape. A small subset of sample (~100 mg) was extracted from the mesh bag, sprinkled onto the grid, and gently pushed into the grid surface to secure the sample prior to loading the stub into the microscope. The samples were coated with a 10 nm carbon layer by thermal evaporation using a 108 C Auto Carbon Coater .2 Silicon Drift Detector (SDD) from Oxford Instruments. This detector is known for high stability and accuracy given its large analytical area and permits high resolution, high-count rate mapping at low and high energies. The SDD can detect lighter elements with accuracy starting from Be. The detector also performs high count rate mapping on rough surfaces and can identify phase composition for nanosize particles as small as 30 nm. The limitation in particle quantification thus is related to the electron beam penetration and excitation volume from the underlying substrate. The secondary electron images were collected at 5 kV voltage and 0.34 nA current at 4 mm working distance with the Everhart-Thornley detector (ETD), which is insensitive to takeoff collection angles. We collected both low and high magnification SEM images to find matching grain locations that were correlated with subsequent HIM micrographs. Briefly, we first located grains of interest using SEM and then located the identical grains for nanoscale imaging with HIM, and then revisited the same interface with SEM. Once HIM-SEM co-location was established for a site of interest, elemental mapping and point analyses were conducted to acquire relevant elemental data. The EDS analyses were performed at 10–20 kV voltage and 2–3 nA current using a backscattering TLD (through-the-lens) detector. Oxford INCA software was used to collect compositional maps and point spectrum analyses.The SEM-EDX technique represents a semi-quantitative approach for identifying the elemental composition of minerals in granular substrates. Sample analyses were conducted with a FEI Helios NanoLab 600i field emission electron microscope. The Helios-EDX detector is an X-Max 80 mm−7 Torr vacuum during the imaging. When necessary, a low energy electron flood gun with beam energy of 500 eV was applied briefly interlaced with the helium ion beam so that charge build up on the surface was eliminated. Secondary electron images were collected using an Everhart-Thornley Detector. The image signal was acquired in line-averaging mode with a dwell time of 1 µs at a working distance of 7 to 8 mm. No post-processing procedures were applied to the digital images besides brightness and contrast adjustment.Sub-micron to nanoscale secondary electron images of the samples were obtained using an Orion Plus helium ion microscope . We used a 25 keV helium ion beam with probe current range of 0.1 to 1 pA to image the samples. Samples were transferred into the HIM via a load-lock system and were maintained at ~4 × 10We also used SEM and HIM to image unreacted control samples for the granular substrates that were sealed into mesh bags, sterilized with the field-deployed samples, and maintained in sealed containers at room temperature in the laboratory for the duration of the one-year field experiment. The control samples were imaged to confirm the absence of biological inputs in the initial substrate rock and to document the types of morphological surface features on the granular substrates at the start of the experiment Figs –S3.Supplementary Information


Statistical analysis was performed with the Student’s t-test and the significance level was set at p-value < 0.01. Resection of parasellar meningiomas is challenging because of the proximity of the optic apparatus. We report a case of unilateral vision loss after resection of a tuberculum sella meningioma in which the impaired eye’s NPi value correlated closely with visual function. NPi values that decrease below 3 predict spikes in intracranial pressure in TBI patients; similarly, increases in the NPi value above 2.5-3 predict improvement in vision in the case reported here. By monitoring the proximal portion of the oculomotor reflex, the NPi can be a marker of visual impairment after surgery.The Neurological Pupil index (NPi) is a standardized method for evaluating pupil reactivity that removes inter-examiner variability. Changes in the NPi can predict clinical deterioration in patients with traumatic brain injury (TBI); however, its use to predict visual impairment after the resection of parasellar meningiomas has not been described. A 71-year-old female underwent a modified expanded bifrontal craniotomy for resection of a 3.1 cm tuberculum sella meningioma that caused compression of the optic chiasm and resulted in left temporal and right superior temporal visual field deficits. Postoperatively, she lost vision in the right eye. Pupillometer measurements demonstrated an asymmetrically low NPi at that time, which improved to normal prior to partial vision recovery. The average NPi in the right pupil was 1.67 during the time of vision loss compared to 3.47 in the left pupil (p=1.7x10 The Neurological Pupil index™(NPi®) standardizes the evaluation of pupil reactivity. Using the NPi-200 Pupillometer System , the NPi  is derived by a proprietary algorithm; values closer to 0 represent “sluggish” or “abnormal” pupils and values closer to 5 represent normally reactive pupils. The NPi is used in trauma settings to quantify pupil reactivity, and changes in NPi over time predict clinical deterioration -3. PupilA 71-year-old female presented with 2.5 years of visual loss in her left eye and was found on Humphrey visual field testing to have left temporal and right superior temporal visual field deficits. An MRI scan revealed a 3.1 cm tuberculum sella meningioma encasing bilateral internal carotid arteries and causing elevation of the optic chiasm Figure . She undPostoperative courseImmediately after surgery, the patient’s vision was at her preoperative baseline and pupils were briskly reactive on the standard penlight exam. Within a few hours of recovering in the intensive care unit (ICU), however, the patient lost vision in her right eye, retaining minimal light perception. At that time, the right pupil was sluggishly reactive with an afferent pupillary defect; thereafter, pupil exams were performed using a pupillometer. The NPi values of the right and left pupils were 1 and 3, respectively; the pupil diameter was 4 and 5 mm, respectively , which was seen in this case. Unfortunately, the pupillometer cannot detect APDs because it only measures pupil reactivity in the eye in which light stimulation is provided. Even so, manual examination of pupil reactivity has high inter-examiner variability compared to pupillometry measurements ,13. ThisPrior studies demonstrate that NPi changes can predict intracranial pressure (ICP) changes in TBI patients. In particular, NPi values <3 correlate with ICP spikes >20 mmHg that can affect the oculomotor nerve during uncal herniation (thereby affecting the efferent limb of the pupillary light reflex) -2. In thFuture prospective studies should determine the overall effectiveness of pupillometry measurements in predicting vision improvement in patients undergoing tuberculum sellae meningioma resections. Though NPi measurements are limited by the fact that they only measure a single pupil at a time (and thus cannot quantify APDs), our report suggests that trending NPi values can predict visual outcome in cases of suspected unilateral optic nerve ischemia/injury after surgical resection of parasellar tumors.Much like its predictive abilities in TBI patients, NPi values may predict vision improvement after suspected vascular-ischemic causes of vision loss following resection of tuberculum sellae meningiomas. Unlike in cases of TBI, we propose that NPi abnormalities likely reflect the afferent arc of the pupillary light reflex in the case described here. Future work should include cohort studies that more thoroughly examine the predictive capacity of pupillary measurements.


To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo.Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans.We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans.Combined intact heart and cellular electrophysiology show that regions of myocardium in the  We utilized a novel approach combining intact heart electrophysiological mapping, targeted myocardial biopsies for ion channel profiling, and computational modelling to study repolarization alternans, an established pro-arrhythmic marker, in the intact human heart.We identified up-regulation of two calcium cycling proteins  as playing a major role in repolarization alternans development.This provides valuable human data to complement experimental studies in animal models, myocardial cells, and computational modelling.,Action potential duration (APD) alternans, an alternation of the APD on an every other beat basis, has long been recognized and linked to arrhythmogenesis.in vivo in order to combat the continuing high mortality due to cardiac arrhythmias. Whereas a great deal of vital information has been obtained from a range of experimental models, extrapolation from these models to the human is by no means straightforward owing to the well-known species differences in cardiomyocyte electrophysiology and species-dependent mechanisms underlying arrhythmias.in vivo approach that enables the investigation of electrophysiological properties relevant to fatal arrhythmias. Myocardial biopsies, guided by rapid on-site analysis of intact heart electrophysiological recordings, enabled us to examine cellular properties in relation to intact heart behaviour. Detailed biophysical cellular and tissue models were then used to elucidate the mechanisms that link the identified cellular properties and electrophysiological abnormalities.The mechanism underlying repolarization alternans has been the subject of much investigation, as has the mechanism by which alternans may initiate arrhythmia. An important challenge remains to advance our understanding of the physiological and pathophysiological function of the human heart Repolarization alternans was initiated by a pacing induced increase in heart rate. Electrophysiological mapping identified areas of myocardium exhibiting APD alternans interspersed with areas showing no alternans. The cellular properties were compared from myocardial biopsies and restitution properties compared using a standard pacing protocol. The role of the cellular changes observed in the alternans regions in the generation of alternans was examined using biophysical modelling. Within this novel framework, our data provide evidence for the mechanisms underlying APD alternans in the intact human heart, and implicate calcium cycling mechanisms, specifically Calsequestrin (CSQN) interactions with Ryanodine (RyR), in the mechanism underlying APD alternans.A detailed description of the methods is provided in the n = 22), aortic valve disease (n = 5), or both (n = 4). Most patients (71%) presented ischaemic cardiomyopathy with multiple vessels disease. Ten patients had a previous myocardial infarction, at least more than 12 months before enrolment. No patient had recent syncope or major arrhythmic event. Detailed patient information is shown in Table S1. The study was approved by the local ethics committee, and all subjects gave written informed consent.Thirty-one subjects  were studied undergoing routine cardiac surgery for ischaemic heart disease . Alternans was induced by incremental pacing. S1 drive trains of 30–50 beats were delivered from one of the electrodes of the multi-electrode sock at the right ventricular (RV) apex at cycle lengths decreasing from 600 ms to 350 ms. Unipolar electrograms were recorded at a sampling rate of 1 KHz and referenced to the rib retractor. Haemodynamic stability during the pacing protocol was closely monitored and pacing discontinued if appropriate. The electrograms were processed on-site and activation-recovery intervals (ARI) derived as a conventional surrogate for APD.Following cannulation for cardiopulmonary bypass, a multi-electrode sock (240 electrodes) was fitted over the epicardium of both ventricles as described previouslyBiopsies were taken from an alternans-susceptible and an alternans-resistant site in 17 patients for RNA profiling, and from a further group of seven patients for western blot analysis. In another group of seven patients, after conducting incremental pacing for inducing alternans electrical restitution properties at alternans-susceptible and alternans-resistant sites were studied using a standard S1S2 stimulation protocol.Figure 1). Action potential duration alternans was identified as being present when beat-to-beat variation of ARI exhibited an alternating pattern  for at least eight beats with a magnitude of variation equal or greater than 4 ms.Immediately following the pacing protocol, data were exported from the recording system to a PC for on-site analysis. Signal processing was performed by custom-designed algorithms. Activation time (AT) and repolarization time (RT) at each electrode site were measured from the unipolar electrograms as the time of the minimum first derivative during depolarization and the time of the maximum first derivative in both positive and negative T-waves, respectively.Additionally, for each cardiac site, spatial gradients of repolarization were measured during pacing as the absolute ARI difference between neighbouring sites divided by their distance, averaged across all neighbouring sites within a search radius of 15 mm see .1–S2 pacing protocol was conducted twice per patient, pacing adjacent to an alternans-resistant and an alternans-susceptible site, respectively. The protocol consisted of eight beat trains at basic cycle length S1–S1 = 600 ms followed by a premature test stimulus S2 with decreasing S1–S2 coupling interval. The S1–S2 interval was decremented by 50 ms steps from 550 ms to 350 ms and then by 10 ms from 330 ms until loss of ventricular capture. Activation time, RT, and ARI were carefully reviewed and corrected if needed. The diastolic interval (DI) preceding the post extra-stimulus beat was calculated as the difference between local cycle length and RT (see Methods for details), and restitution curves relating DI and post extra-stimulus ARI were created. Maximal restitution slope, α, was calculated using a piecewise linear fitting strategy by performing linear regression in sliding windows 70-ms wide and recording the line with the maximal slope.To study the interaction between APD alternans and APD restitution properties,2 snap frozen biopsy tissue using a mirVana kit (Applied Biosystems) according to the manufacturer’s standard protocol and DNase I treatment. Samples were homogenized for 1 min with an Ika T10 homogenizer (IkaWerke). The concentration of total RNA obtained from each sample was measured using a Nanodrop ND1000 spectrophotometer (Thermo Scientific).Total RNA was isolated from liquid NSingle-stranded cDNA was synthesized from 144 ng total RNA using High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems) in a 20 μL reaction according to the manufacturer’s protocol. Samples were run on a qPCRVeriti 96 well thermal cycler (Life Technologies).qPCR was performed using Taqman low density array (TLDA) microfluidic cards with 48 targets per sample (48a format) (Applied Biosystems) and QuantiTect primer assays (Qiagen) with Power SYBR green fluorescent reporter (Applied Biosystems). Details are given in the Immunohistochemical staining was performed on frozen sections and western blotting on frozen ventricular biopsy samples. Western Blots were conducted on samples from seven patients for Phospholamban and SERCA2 and from a subset of five patients for CSQN.2+ cycling by altering RyR inactivation kinetics (see Methods for details and references). This modified O’Hara–Rudy model was then taken as control for the alternans-resistant cells. For simulation of alternans-susceptible cells, the model was further modified to incorporate statistically significant changes in RNA observed in vivo associated with IK1, sarcoplasmic Ca2+ release (flux) (Jrel), Ito, the RyR, and CSQN (Table S3). It was then assumed that a difference in RNA expression between the two tissue regions would result in a corresponding change in the conductance (or maximum activity) of the corresponding ionic channel (or intracellular concentration flux). The single cell model was then incorporated into a two-dimensional ventricular tissue model, taking into consideration the electrotonic coupling between cells. In the tissue model, patches of alternans-susceptible cells were coupled to surrounding patches of alternans-resistant cells. The size of the alternans-susceptible patches were chosen based on the experimental observation, and the patch size was varied to ascertain if a minimum patch size was necessary for production of alternans.The O’Hara–Rudy model of an undiseased human ventricular cellP < 0.01 for at least two out of three housekeepers  were considered as significant.Results of qPCR analysis were divided in alternans-susceptible and alternans-resistant groups. No RNA group of data was normally distributed (Lilliefors test and one-sample Kolmogorov–Smirnov test). Differences in any single RNA between these two groups were therefore quantified with the two-sided Wilcoxon signed-rank test. In order to select the main determinants of APD alternans, only RNA with Differences in the magnitude of repolarization gradients between alternans-susceptible and resistant sites were assessed with the two-sided Wilcoxon signed-rank test.Differences in restitution properties between alternans-susceptible and alternans-resistant sites were quantified by comparing median restitution slope α at alternans-susceptible and alternans-resistant sites with the two-sided paired Wilcox signed-rank test. Differences were assessed by pooling data from the two restitution protocols per patient together and the Holm–Bonferroni correction was used to reduce the probability of Type I errors.1) and third (Q3) quartiles and the Whiskers extend to the most extreme data points not considered outliers. Values lower than Q1 − 1.5 × (Q3 − Q1) and higher than Q3 + 1.5 × (Q3 − Q1) are considered outliers.Standard box-plots were used to describe data distribution, where central line is the median, the edges of the box are the first . There was a trend for the number of sites exhibiting alternans as well as for the magnitude of alternans relative to the mean ARI to increase at shorter cycle lengths . Furthermore, the mean number of alternans-susceptible sites and the average magnitude of alternans relative to the mean ARI was higher for cycle length ≤450 ms than for cycle length ≥500 ms . The prevalence of APD alternans presented a border-line significant trend to increase with shorter APD , as differences in the number of alternans susceptible-sites within ARI quintiles was significant for cycle lengths ≥500 ms  and elevated but non-significant for cycle lengths ≤450 ms . No significant differences were found in the prevalence of APD alternans within quintiles of AT  demonstrating that sites activating early showed similar probability of developing APD alternans than sites activating late. No left ventricle/right ventricle, base/apex, or anterior/posterior differences were found in the number of alternans-susceptible sites or in the alternans and gradients magnitudes.Representative examples of activation and repolarization maps as well as of epicardial recordings exhibiting repolarization alternans are shown in Figure S8).No differences were observed in the prevalence of repolarization alternans between patients with and without reduced LV ejection fraction or between patients with and without previous myocardial infarction . The mean spatial gradients were significantly larger in alternans-susceptible  than in alternans-resistant sites , confirming a potentially pro-arrhythmic interaction between APD alternans and spatial heterogeneity of repolarization.The magnitude of spatial gradients of repolarization at alternans-susceptible and resistant sites was compared , indicating that APD restitution slope higher than one was prevalent. There was no difference either in the steepness of the restitution curves  or in the number of curves with maximum slope α >1 between alternans-resistant and alternans-susceptible sites  with α equal to 1.28 (0.84–1.41) vs. 1.20 (0.88–1.48), P = 0.47, [median (Q1–Q3)], respectively, and the proportion of sites showing α >1 equal to 88% (19–100%) vs. 71% (31–100%), P = 0.81, respectively.Total RNA was isolated from pairs of alternans-susceptible and alternans-resistant biopsies from the left ventricle of 17 patients. Biopsies from four patients were excluded from the analysis because of insufficient signal quality in at least one of the two sites.Biopsies were equally distributed between apical and basal regions, indicating that the results reported below stem from an alternans susceptible/resistant difference and not intrinsic base/apex differences.2+ handling proteins RyR and CSQN as well as in Kir2.1 and Kv4.3, whose expression was higher in alternans-susceptible than in alternans-resistant samples . These differences were highly significant and consistently maintained over different housekeepers (Table S2). The median relative increase in the alternans-susceptible sample with respect to the alternans-resistant one was always higher than 50%. RyR is the Ca2+ release channel of the sarcoplasmic reticulum (SR), whereas CSQN is an SR Ca2+ binding protein. Kv4.3 is a voltage-gated channel involved in the regulation of the transient outward K+ current, and Kir2.1 is the principal inward-rectifier K+ channel responsible for the inward-rectifier K+ current.The most significant changes were observed in the CaFigure S9), but the number of samples analysed (N = 5) was insufficient to reach statistical significance . No significant differences were found in phospholamban (P = 0.38) and SERCA2 (P = 0.22) protein expression.Western blot analysis was performed for CSQN, SERCA2, and phospholamban. All samples from alternans-susceptible sites had higher CSQN expression than samples from alternans-resistant sites (Table S2). This suggests that the amount of extracellular matrix and the degree of any fibrosis in the alternans-susceptible and alternans-resistant regions was similar. This was confirmed by histology: tissue sections through alternans-susceptible and alternans-resistant biopsies were stained with Masson’s trichrome and picrosirius red and there was no difference in the amount of extracellular matrix in the two .Expression of RNAs for some extracellular matrix components  was measured, but no significant differences were observed  may account for the production of alternans, as modification of these two substrates alone produces alternans of the same magnitude as the full modification condition . Greater CSQN expression is determined to be the critical factor in producing alternans, with an increase in Jrel enhancing the susceptibility to alternans in combination with CSQN increase, but insufficient to produce alternans on its own . Ionic current modifications accounted for the difference in APD observed between the full modification and modified CSQN-RyR only conditions , but did not significantly affect alternans.In single cell simulations, implementation of all four of the observed changes in RNA expression associated with alternans-susceptible tissue resulted in alternans across a range of cycle lengths, between 280 ms and 480 ms. on Figure.A2. Greans Figure.A2, but 2+ alternans . As a result, electrotonic interactions did not significantly affect the vulnerability to alternans, and in tissue simulations, significant alternans was observed throughout the patch of alternans-susceptible tissue, uninhibited by the absence of alternans in the surrounding alternans-resistant tissue . Alternans was not significantly influenced by the size of the alternans-susceptible tissue patch.Observed APD alternans were driven by secondary effects on ionic current kinetics resulting from intracellular Cans Figure.B1. As aFigure 8). Greater abundance of SR Ca2+ release mediated by the RyR causes greater SR Ca2+ depletion during excitation, resulting in a larger fraction of CSQN monomers, and a prolonged RyR recovery period. Greater expression of CSQN results in a greater number of CSQN monomers at low [Ca2+]jsr. Thus during Ca2+ influx the time taken for polymerization of CSQN is longer with the concomitant increase in the refractory period of the RyR. At shorter cycle lengths this incomplete recovery from inactivation of the RyR leads to incomplete Ca2+ release from the jSR. Both of these effects combined to promote established mechanisms of intracellular Ca2+ alternansFigure 8).Greater expression of both CSQN and RyR plays a synergistic role in alternans production (in vivo. The main findings were (i) following an incremental pacing protocol electrophysiological mapping revealed a patchy distribution of alternans with areas of alternans interspersed with areas showing no alternans. (ii) The myocardial tissue collected in vivo demonstrated significantly greater expression in the calcium handling RNA transcripts CSQN and RyR in alternans-susceptible as compared to alternans-resistant sites, and CSQN protein expression of a limited number of separate samples showed a parallel trend. (iii) The biophysical model utilising the RNA expression data predicted alternans of similar magnitude and in the same range of cycle lengths observed in vivo, principally determined by local greater abundance of CSQN and RyR. (iv) Electrical restitution properties (i.e. steepness APD restitution slopes >1) as well as the degree of histological fibrosis between alternans-susceptible and resistant sites were not different, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites.Integrating human intact heart mapping with targeted ion channel profiling provided insights into mechanisms of repolarization alternans in the human heart Figures S1–S4) was consistent with previous detailed studies on Langendorff perfused human hearts.Overall, the distribution of AT and RT  suggesting that this mechanism is unlikely to be of prime importance. This is in agreement with studies in humans showing no relation between T-wave alternans in ECG and APD restitution slope in LV and RV endocardial sites,in silico,in vivo, which coincided with the plateau rather than the steep portion of the restitution curve.Theoretical studies and experimental models have suggested the existence of a causal link between cardiac restitution properties and repolarization alternans.2+ release to whole cell Ca2+ alternans referred to as 3R theory.2+ proteins, RyR, SERCA, NCX L type Ca2+ channels and mitochondria, and Ca2+ load interact with the parameters in the model and the initiation of APD alternans, one of the key components being RyR refractoriness. A recent study using optical mapping of SR Ca2+ in Langendorff rabbit hearts also identified RyR refractoriness as a major mechanism in alternans.in vivo mapping, RNA expression and in silico modelling data predict that APD alternans is partially due to the greater expression of CSQN and RyR and the modulatory role of CSQN on RyR activation kinetics , i.e. refractoriness, and are consistent with these studies.,2+ alternans and repolarization alternans at the tissue level. Furthermore, elegant animal studies have demonstrated that pharmacological restoration of RyR function reduces alternans susceptibility. For instance, Zamiri et al.Previous studies have linked repolarization alternans with calcium handling proteins. For example, a theoretical model has been advanced which relates the properties of local intracellular Ca2+-binding protein that can store Ca2+ within the SR. Each molecule of CSQN can bind 18–50 Ca2+ ions. CSQN is also believed to regulate the activity of RyR Ca2+ release channels by controlling the local luminal Ca2+ concentration in the vicinity of the RyR channels.2+ alternans have also been described in adenoviral gene transfection experiments promoting cellular up-regulation of CSQN in the endoplasmic reticulum and perinuclear regions of cardiomyocytes.2+ uptake and release, the modulation of RyR kinetics mediated by CSQN can also play a role in generating alternans.CSQN is a low-affinity, high-capacity Ca2+ handling proteins and a number of technical challenges. Although biopsies were collected transmurally, whereas electrograms were recorded epicardially, we took care to identify the subepicardial portion of the biopsy at the time of taking it, and only this portion was used for the laboratory analysis. Although the dynamic restitution protocol has been shown to better correlate with APD alternans than the standard S1–S2 protocol performed in this study,,This study presents limitations. The results are based on an heterogeneous population and epicardial mapping and did not include transmural conduction and repolarization dynamics, as simultaneous high-density mapping of epicardial, endocardial, and transmural extracellular potentials is currently not feasible in the context of cardiac surgery. We have primarily focused on transcript data, with borderline supportive protein analysis for CSQN from a limited number of samples, as there are only limited antibodies available to study CaOur data obtained by combining epicardial intact heart electrophysiology, ion channel expression, and computational modelling in human implicate calcium cycling mechanisms, specifically CSQN interactions with RyR, in the mechanism underlying APD alternans. We were unable to find evidence for a role of APD restitution and documented an interaction between repolarization alternans and spatial gradients. These results add human data to experimental models in the search for therapeutic targets to prevent alternans and sudden cardiac death.euz007_Supplementary_MaterialClick here for additional data file.


Cystoid macular oedema (CMO) is one of the most frequent postoperative macular complications to cause partial visual recovery after successful retinal detachment (RD) repair. Refractory CMO is difficult to treat and many strategies have been employed with varying degrees of success. We report for the first time the use of ILUVIEN implant to treat refractory CMO after successful RD repair. A 65-year-old female presented with right eye full-thickness macular hole and underwent pars plana vitrectomy, internal limiting membrane peeling and cryotherapy with gas tamponade with 12% C3F8. She subsequently developed right eye macula-on RD and proliferative vitreoretinopathy and required multiple procedures for successful retinal reattachment. Later, she developed CMO that responded to intravitreal triamcinolone injections and intravitreal dexamethasone 0.7-mg implants but recurrence of CMO continued to be a problem. After receiving ILUVIEN intravitreal implant, her visual acuity improved and CMO resolved without recurrence for 13 months. Refractory CMO after RD repair is difficult to treat and in a quarter of cases will not improve without treatment. Our case shows that a single ILUVIEN implant maintained anatomical dry fovea and improved vision. This also demonstrates that ILUVIEN is an effective management strategy to reduce the need for repeated treatments. The most important preoperative factors are visual acuity (VA) and the duration of the RD. Cystoid macular oedema (CMO) and epiretinal membranes are the main postoperative factors and CMO appears to be the most frequent postoperative macular complication to cause partial visual recovery after successful RD repair.3 Spontaneous resolution of CMO within 2 years postoperatively has been reported in up to 76% of cases.4 Many strategies have been employed to manage CMO after RD surgery, with varying degrees of success. Different anti-inflammatory medications have been used, including non-steroidal anti-inflammatory medications and topical, periocular and intravitreal corticosteroids.6The exact aetiology of CMO after RD repair is unclear but inflammation is thought to be an important mechanism.7ILUVIEN implant  is a sustained-release intravitreal steroid lasting up to 36 months that has been approved in the UK to treat chronic refractory CMO in pseudophakic eyes unresponsive to available therapies.We report for the first time the use of ILUVIEN implant to treat highly refractory CMO after successful RD repair and the outcomes of 20-month follow-up period after ILUVIEN implant.A 65-year-old female presented to our tertiary eye centre with 6 weeks’ history of painless right eye vision distortion and no history of eye injury or trauma. On examination, VA was 6/24 in the right eye and 6/5 in the left eye. Following slit-lamp biomicroscopy and fundoscopy a diagnosis of right eye full-thickness macular hole was made and optical coherence tomography showed right eye cuff of subretinal fluid, left eye epiretinal membrane and posterior vitreous detachment. Ten years before presentation she had uneventful bilateral phacoemulsification and intraocular lens implantation with no other significant past ocular or medical history.Eight weeks later, she underwent right eye pars plana vitrectomy, internal limiting membrane peeling and cryotherapy with C3F8 12% gas tamponade. Two weeks postoperatively, she exhibited a flat retina, closed macular hole and her VA had improved to 6/18 with normal intraocular pressure (IOP). Unfortunately, 7 weeks postoperatively, she developed right eye macula-on RD due to proliferative vitreoretinopathy (PVR) in the inferior retina. RD repair was done within 3 days with silicone oil (Densiron 68) tamponade and retinectomy to release the PVR. After 4 months, VA of the right eye after removal of silicone oil was 6/12 with flat retina and closed macular hole. Four months later, her VA declined to 6/36 in the right eye and remained 6/5 in the left eye and fundus fluorescein angiogram confirmed severe right eye CMO. She underwent right eye posterior sub-Tenon’s triamcinolone injection and was started on ketorolac trometamol eye drops (Acular) 3 times/day and oral acetazolamide 250 mg slow-release 2 times/day. Treatment of the CMO during the follow-up period is summarised in Table 1. She received 3 posterior sub-Tenon’s triamcinolone injections and 2 intravitreal triamcinolone injections within 14 months with no complications. The CMO initially responded to each triamcinolone injection but later recurred . The patient then received 4 intravitreal dexamethasone 0.7-mg implants  uneventfully within 15 months, which maintained a dry fovea for a longer period but the CMO recurred again . She alsHer refractory CMO persisted after 2 years without treatment. Finally, she received ILUVIEN intravitreal implant. In the first week she developed mild right eye anterior uveitis; IOP was 27 mmHg in the right eye and 18 mmHg in the left eye. These markedly regressed within a week on dexamethasone drops and latanoprost drops and topical medications were stopped within 4 weeks. At the time of this report, it is 20 months since receiving the ILUVIEN implant and she still has a dry fovea with right eye VA of 6/18 .Refractory CMO after RD repair is difficult to treat and in a quarter of cases it will not improve without treatment. Intravitreal corticosteroid injections have shown to be an effective treatment option. We are not aware of any published literature in which ILUVIEN was used to treat this condition. This approach not only maintained an anatomical dry fovea but also provided visual improvement with a single ILUVIEN implant. This also demonstrates that ILUVIEN is an effective management strategy to reduce the need for repeated treatments. There is a risk of IOP elevation after receiving ILUVIEN intravitreal implant but in our case IOP was well controlled with short-term treatment. Further investigation of more cases with longer follow-up is needed. Better understanding of the exact aetiology of CMO after RD repair will lead to the development of more targeted treatment options.


Atypical antipsychotic medications such as risperidone are widely prescribed for diverse psychiatric indications including schizophrenia, bipolar disorder and depression. These medications have complex pharmacology and are associated with significant endocrine and metabolic side effects. This class of medications also carries FDA black box warnings due to increased risk of death in elderly patients. Clinical reports indicate that patients treated with these medications are more susceptible to infections; however, the underlying mechanisms/pharmacology are unclear. We have previously reported that risperidone and it’s active metabolite distributes to the bone marrow in clinically relevant concentrations in preclinical species, leading us to hypothesize that the hematopoietic system may be impacted by these medications. To test this hypothesis, using proteomic and cytokine array technology, we evaluated the expression of genes involved in inflammatory and immune function following short term (5 days) and longer term (4 weeks) treatment in healthy animals. We report that low-dose risperidone treatment results in global immunosuppression in mice, observed following 5 days of dosing and exacerbated with longer term drug treatment (4 weeks). These data are consistent with increased susceptibility to infection in patients administered these medications and have profound implications for the increasing off-label prescribing to vulnerable patient populations including children and the elderly. Antipsychotic medications are FDA approved for treatment of psychosis associated with schizophrenia and bipolar disorder as well as severe depression and autism-related irritability. These medications have complex pharmacology, antagonizing myriad G protein coupled receptors (GPCRs) including dopamine D2, serotonin 5HT2, alpha adrenergic, histaminergic and muscarinic receptors. It is thought that antipsychotic efficacy is linked primarily to central antagonism of dopamine D2 and 5HT2c receptors –2. Over per se, on the increased susceptibility to infections in this population has not been clearly delineated. Off-label antipsychotic use in elderly patients is associated with a higher incidence of urinary tract infections ). Significant differences between groups for each immune function marker were determined by two-tailed T test using GraphPad Prism. Responses were categorized as follows: acute onset/transient effects (those present at 5 days plus 1 hour of dosing only), early onset/transient effects (those present at 5 days plus 3 hours of dosing only), early onset/persistent effects (those present at 5 days plus 3 hours of dosing and 4 weeks of dosing), late onset effects (those present at 4 weeks of dosing), or chronic effects (early onset/persistent effects plus late onset effects).Serum concentrations of forty immune markers  were measured in mice treated with RIS or VEH for 5 days or 4 weeks using the Proteome Profiler Mouse Cytokine Array  according to the manufacturer’s procedures. Arrays were imaged using a FluorChem Q instrument and quantified using ImageQuant v. 8.2. Each duplicate array spot representing a unique immune marker was normalized to each of 3 reference spots per array (N = 3 measurements per spot). Normalized measurements were averaged for each spot, and the duplicate spots for each feature were then averaged to generate a total measure for each immune marker. Unique arrays were run for each animal  in order to qualitatively assess changes in pathology. Two sections from each animal were examined via brightfield microscopy at 4x, 10x, and 40x magnifications using a Keyence BZ-X710 inverted widefield digital microscope. Images were collected using BZ-X analyzer software.i.e., that of VEH-treated mice) were tabulated in VEH and RIS treatment groups at each effect stage . Chronic exposure values reflect the total cytokines that were altered at both 5 days and 4 weeks. Heat maps reflecting the total altered immune factors for each pathway at each effect stage were generated. Each immune factor present at significantly higher levels in one group was allotted proportional wavelength in their assigned color on a scale from 55 nm to 255 nm  . Multiple factors present at significantly higher levels contribute additively to color intensity. Absence of change between groups in immune mediator levels in an effect category are colored black (λ = 0 nm).Entries for all measured cytokines were accessed from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Database . Pathway2, 10% glycerol, 1% Triton‐X 100 1mM EDTA, protease‐inhibitor cocktail . Protein concentration of the supernatant was determined by BCA (pierce). 40 μg of protein was used from each sample. Tryptic digests of protein samples were performed using the ProteoExtract digestion kit . Tryptic peptides were then separated on a Ultimate RSLC system 3000 (ThermoFisher/Dionex) nanoscale liquid chromatograph and infused onto a 5600 TripleTOF mass spectrometer (Sciex). Sequential window acquisition of all theoretical spectra (SWATH) was used to profile all proteins in each sample using a data-independent acquisition method. A human-specific ion library comprising 4091 proteins was constructed using ProteinPilot software (Sciex). For identification of peptides, multiple fragment ion chromatograms were retrieved from the spectral library for each peptide of interest. These spectra were compared with the extracted fragment ion traces for the corresponding isolation window to identify the transitions that best identify the target peptide. SWATH analysis was performed using PeakView software, and MarkerView software was utilized for principal component analysis and T-test comparisons [A proteomic analysis of heart tissue was performed as part of a larger study focused on side effects of antipsychotic drugs. Whole hearts and portions of liver from RIS-treated (N = 5) and VEH-treated (N = 4) male mice were homogenized in HTNG lysis buffer  distributed to marrow of male mice following 5 days of dosing, and concentrations were higher in marrow than in plasma at 1 and 3 hours post-dose  in the heart during RIS treatment relative to VEH-treated controls after 4 weeks  are consistent with those seen in natural thymic involution and atrophy due to aging [Histopathologic changes observed within the thymus  and spleto aging ; howeverto aging . Immune e.g., TNF-α) in RIS-treated mice. It is notable that many of the clinical features of these diseases such as insulin resistance and dyslipidemia are known side effects of long-term antipsychotic use [Staphylococcus aureus infection, all of which are significant causes of morbidity and mortality in nursing home residents [The effects observed in the current study are seen in a pre-clinical model using unchallenged animals without a confounding disorder that impacts immune signalling. Impacts on immune function by RIS treatment seen here are predictive of impacts on non-schizophrenic patients who are prescribed RIS off-label. Interactions between immune function pathways and those describing the pathophysiology of chronic diseases such as type II diabetes mellitus and nonalcoholic fatty liver disease are likely to be similarly impacted by the observed reduction of certain inflammatory mediators  and Liver (Table B) Following RIS Treatment (4 Weeks).(DOCX)Click here for additional data file.


Citrus sinensis L. Osbeck) and its mutant fruit Pinalate, which presents a severe blockage at early steps of the carotenoid biosynthetic pathway and consequently reduced ABA levels. Peel ripening involved the decrease of the photosynthetic activity and the transmembrane transport processes, as well as the buildup of starch and cuticular waxes and the cell wall modification. In addition, a number of biotic and abiotic stress responses, including the defense response, and the response to blue light, water deprivation and abscisic acid stimulus were modulated in a ripening-stage specific manner. The regulation of energy-related processes and secondary metabolism pathways was attenuated in Pinalate, while the molecular mechanisms underlying stress responses displayed dependency on ABA levels. These results indicate that ABA is a key signal inducing stress responses along orange peel ripening, which might determine the fruit postharvest performance.Citrus fruit ripening is a complex process involving biochemical, physiological and molecular events that differ between the flesh and the peel of the fruit. We characterized sweet orange peel maturation by means of a comparative transcriptomic analysis between Navelate orange ( Citrus are of main relevance worldwide because they are grown in more than 130 countries, covering more than 9.5 million ha harvested area and with an estimated production over 125 million Tm per year .Fruits from the genus 3. Among these changes, the main affected processes include the increase in sugars and other soluble solids, and the reduction of organic acids2. Peel pigmentation develops as a result of chlorophyll degradation and activation of carotenoid biosynthesis, while the increase of specific volatile compounds, and an exhaustive modification of the cell wall metabolism occurs5. However, defining the criteria to uniformly determine citrus fruit maturation may not be simple because physiological changes and metabolic processes occurring in peel and pulp during maturation are autonomous and independent, mainly due to the lack of vascular connections between both tissues6. In spite of flavedo (outer colored cell layers of the fruit peel) is the first tissue of citrus fruit interacting with the environment and the first protection barrier of the fruit against biotic and abiotic stresses, most of the studies have been focused on the molecular processes underlying flesh maturation, and a limited number of reports investigated the molecular changes occurring along peel ripening, being most of them centered on changes in pigmentation12.Biochemical and physiological events involved in citrus fruit development and ripening have been extensively studied for decades. Citrus fruit growth and development follows three stages: first, a cell division phase; then, a cell expansion stage of major increase in size and weight; and the third phase during which fruit growth bottoms down and most of the characteristic events of fruit maturation or ripening takes place13, reports pointing to abscisic acid (ABA) as a major regulator of non-climacteric fruit ripening are accumulating in last years. ABA biosynthesis and the expression of the components of the ABA-signalosome have correlated with the progress of fruit ripening in strawberry, grape and sweet orange, among others17. In the peel of citrus fruits, the ABA content increases during maturation and the expression of the ABA perception system components is dependent on the hormone accumulation profile in this tissue21. It has been also demonstrated that alteration of the ABA perception system modifies sugar accumulation and sugar-responsive genes along strawberry fruit ripening15. In contrast, ABA regulated anthocyanin accumulation and cell wall modification in bilberry, but sugars appeared to play a less relevant role modulating ripening-related processes in this fruit22. In addition, Koyama et al.14 found that exogenous ABA application induced pigmentation in the skin of Cabernet Sauvignon grape berries stimulating anthocyanin and flavonols biosynthesis. In citrus fruit conflicting information has been reported regarding ABA role on peel fruit pigmentation. Rehman et al.23 showed that pre-harvest ABA spray regulates fruit color development in Navel orange, whose pigmentation is due to carotenoids, suggesting the involvement of ABA in the regulation of ripening-related processes. However, in Valencia sweet oranges the ABA level in flavedo increases as color develops but ABA did not trigger color break24. On the other hand, it has been suggested that ABA would enhance tissue sensitivity to ethylene, whereas exogenous ethylene application stimulates ABA accumulation and expression of the corresponding biosynthesis genes26, indicating that ABA takes part of a complex hormonal interaction in the regulation of non-climacteric fruit maturation.Citrus are defined as non-climacteric fruit, since respiration rate declines progressively during fruit development and full ripen fruit produce a very low and constant amount of ethylene. In contrast to the well-known role of ethylene controlling most of the maturation processes in climacteric fruits32. In citrus, spontaneous bud mutants occur frequently and are a valuable source of variability of important phenotypic traits. These mutants provide a promising platform to investigate the regulatory network of citrus fruit development and ripening, as demonstrated by comparative studies of spontaneous late-ripening and stay-green mutants with their parental cultivars36. In the context of this study, it has been described a spontaneous fruit-specific mutant, named Pinalate, derived from Navelate sweet orange37. Pinalate has a fruit-specific biochemical blockage upstream of the carotenoid biosynthetic pathway, at the level of ζ-carotene desaturation, resulting in fully ripe yellow-pigmented fruit19 . Moreover, the level of the β,β-xanthophyll 9-Z-violaxanthin, which is the main carotenoid and the precursor of ABA in ripe sweet orange fruit, is highly reduced in Pinalate fruit tissues38. Consequently, Pinalate presents reduced ABA levels in the peel along maturation as compared to its parental Navelate. In contrast to mutants presenting delayed fruit ripening, Pinalate fruit reach the same maturity index (TSS/acidity) than its parental Navelate at the end of the harvesting season. Therefore, Pinalate represents an exceptional experimental system to understand the molecular events governing peel ripening and how alteration in carotenoid biosynthesis resulting in reduced ABA levels may interfere in their regulation. In this work, special attention has been paid on how ABA might regulate stress responses along fruit maturation, even before fruit exposure to the stress.Comparative transcriptomic analyses between genotypes with differential performance along maturation or responsiveness to ABA applications have proven to be efficient approaches to unravel the molecular mechanisms underlying developmental events in citrus fruitsPhysiological and biochemical data suggest that Breaker (Bk) stage is a turning point in the regulation of ordinary sweet orange ripening not only because of the peel color change but also because the sharp increase in ABA content and the later increase in maturity index . Differences between genotypes were lower at FC and MG stages but remarkable at Bk stage . Accordingly, HCA revealed that samples were grouped in a ripening stage- and genotype-dependent manner  and Hierarchical Cluster Analyses (HCA) were performed to validate the repeatability of the microarray data across replications and to cluster samples according to their global gene expression profiles. Under all conditions, the transcriptional profiles of the three separate RNA replicate samples were tightly clustered together Fig. . The relner Fig. . The genner Fig. . Heatmapner Fig. . Some clner Fig. . At FC sner Fig. .Functional categorization analysis on the DEG identified biological processes, molecular functions, cellular components and KEGG pathways significantly (P < 0.05) under- or over-represented along peel maturation in the parental and its mutant. Gene ontology analyses revealed that the high number of DEG in Navelate and Pinalate Bk fruit were only enriched in the chlorophyll binding molecular function and in the fructose and mannose metabolic pathway, both repressed in either genotype Table . The numTo further uncover biological processes related to peel maturation and how Pinalate mutation might interfere in their regulation, we performed a more restrictive ontology analysis, considering only those DEG responsible for the separation of genotypes and ripening stages observed in the multivariate analyses. The 1116 genes satisfying the ANOVA test (P < 0.001) were enriched in a high number of biological processes that might be classified into two different groups. The first group would include biological processes related to primary and secondary metabolism and those involved in the redox potential and energy generation. This group is composed of the lignin, wax, and starch biosynthetic processes and of the transmembrane and photosynthetic electron transport categories. Secondly, we identified 12 different biological processes that may be grouped as biotic and abiotic stress responses , CUMARATE-3-HYDROXYLASE (C3H), CINNAMOYL-COA REDUCTASE (CCR2), O-METHYLTRANSFERASE1 (OMT1) and FERULIC ACID 5-HYDROXYLASE1 (F5H) genes. Only minor differences between genotypes were found among the expression values of these genes. In fact, OMT1 and F5H, acting downstream in the pathway, were repressed in both cultivars and no significant differences were observed when compared Navelate and Pinalate FC fruit , 3-KETOACYL-COA SYNTHETASE6 (KCS6), 3-KETOACYL-COA SYNTHETASE1 (KCS1) and ECERIFERUM1 (CER1) genes identified in this category, only KCS1, which participates in the catalysis of several steps for the formation of very long chain fatty acids, showed opposite regulation between genotypes and a two-fold induction in Navelate compared to Pinalate FC fruit  and starch branching  activities, involved in the production of ADP-alpha-glucose and in the formation of branches in amylopectin and alpha-amylose, respectively, were induced in the parental but not in the mutant FC fruit , the delta subunit of the ATP synthase (ATPD) and two proton pumps  involved in the electron flow in the photosystem I (Table MULTI-ANTIMICROBIAL EXTRUSION (MATE), ABC TRANSPORTER39 (ABCG39) or VACUOLAR ATP SYNTHASE F and H  genes, and showed higher repression values than its parental for the ABC TRANSPORTER32 (ABCG32) and a PUTATIVE SUGAR TRANSPORTER 1 (ESL1) , DEOXYXYLULOSE 5-PHOSPHATE SYNTHASE2 (DXS2) and GERANYLGERANYL PYROPHOSPHATE SYNTHASE1 (GGPPS1), and to the carotenoid metabolism such as PHYTOENE SYNTHASE (PSY) and PHYTOENE DEHYDROGENASE (PDS), genes related to wax and cutin biosynthesis such as LACS2, KCS6, 3-KETOACYL-COA SYNTHETASE10 (KCS10) and SQUALENE SYNTHASE1 (SQS1), genes encoding terpene synthases involved in the generation of volatile compounds , and genes involved in the catabolism of fatty acids to generate jasmonic acid such as ALLENE OXIDASE CYCLASE4 (AOC4), ALLENE OXIDASE SYNTHASE (AOS), JASMONIC ACID CARBOXYL METHYLTRANSFERASE (JMT), FATTY ACID OXYGENATION UPREGULATED2 (FOU2) and LIPOXYGENASE1 and 2 (LOX1 and LOX2).Taking into account the partially overlapping results obtained separately after the two functional categorization analyses performed in this work Tables  and 2, wge Table , althougThe response to biotic and abiotic stresses group included 12 different biological processes Table . NotablyCYP707A1), components of the ABA-signalosome , several transcription factors  and a number of loci described as ABA-responsive genes in the Arabidopsis model plant   and conducted their expression analysis by qRT-PCR on Navelate and Pinalate fruit harvested at 6 ripening stages in order to investigate how Pinalate mutation affects their regulation along fruit maturation  described as ABA-independent and belonging to the response to water deprivation biological process  and about a 10-fold in the mutant FC fruit (from 0.2 to 1.93 μg/g fresh weight), which means that ABA was efficiently uptaken by the fruit and similar levels of the hormone were reached in fruit of both cultivars upon 1 week of treatment. Consistently with data reported in Fig. Citrus sinensis L. Osbeck) displaying a blockage upstream of the carotenoid biosynthetic pathway and reduced ABA levels, to perform transcriptomic comparisons at different peel maturation stages. Under our experimental conditions, we confirmed the suitability of Pinalate for this study based on its reduced ABA accumulation in the flavedo along fruit ripening  and the highest accumulation of ABA in the flavedo of parental fruit was found at Bk stage, showing more than 4-fold ABA levels than Pinalate  provokes a massive accumulation of colorless carotenes (phytoene and phytofluene) in chromoplasts of the mutant and the presence of atypical suborganellar structures38 which may result in altered plastid organization and enhanced repression of all those photosynthesis-related functional categories as compared to its parental fruit  in the flavedo42, named CaraCara, does not display an altered developmental phenotype although, in this case, ABA levels in the flavedo are very similar between the mutant and the parental fruit, and molecular analyses on this mutant along fruit maturation have been limited to pulp tissues43. In tomato fruit, VIGS mutants with altered ZDS (ζ-carotene desaturase) or Z-ISO (ζ-carotene isomerase) activities causing a similar chemotype to Pinalate in terms of carotenoids accumulation indicate that no other trait but color and carotenoid accumulation are affected44. Therefore, we cannot rule out the possibility that carotene-derived signaling molecules might participate somehow in the regulation of the ripening process, but present results still support the hypothesis that ABA might also play a role controlling photosynthesis, as previously suggested by Farnsworth45. In line with this, there is long compelling evidence that the biosynthesis of flavonoids, specifically repressed in Pinalate FC fruit , participating in the influx of substances through ATP consumption, were induced in Navelate but not in the mutant FC fruit. Also, the repression of a putative sugar transporter (ESL1) that imports glucose to the cytosol was enhanced in Pinalate mutant FC fruit  was abolished in the Pinalate mutant . However, since our GO analyses did not reveal significant lipid metabolism-related processes at Bk stage either in Navelate or Pinalate fruit, we selected the FC fruit for the global transcriptional analyses comparing both genotypes  orange and its spontaneous fruit-specific Pinalate mutant were randomly harvested from adult trees grown in experimental orchards under normal cultural practices at Citrus Germoplasm Bank at Instituto Valenciano de Investigaciones Agrarias . Both parental and mutant trees are subjected to the same environmental conditions and agronomical practices. Fruit from each cultivar were harvested at different ripening stages: Immature Green ), Mature Green , Mature Green 2 , Breaker , Colored  and Full Colored , and immediately delivered to the laboratory. Fruit maturity index was calculated by dividing the °Brix of the extracted juice by its acid content. Peel color was expressed as the a/b Hunter ratio and analyzed by using a Minolta CR-300 Chromameter  at three locations around the equatorial plane of the fruit. For the ABA treatment assay, fruits from Navelate and Pinalate cultivars were harvested at FC stage and divided into two groups. The first group was treated with ABA (Sigma-Aldrich) by dipping the fruits for 1 min in an aqueous solution of 1 mM ABA containing 0.7% ethanol to dissolve the hormone. Control non-treated fruits (second group) were dipped into 0.7% ethanol following the same procedure. Fruits were dried at room temperature and stored for one week in chambers simulating temperature field conditions at this time of the season (12 °C) and avoiding dehydration stress (90–95% relative humidity). Flavedo samples for both the ripening and the ABA treatment assays were collected from the total surface of fruit, frozen and homogenized in liquid nitrogen, and kept at −80 °C until analysis. Three biological replicates of at least 5 fruit each were collected at each sampling period.Fruits of Navelate  following the manufacturer’s instructions for removing possible genomic DNA contaminations. For microarray purposes, total RNA (30 μg) samples from each biological replicate were labeled with the indirect method by incorporation of 5--2-deoxi-UTP into single-stranded cDNA during reverse transcription. cDNA synthesis and purification, dye coupling, and labeled-cDNA purification were accomplished according to the method described by Forment et al.72. cDNA samples were Cy5-labeled and co-hybridized with a Cy3-labeled cDNA reference pool from a mixture containing equal amounts of RNA from all experimental samples assayed. Microarray hybridization and slide washes were performed as described by Romero et al.28. The cDNA microarrays used were developed in the framework of the Spanish ‘Citrus Functional Genomics Project’ (http://bioinfo.ibmcp.upv.es/genomics/cfgpDB/), and contained 21081 putative unigenes (20 K) isolated from 52 cDNA libraries of citrus from a wide range of varieties, developmental and fruit ripening stages, and from different tissues subjected to biotic and abiotic stress conditions73, covering more than two thirds of the Citrus genome.Total RNA was extracted from frozen flavedo samples as described in Romero http://www.applied-maths.com). ANOVA test (Benjamini-Hochberg FDR < 0.001) and Pearson’s product-moment correlation coefficient were used to identify DEG and to measure gene-to-gene correlation, respectively. FatiGO+ tool  was used to identify biological processes, molecular function, cellular components and KEGG metabolic pathways significantly under- or over-represented in a set of DEG relative to a reference group containing all genes present in the microarrays. In these analyses, a Fisher two tailed test with a p-value lower than 0.05 was applied and the specificity of the process increases with the GO level from 3 to 10.Hybridized microarrays were scanned by using a GenePix 4000A scanner (Axon Instruments) equipped with GenePix Pro 6.0 image acquisition software, following manufacturer’s instructions to adjust the channels intensity ratio to 1.0 and the percentage of saturated spots close to 1%. Non-homogeneous and aberrant spots were discarded. Only spots with background–subtracted intensity greater than 2-fold the mean of background intensity were used for normalization and further analysis. In order to compensate labeling differences among samples and other non-biological sources of variability, results were normalized by using Print-Tip-Lowess method, included in the Acuity 4.0 software (Axon Instruments) by using background subtracted median values and an intensity-based Lowess function within and among microarrays. Thereafter, differentially expressed genes for all possible pairwise comparisons were determined by applying the Significant Analysis of Microarrays (SAM) program from the TM4 Microarray Software Suite. Genes that satisfied a statistical threshold  lower than 0.01 were identified as differentially expressed genes (DEG) and represented in Venn diagrams. Principal Component (PCA) and Hierarchical Cluster (HCA) Analyses, and the associated expression Heatmap were performed by using the GeneMaths XT software package  and reverse (R) sequences for specific primers are shown in Supplementary Table CsACT, CsGAPDH and CsTUB) were used for data normalization. Statistical analysis was carried out by using the Relative Expression Software Tool . Each sample was analyzed in triplicate and mean ratios ± SE were calculated.Reverse transcription followed by quantitative polymerase chain reaction analysis was performed to validate microarray results and to examine the time-course expression pattern of selected genes along fruit ripening or in response to ABA treatment as described by Romero −1 citric acid and 100 mg L−1 of butylated hydroxytoluene as previously described by Lafuente et al.18. After centrifugation the supernatant was diluted in 3 serial dilutions in ice-cold TBS  and 3 samples for each dilution were analyzed by indirect ELISA. The results are the means of three replicate samples ± SE.The ABA was extracted from 1 g fresh weight frozen flavedo with 80% acetone containing 0.5 g LAll values are given as the mean of the three replicate samples ± SE, and a mean comparison using the Tukey’s test was performed to determine significant differences (P ≤ 0.05). The analyses were performed with the Statgraphics Plus 4.0 Software .Supplementary Information