Drought and flooding occur at opposite ends of the soil moisture spectrum yet their resulting stress responses in plants share many similarities. Drought limits root water uptake to which plants respond with stomatal closure and reduced leaf gas exchange. Flooding limits root metabolism due to soil oxygen deficiency, which also limits root water uptake and leaf gas exchange. As drought and flooding can occur consecutively in the same system and resulting plant stress responses share similar mechanisms, a single theoretical framework that integrates plant responses over a continuum of soil water conditions from drought to flooding is attractive. Based on a review of recent literature, we integrated the main plant eco‐physiological mechanisms in a single theoretical framework with a focus on plant water transport, plant oxygen dynamics, and leaf gas exchange. We used theory from the soil–plant–atmosphere continuum modeling as “backbone” for our framework, and subsequently incorporated interactions between processes that regulate plant water and oxygen status, abscisic acid and ethylene levels, and the resulting acclimation strategies in response to drought, waterlogging, and complete submergence. Our theoretical framework provides a basis for the development of mathematical models to describe plant responses to the soil moisture continuum from drought to flooding. Our study aims to develop a conceptual framework to describe plant responses across a range of soil moisture conditions that span from drought to flooding. We use theory from the soil–plant atmosphere continuum modeling as “backbone” for our framework, coupling plant oxygen status, and the mechanistic processes of abscisic acid and ethylene signaling to describe plant responses to drought, waterlogging, and complete submergence. Our theoretical framework provides a basis for the development of mathematical models to describe plant responses to the soil moisture continuum from drought to flooding. These stress responses to opposing water conditions can be understood from the fact that the functioning of these plants requires sufficient liquid water uptake from the soil as well as rapid exchange of CO2 diffusion across the stomata and thereby photosynthesis under conditions when light and other factors are not limiting assimilation of CO2  is determined by stomatal conductance and the partial pressure deficit inside and outside the leaves.The plant response to drought has been well captured by existing soil–plant–atmosphere continuum models   protein that converts molecular oxygen to apoplastic reactive oxygen species (ROS)  barrier, a suberin‐rich structural layer at the exodermis to prevent radial oxygen loss from the root to the waterlogged soil Table . AlthougSNORKEL (SK) locus encoding two ethylene inducible group VII ERFs, SK1 and SK2,  locus encodes a group VII ERF, SUB1A, induced by accumulated shoot endogenous ethylene, and which represses energy and carbohydrate consumption, including the “escape” processes via gibberellin signaling  responses to drought and flooding stress. A key hypothesis we propose this framework can test is that prior exposure to water stress alters future stress responses through the occurrence of a “memory,” which can be formed through both plant morphological changes and preconditioning of hormone levels. For instance, flooding‐induced aerenchyma formation may promote plant tolerance to subsequent drought due to a reduction in root metabolic cost (Klein et al., A possible future extension of the mechanistic framework is to incorporate the life history of plants and the changes in plant architecture and physiology this results in, thus enabling the simulation at longer time scales, say months to years or even decades. Besides this extension on the temporal scale, we also envision straightforward extensions on the spatial scale. This proposed framework can serve to represent individuals within individual‐based models that scale up to the community and ecosystem level. Incorporating only horizontal water transport would already enable one to investigate competition for water and the impact of species composition and distribution on ecosystem water stress responses. Additionally, these models can be extended to incorporate, for example, interplant differences in their efficiency of nutrient and light acquisition and their competition for these resources.A natural application for our model framework is its incorporation into land‐surface models. So far, the incorporation of plant hydraulics into land‐surface models has been used to investigate the feedback between climate and vegetation in terms of water cycle during non‐stressed conditions and drought, yet mechanistic plant responses to excessive soil water content are still lacking in these models (Li et al., All co‐authors have no conflict of interest to declare.


Mycoplasma genitalium (MG). Only 11.4% of 299 women were tested for MG despite 74.2% being tested for Chlamydia trichomonas (CT) and Neisseria gonorrhoeae (NG). Only 9% of the women were treated with antibiotics which would treat macrolide‐sensitive MG infection. Increasing education and awareness of MG and utilising reflex macrolide testing for MG will help direct effective antibiotic therapy and prevent the long‐term sequalae of PID.The records of women attending Gold Coast health hospital sites were retrospectively analysed to determine if women diagnosed with pelvic inflammatory disease (PID) were being tested for  Pelvic inflammatory disease (PID) is a syndrome comprising of a spectrum of inflammatory disorders of the upper female genital tract, including any combination of endometritis, salpingitis, tubo‐ovarian abscess and pelvic peritonitis.The long‐term sequalae of PID includes ectopic pregnancy, tubal factor infertility and chronic pelvic pain.Chlamydia trichomonas (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG) have been implicated.Although up to 70% of cases have an unidentified cause, sexually transmitted infections (STIs) including Investigating for MG infection is important as MG is not always adequately treated with principal PID antibiotic regimens, leading to persistent infection in patients with MG‐associated PID.The primary aim of the project was to establish if patients diagnosed with PID are being tested for MG as per Therapeutic Guidelines (eTG)A clinical audit was performed of patients presenting to Gold Coast health hospital sites from January 2019 to January 2020.All data was extracted from the integrated electronic medical records (iEMR) which contains all clinical data including investigations and medications.A list of eligible patients was generated from a report of all inpatient and outpatient presentations given a coded diagnosis which satisfied the spectrum of PID .Our findings are summarised in Table There were 222/299 (74.2%) patients diagnosed with PID tested for CT or NG, but only 34/299 (11.4%) of patients were tested for MG. This highlights an awareness of testing for STIs, but a missed opportunity to appropriately include MG testing in patients with PID. All patients who were tested for MG had CT and NG tests.In our study, only 9% of patients treated for PID received antibiotics which would have treated macrolide‐sensitive MG infection.Our study demonstrates that although healthcare professionals are aware of the need to test for STIs (CT and NG) in PID they are less aware of the need to request MG tests. It suggests that education and a review of guidelines used in our emergency and gynaecology departments is required to increase MG testing rates for patients diagnosed with PID.Diagnosing MG‐associated PID is important because: (1) empirical principal antibiotic regimens for PID do not adequately treat MG infections, potentially leading to long‐term sequelae;There are several limitations to our study. The diagnosis of PID includes a spectrum of presentations and hence it is difficult to determine the completeness of our patient list. However, all notes that were included were reviewed to ensure that patients who were included fitted a clinical diagnosis of PID as per the Australian STI guidelines. Also, our study is limited to a single site, and hence larger studies would be required to determine the scale and potential impact of undertested MG in patients with PID. Further qualitative studies would be required to determine the barriers and reasons for low MG testing rates and to inform intervention strategies to improve testing rates for MG in patients diagnosed with PID.Testing for MG in patients diagnosed with PID remains low despite recommendations in current guidelines. This is a missed opportunity for prompt diagnosis and effective treatment, not only for positive patient outcomes but also to reduce the cost of managing the long‐term sequalae of PID.The current prevalence of MG, which is relatively low,


Sceloporus undulatus is a female‐larger, sexually size dimorphic lizard species that is heavily parasitized by chigger mites . In particular, the intensity of mite parasitism is higher in male than in female juveniles during the period of time when sex differences in growth rate lead to the development of sexual size dimorphism (SSD). Sex‐biased differences in fitness costs of parasitism have been documented in other species. We investigated whether there are growth costs of mite ectoparasitism, at a time coinciding with sex differences in growth rate and the onset of SSD. If there are sex‐biased growth costs of parasitism, then this could suggest a contribution to the development of SSD in S. undulatus. We measured growth and mite loads in two cohorts of unmanipulated, field‐active yearlings by conducting descriptive mark‐recapture studies during the activity seasons of 2016 and 2019. Yearling males had consistently higher mid‐summer mite loads and consistently lower growth rates than females. However, we found that growth rate and body condition were independent of mite load in both sexes. Furthermore, growth rates and mite loads were higher in 2019 than in 2016. Our findings suggest that juveniles of S. undulatus are highly tolerant of chigger mites and that any costs imposed by mites may be at the expense of functions other than growth. We conclude that sex‐biased mite ectoparasitism does not contribute to sex differences in growth rate and, therefore, does not contribute to the development of SSD.Parasitism is nearly ubiquitous in animals and is frequently associated with fitness costs in host organisms, including reduced growth, foraging, and reproduction. In many species, males tend to be more heavily parasitized than females and thus may bear greater costs of parasitism.  We tested whether a sex‐biased growth cost of parasitism contributes to the development of SSD in Sceloporus undulatus, a female‐larger lizard species. Yearling males had higher mite loads and lower growth rates than females, but growth rate and body condition were independent of mite load in both sexes. We conclude that sex‐biased mite ectoparasitism does not contribute to sex differences in growth rate and the development of SSD. Our study site was a 0.045 km2 patch of oak‐pine forest bounded by open, mowed fields and human‐made sand paths, with a variably open canopy, sparse understory due to prescribed burns, and abundant tree debris on the forest floor.We investigated relationships between chigger mite loads and lizard growth rates in yearling juveniles of Eggs in this population are laid from late May into June and begin to hatch in late July. Depending on weather, the neo‐natal activity season for hatchlings can extend into mid‐November. Lizards emerge from winter inactivity as yearlings in March of the next year and grow rapidly to reach the size of reproductive maturity by the end of the first full activity season. Following a second winter of inactivity, lizards re‐emerge as reproductively mature adults at approximately 20 months of age, in the spring of their second full activity season  to prevent possible measurement biases in SVL, body mass, or mite counts. Although measurements were performed by a different investigator in 2016 and 2019, it is unlikely that an investigator bias would have influenced calculated growth rates because any differences between measurements would be consistent across the entire growth interval and as such, the calculated growth rates (change in length per time) would not be affected. We captured lizards with a slip‐noose or by hand. Upon capture, we measured snout‐vent length  using a ruler and body mass to 0.1 g using a Pesola® spring scale. We determined sex by the presence  or absence  of enlarged post‐cloacal scales, and we unambiguously distinguished yearlings from older age‐classes by their smaller size during the month of June .In 2019, we assessed the precision of mite counts by comparing the number of mites counted by eye in the field to the number of mites counted by using ImageJ  to analyze photographs of the same lizards taken at the time of mite counts in the field with a Nikon D7200 digital camera and a Sigma 105 mm F2.8 EX DG OS HSM Macro lens. Mite loads obtained by these two methods ranged from 7 to 424 mites/lizard and were highly correlated /(time in days)) was equal to growth rate computed as the slope of SVL plotted as a function of date of recapture for each lizard . In 2019, we demonstrated that growth rate calculated as ((SVL2–SVL1)/(time in days)) was independent of the number of days between measurements of SVL . To analyze relationships between growth rate and mite load separately for the months of June and July, we averaged each lizard's mite counts taken within the focal periods for each month. For example, June's mite count in 2016 was the average of mites counted in weeks 3 and 4 of that study. We calculated body condition separately in June and in July as the residual of log mass regressed on log SVL using measurements taken in the first and last weeks of the intervals defined above.For a direct comparison of 2016 versus 2019, we selected comparable 3‐week periods: 22–24 June through 13–14 July 2016 versus 24–27 June through 15–17 July 2019. These three‐week periods included measurements of SVL and mite counts taken in weeks 3, 4, 5, and 6 of the 2016 study  to analyze effects of mite load on body condition and also to analyze effects of body size, sex, and year on growth rate and mite load, where the earliest measurement of SVL was used for body size. We log‐transformed growth rate and body size to linearize the relationship between the two. Analyses evaluating the effects of sex and year on mite loads in June and July had logSVL entered as a covariate to remove potentially confounding effects of SVL. Analyses evaluating the effects of mite load on body condition in June and July had sex and year entered as covariates to remove potentially confounding effects. All To determine if mite load impacted growth rate, we used R statistical software (version 4.3.1) to generate a series of generalized linear mixed models . Sex differences in growth rate were similar in 2016 and 2019, resulting in the development of significant sexual size dimorphism (SSD) early in July of both years. In 2019, for example, the difference between female and male SVL, and thus the development of SSD, had become statistically significant by 14 July .Mite loads, mite load ranges, and growth rates of male and female yearling lizards are summarized in Table F3,138 = 3.33, p = .022; July: F3,97 = 3.13, p = .029). Mite loads tended to increase with body size (SVL) in both months, but these size effects failed to achieve conventional statistical significance . Controlling for SVL, mite loads were significantly higher on males than on females in July , but not in June , and were higher in 2019 than in 2016 in June  but not in July .Generalized linear models evaluating the effects of sex and year on mite loads in June and July were overall significant .Overall, mite load does not appear to impact growth rate Figure . None of4E. alfreddugesi) are highly abundant in the environment, and all age‐ and sex‐classes  of S. undulatus are heavily parasitized  during the peak growing season. This study reported high mite loads, an average of 178.3 ± 65.2 mites on yearling male lizards, which are comparable to what we found on S. undulatus in the present study. It is worth mentioning that the mite loads on lizards in the studies finding no growth cost of mite parasitism were substantially lower than those on S. undulatus in the present study . Despite our higher mite loads, however, we still found no effect on growth.The general narrative on whether mite parasitism affects growth has been mixed. Studies on p Abell, . Howeverp Abell,  found a p Abell,  found evMimus parvulus) showed that tolerance to parasites and effects of parasites varied through the years based on rainfall and food abundance. The effects of parasites on the hosts were not because the parasites caused more damage, but were because the hosts could not compensate for the parasite‐induced costs  can fluctuate across the years and have different effects on life history traits. Tolerance to parasites is greater in populations that experience higher variation in environmental conditions  is typical of a host–parasite system in which the host would be expected to have evolved tolerance to parasites  have also been found to be highly tolerant of mite parasites  and Western fence lizards   and is not correlated with growth rate , even while mite loads are greater in males than in females , and we saw an increase in both environmental mite abundance and lizard growth in 2019 as compared to 2016. Increased precipitation would have increased soil moisture and humidity, which are positively correlated with the abundance of arthropods, including mites ; data curation ; formal analysis (supporting); funding acquisition (supporting); investigation ; methodology ; project administration ; supervision ; writing – original draft ; writing – review and editing . Hailey Conrad: Conceptualization (supporting); data curation ; formal analysis (supporting); investigation ; methodology ; project administration ; writing – original draft ; writing – review and editing . Henry John‐Alder: Conceptualization ; data curation ; formal analysis (lead); funding acquisition (lead); investigation ; methodology ; project administration ; supervision ; writing – original draft (supporting); writing – review and editing (supporting).Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.Appendix S3.Click here for additional data file.Appendix S4.Click here for additional data file.Appendix S5.Click here for additional data file.


Carex filispica was related to the advantage of multiple functional traits after trampling, reflecting short‐term resistance. However, the long‐term response of dauciform roots to trampling and the recovery of C. filispica with and without dauciform roots to trampling require further studies. In this study, different intensities of trampling  were performed in an alpine meadow. One year later, individuals with and without dauciform roots were separated and their functional traits related to the economic spectrum of leaves and roots were measured as a reflection of recovery from trampling. The results showed that: (1) 1 year after trampling, the number of dauciform roots showed an increase with trampling intensity; (2) 1 year later, there was no significant difference in the response of economic spectrum traits among trampling intensities, or between plants with and without dauciform roots; (3) the number of dauciform roots was positively correlated with the leaf area of both individuals with and without dauciform roots, as well as with the biomass of those without dauciform roots; and (4) plants with more resource‐conservative roots showed an advantage after trampling recovery: specifically, plants with dauciform roots showed such an advantage in the control group, which was lost with a leaning towards resource‐acquisitive roots and an increased density of dauciform roots once trampled. In contrast, plants without dauciform roots showed a significant advantage of conservative roots only after trampling. In conclusion, the presence of dauciform roots is related to the plants' position on the root economic spectrum, thereby influencing the recovery of C. filispica from trampling. Carex filispica showed strong recovery from trampling after 1 year, which makes it an adequate choice for ecological restoration in alpine meadows. Dauciform roots showed a positive correlation with the aboveground growth of both plants with and without them, however, it requires a lab‐controlled study to confirm whether there is indeed a positive effect on the growth of neighbouring plants.In the natural habitats of China, dauciform roots were only described in degraded alpine meadows. It was found that the presence of dauciform roots of  Carex filispica to trampling.The number of DRs increased with trampling intensity after a long‐term recovery, which has a positive relationship with the aboveground growth of both individuals with and without DRs. The presence of DRs may be related to the plants' position on the economic spectrum, thus influencing the resilience of  This area has a cold‐temperate mountain monsoon climate with a mean annual temperature of −1.0°C and precipitation of 600–650 mm  in each set, while the sets were not placed in a line to avoid positional effects and separated from each other by at least 1 m to avoid interaction effects. The experimental trampling was applied based on standard experimental procedures recommended by Cole , specific leaf area (SLA), leaf dry matter content (LDMC) and leaf nitrogen (N) content were selected for the leaf economic spectrum (LES) traits, while root area (RA), specific root area (SRA), root density (RTD) and root nitrogen (N) content were selected for the root economic spectrum (RES) traits. Previous studies showed that plants with smaller leaf area (LA) tended to have higher resistance towards trampling; specific leaf area (SLA) reflects the balance of investment in growth and defence, which tends to have a positive relation with trampling resilience; lower leaf dry matter content (LDMC) and higher leaf N content are associated with faster growth , the plants and surrounding soil plot of 10 × 10 × 10 cm were collected from each lane, and the 2SO4‐H2O2  of DRs per individual were calculated using an XTL‐3B stereoscopic microscope. All the leaves of three individuals with DRs and three without DRs in each lane were removed and measured by LI‐COR portable leaf area meter to determine the total leaf area of each plant. The whole root systems of these individuals were then scattered in the water carefully without overlapping, and their pictures were scanned and analysed by LA‐S root analyser to determine the root surface area of each plant. Afterwards, their above‐ and belowground parts were separately dried to constant weight and weighed for biomass. The nitrogen content was determined by the Indophenol blue colorimetric method after digestion with H2O2 Bao, . The spe2.4C. filispica after different trampling treatments, if any, using LSD post hoc tests to indicate the significant difference between two treatments. Two‐way ANOVA was used to analyse the interaction between the presence of DRs and trampling intensity on each functional trait. Pearson correlation analysis was used to analyse the correlation between the amount of DRs and the functional traits of individuals with and without DRs. Principal component analysis (PCA) was used to downscale the standardized functional traits related to the economic spectrum of leaves and roots and calculate the score of their first axis respectively. General linear regression model (GLM) was used to simulate the correlation between the principal component scores of both the leaf and root economic spectrum of individuals with and without DRs after different treatments and their correlation with biomass.All analyses were performed using SPSS  and the figures were produced using Origin . One‐way ANOVA was used to analyse the differences in the amount of DRs and the functional traits of 33.1p = .033, F = 3.038, df = 103) among different treatments . Post hoc test indicated that the DR number after 500 passages was significantly higher than that of 50 passages and the control group without trampling  and leaf dry matter content (LDMC), and the first axis of the root economic spectrum was dominated by specific root area (SRA) and root density (RTD). From Figure r = .459, p = .031), indicating a co‐variation with the resource‐acquisitive root traits.Principal component analyses (PCA) were conducted separately for leaf economic spectrum traits and root economic spectrum traits of Therefore, in Figure r = −.657, p = .001 for individuals with DRs and r = −.605, p = .004 for those without respectively. Plants with resource‐conservative roots showed better growth after trampling, which is consistent in both individuals with and without DRs. Interestingly, further analysis showed that individuals with DRs only showed a significant resource‐conservative advantage to biomass in the control group, while individuals without DRs showed only after trampling of different degrees ; formal analysis (lead); investigation ; writing – original draft (lead). Wanting Liu: Investigation . Songlin Jiang: Investigation . Yulin Huang: Investigation . Wenli Ji: Conceptualization ; funding acquisition (lead); supervision (lead).This manuscript is approved by all authors for publication with no conflict of interest. The work described was original research that has not been published previously, or is it under consideration for publication elsewhere.


However, to fully benefit from this promising advance, LAED regimens must be paired with care delivery innovations that facilitate their access and use. These integrative approaches will require a multi‐disciplinary research agenda that generates additional long‐acting treatment options as well as strategies that support the effective and equitable use of these novel therapeutics.HIV treatment regimens that require lifetime daily administration present challenges, including treatment fatigue, privacy concerns and suboptimal adherence . LAED reMultiple LAED therapeutics and drug delivery systems are being developed, and some have regulatory approvals for select populations with HIV. The possibilities of LAED regimens can be glimpsed in lenacapavir, a first‐in‐class capsid inhibitor approved for heavily treatment‐experienced patients with multi‐drug resistant HIV‐1 that can be administered subcutaneously every 6 months . Beyond These new therapeutic options also drive a need for innovations in HIV clinical practice and monitoring. Clinical strategies to enable treatment experienced PWH to benefit from LAEDs, including LAED treatment initiation without prior viral suppression using oral regimens, would have a profound impact on the health outcomes of many PWH, and research towards this end is urgently needed. Research can additionally improve clinical care through the development of tools that facilitate mutual decision‐making between patients and providers on LAED treatment options. Rapid point‐of‐care assays to measure LAED drug levels and HIV viral load would be valuable for monitoring periods of LAED treatment discontinuation or interruption, when drug levels may be sub‐therapeutic and risks for developing HIV drug resistance increase . The phaWhile LAIs open new possibilities for patient care, they have also challenged the operational and workforce models of many HIV clinics. Strategies to overcome these challenges and associated infrastructural hurdles must be developed, tested and applied, including strategies to manage cold‐chain requirements for drug shipping and storage, intensified staffing needs for treatment injection administration and scheduling, and complex billing and reimbursement processes for LAIs . Early eNew LAED therapeutic options should leverage and spur innovations in HIV treatment and service delivery to fully realize their potential impact. Models of healthcare delivery that provide low‐barrier care and integrated services for HIV, chronic conditions and unmet basic needs have improved HIV care engagement and clinical outcomes in populations that experience complex adherence challenges , 10. TheAdvancing the equitable impact of long‐acting therapeutics will require concurrent consideration of broader social determinants of health, such as intersectional HIV stigma and discrimination, that can impede HIV treatment adherence, retention and viral suppression , 14. FurNOT‐AI‐22‐042), clinical monitoring (PAR‐21‐070), behavioural support interventions  and implementation science (RFA‐AI‐21‐024). Given the need for multi‐sectoral work, academic and commercial partnerships through small business research grants  could additionally develop and test innovations that may facilitate LAED uptake. Advancing a robust and multi‐level LAED research agenda will better capacitate our ability to overcome inequities in HIV care access and outcomes and bring the HIV pandemic to an end.LAEDs are a technological advance with the ability to significantly improve patient outcomes for all PWH. However, research is still needed to realize the full potential of this technology. The research needs span the research pipeline from the discovery and development of new therapeutic entities and diagnostic assays to the elucidation of novel clinical strategies that address the many interacting levels of healthcare delivery and patient care. NIAID and NIMH have called for research on these fronts, including innovations in long‐acting regimen development  contributed to the conceptualization, writing and editing of this document.Nothing to declare.The opinions expressed in this viewpoint do not necessarily represent the views of the National Institute of Allergy and Infectious Diseases, the National Institute of Mental Health, the National Institutes of Health, the Department of Health and Human Services or the United States Government.


Implementation of chemical energy storage for a sustainable energy supply requires the rational improvement of electrocatalyst materials, for which their nature under reaction conditions needs to be revealed. For a better understanding of earth‐abundant metal oxides as electrocatalysts for the oxygen evolution reaction (OER), the combination of electrochemical (EC) methods and X‐ray absorption spectroscopy (XAS) is very insightful, yet still holds untapped potential. Herein, we concisely introduce EC and XAS, providing the necessary framework to discuss changes that electrocatalytic materials undergo during preparation and storage, during immersion in an electrolyte, as well as during application of potentials, showing Mn oxides as examples. We conclude with a summary of how EC and XAS are currently combined to elucidate active states, as well as an outlook on opportunities to understand the mechanisms of electrocatalysis using combined operando EC–XAS experiments. Electrochemical methods and X‐ray absorption spectroscopy are powerful tools to understand electrocatalysis on an atomistic level, yet the combination of these methods still holds untapped potential. In contrast to spectroscopic and diffraction methods, XAS is element‐specific, which allows correlating EC processes to oxidation state changes of specific elements and which further allows elucidating the changes in the coordination environment of these elements. For the latter, no long‐range order is required when using XAS as compared to XRD. This renders XAS suitable for the study of electrocatalysts, which lose their crystallinity during the formation of the active state.10, 11What is measured in electrochemical experiments? In these experiments, electric current and voltages  are measured, where the former is the flow of electrons and the latter quantifies the capacity of the electrochemical system to do work. Common pitfalls in the physical interpretation of typical electrode potentials used in electrochemistry were recently discussed by Boettcher et al.What is measured by XAS? In these experiments, a core electron is excited leaving a so‐called core hole in an inner shell, which makes the method highly sensitive to the local geometric and electronic structure through various secondary processes. The X‐ray absorption near‐edge structure (XANES) correlates with the number of electrons in the outermost shell  via screening effects of the created core hole. Structural information is more straightforwardly extracted from the extended X‐ray absorption fine structure (EXAFS), which arises due to interference of the ejected core electron with itself. A higher energy of the X‐ray photon results in a larger penetration depth in the material and a larger escape depth being relevant for proxies of the X‐ray absorption measured, e.g., by the X‐ray fluorescence. For the materials and absorption edges covered herein, these depths  are of the order of 0.3 μm  to 60 μm  for simple Mn oxides. Since nanosized materials are attractive electrocatalysts due to their high surface to volume ratio, the XAS discussed herein probes, with some exceptions discussed later, the entire electrocatalyst material.Figure In this Minireview, we concisely introduce the basics of EC and XAS, providing the necessary framework to discuss changes of the electrocatalyst material. As a way of example, we focus on manganese oxides as electrocatalysts for the oxygen evolution reaction (OER), which is the anodic reaction in water electrolyzers and currently considered the efficiency‐limiting process in these devices.2Electrochemistry relates chemical reactions to moving charges in response to gradients in the electric or chemical potential.Several proposals exist for possible intermediates.Electrochemical experiments, in particular those during combined EC and XAS experiments, are either performed by holding the potential at the working electrode for a certain time and measuring the current (chronoamperometry) or by holding the current at the working electrode and measuring the voltage (chronopotentiometry). In addition, cyclic voltammetry (CV) is a convenient potentiodynamic electrochemical method to probe and possibly distinguish processes with charge transfer (Faradaic processes) and those without charge transfer (non‐Faradaic processes).2+ to Mn3+ in the anodic direction, and the corresponding reduction of Mn3+ to Mn2+ in the cathodic direction  to localized Mn3+ (d4 high spin), thus leading to a change in apical bond lengths. Jahn–Teller distortions have been well studied, e.g., for LiMn2O4,Figure n Figure . The potThe potential region indicated with a yellow background in Figure i) and their corresponding electrode potentials (E) are used for determination of the electrocatalyst's activity and stability for benchmarking or for determination of mechanistic parameters such as the Tafel slope, reaction order and Nernst slope , reaction order with respect to pH (∂pH/∂logi) and Nernst slope (∂E/∂pH) are determined and compared to predictions from the proposed reaction paths. 0.2Ca0.8Mn3.8+O3 reacted with water vapor to produce a Mn2+ species  to elucidate possible changes prior to electrochemical experiments.The as‐prepared electrodes may further react prior to the desired electrochemical experiment due to immersion in the electrolyte. Many transition metal oxides are prone to react chemically with water . For example, Prs Figure c. Other 4.2To investigate how the state of an electrocatalyst changes due to applied potential, i.e., due to electrochemical reactions, it is common to resort to post‐mortem experiments as some electrochemical changes can be resolved in this kind of investigations, namely those that result in the formation of stable phases. XAS is particularly useful for this purpose, as it allows to elucidate phase changes even in cases where amorphization takes place, which is not the case for techniques in which crystallinity is a prerequisite, e.g., diffraction‐based techniques.2, LiMPO4  and LiMn2O4 have been investigated as electrocatalysts for the OER.[xMn2O4 . Large redox peaks were observed in the CV at pH 12 while they were absent at pH 14  with a spectrum of Mn4+O2. We concluded that the activation procedure led to oxidation of the electrocatalysts, with the changes being restricted to near the surface where catalysis occurs.Conditioning by potential cycling is a common procedure to activate catalysts prior to electrocatalytic studies.121 The e Figure f showed 4.3In situ or even operando experiments are necessary to understand both reversible and irreversible changes of the electrocatalyst as well as reactive transient states (if the time resolution is appropriate). Any combined EC and XAS experiment on transition metal oxides for studying the OER at room temperature is an in situ spectroscopic experiment as it needs to be conducted in an electrochemical cell including electrolyte and electrodes. In our definition, the reaction  must be taking place during a spectroscopic operando experimentE–pH diagrams  recorded at 1.5 and 1.65 V vs. RHE and that of Mn3O4 (spinel) recorded at 0.5 and 0.8 V vs. RHE. Note that the spectra are susceptible to bubble formation during the OER at high potentials . The spectra collected at 1.2 V vs. RHE look similar to either that of birnessite or spinel depending on which was measured before. This indicates hysteresis due to slow kinetics of the phase changes and highlights the importance of the history of the electrode. The hysteresis can also be seen clearly when the oxidation state is calculated by calibration of the centroid under the Mn‐L3 edge with respect to reference materials  resolution to follow expected changes. Moreover, in situ XAS enables the study of metastable phases, as well as the investigation of reversible and reactive processes. In the following paragraphs, we will give a perspective on the operando XAS studies needed to close current knowledge gaps.In this Minireview, we reviewed what XAS can tell us about the active state of Mn oxides during the OER with references to other first‐row transition metal oxides. In the introduction, we highlighted that the macroscopic insight from electrocatalysis can be combined with the atomistic insight from XAS, such as the local coordination environment or polyhedral connectivity. The formation of the electrocatalytic product creates a flow of electrons, i.e., an electric current. XAS measures the electrons in the outer shell of atoms, i.e., the oxidation state of the metal, so that the two can be correlated. In order to put the desired correlations on a sound scientific basis, we briefly introduced the fundamentals of electrochemistry where we discussed the key processes of redox changes, double layer charging and electrocatalysis, all of which may contribute to the measured currents. E–pH diagrams. While scan durations of the order of minutes are sufficient to study slow processes such as corrosion and bulk phase changes, the expected changes may occur on time scales longer than typical synchrotron experiments of one to two weeks, which is not compatible with usual synchrotron operation where users apply for an allotment of one or few measurement weeks. A possible solution might be implementing alternative operation modes where one applies for an allotment of a few hours each week over a long duration. Another solution could be the use of lab‐based XAS systems, albeit at the cost of lower X‐ray intensity. It will be crucial for the optimization of electrocatalytic materials with relevance for practical applications to understand the origins of degradation that often occur on time scales much longer than those currently investigated in most academic reports.The relevant time scales of solid state and electrocatalytic processes differ drastically and both are not matched by most current XAS studies. Current conventional in situ XAS studies are performed with durations of a few minutes to an hour per spectrum, which is appropriate to study the faster changes in Typical current XAS scans are also too slow to resolve changes with relevance to the catalytic mechanism such as Mn redox changes of the order of tens to hundreds of milliseconds for electrodeposited Mn oxide.Q). The time derivative of the signals (dQ/dt) is thus the redox current.3+/4+ redox couple is essential for the evolution of oxygen in natural photosynthesis as well as for electrodeposited Mn oxides4+ together with Mn3+ has been proposed as a necessity for the OER and because its midpoint potential is similar to that of the OER. The M3+/4+ redox couple is likewise important for the OER on other transition metal oxides,4+ has been correlated with oxygen evolution for a Co oxide.3+/4+ redox couple was also used to rationalize electrocatalytic trends of the ORR.The final point that can advance future combined EC and XAS experiments is the study of states rather than applied potentials. Relevant states are the redox changes of transition metals such as Mn. They may be identified by electrochemical features in cyclic voltammograms Figure a but espThe authors declare no conflict of interest.6Marcel Risch leads the Young Investigator Group NOME at Helmholtz‐Zentrum Berlin cofunded by the ERC Starting Grant ME4OER. He earned his PhD from Free University Berlin and performed interdisciplinary postdoctoral work at MIT. Marcel is enthusiastic about the knowledge‐guided design of electrocatalysts for oxygen and nitrogen electrocatalysis for sustainable fuels such as green hydrogen harnessing insights from innovative operando experiments.Dulce M. Morales is currently deputy head of the Young Investigator Group NOME led by Dr. Marcel Risch at Helmholtz‐Zentrum Berlin. She has a background in Industrial Chemical Engineering from Instituto Politécnico Nacional (Mexico), and earned her doctoral degree in Chemistry in 2019 mentored by Prof. Wolfgang Schuhmann at Ruhr‐Universität Bochum. Her research focuses on the development of multi‐metallic composites as electrocatalysts for sustainable energy conversion and storage applications, and presently aims for their study under industrial‐relevant conditions.Javier Villalobos is currently a Ph.D. student in the Young Investigator Group “Oxygen Evolution Mechanism Engineering” led by Dr. Marcel Risch at Helmholtz‐Zentrum Berlin. He has a background in Chemistry from the University of Costa Rica, where he obtained his master's and bachelor's degrees. His research focuses on studying the stability and activation of Co‐ and Mn‐based electrocatalysts for oxygen evolution reaction by X‐ray spectroscopy, including the development of operando experiments.Denis Antipin graduated from Lomonosov Moscow State University with a master's degree in Materials Science. He is currently a Ph.D. student in the Young Investigator Group “Oxygen Evolution Mechanism Engineering” led by Dr. Marcel Risch at Helmholtz‐Zentrum Berlin. His current research interests include simulations and experimental investigation of the mechanistic parameters of the oxygen evolution reaction.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


To test for evidence of statin‐mediated effects in patients with castration‐resistant prostate cancer (CRPC) as post‐diagnosis use of statins in patients with prostate cancer is associated with favourable survival outcome.The SPECTRE trial was a 6‐weeks‐long proof‐of‐concept single‐arm Phase II treatment trial, combining atorvastatin and androgen deprivation therapy in patients with CRPC (regardless of metastatic status), designed to test for evidence of statin‐mediated effects in patients with CRPC. The primary study endpoint was the proportion of patients achieving a ≥50% drop from baseline in prostate‐specific antigen (PSA) levels at any time over the 6‐week period of atorvastatin medication (PSA response). Exploratory endpoints include PSA velocity and serum metabolites identified by mass spectrometry .At the scheduled interim analysis, one of 12 patients experienced a ≥50% drop in PSA levels (primary endpoint), with ≥2 patients satisfying the primary endpoint required for further recruitment. All 12 patients experienced substantial falls in serum cholesterol levels following statin treatment. While all patients had comparable pre‐study PSA velocities, six of 12 patients showed decreased PSA velocities after statin treatment, suggestive of disease stabilization. Unbiased metabolomics analysis on serial weekly blood samples identified tryptophan to be the dominant metabolite associated with patient response to statin.Data from the SPECTRE study provide the first evidence of statin‐mediated effects on CRPC and early sign of disease stabilization. Our data also highlight the possibility of altered tryptophan metabolism being associated with tumour response. For advtoxicity , 8. Howein vitro and in vivo preclinical research further implicated altered cancer cholesterol metabolism in progressive disease, with statins enhancing tumour response to ADT [de novo androgen biosynthesis to overcome ADT [The role of statins in prostate carcinogenesis and patient outcome is hotly debated. While there is significant controversy concerning whether statins might reduce the overall risk of prostate cancer, men taking statins for at least 3 years post diagnosis of non‐metastatic prostate cancer have 39% lower cancer mortality rates and a 23% lower chance of developing distant metastases . Recent e to ADT , 12. Specome ADT .Statins are currently licensed for and widely used in the treatment of hypercholesterolaemia and the prevention of cardiovascular disease by inhibiting the function of 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG CoA) reductase, the rate‐controlling enzyme of the mevalonate pathway required for cholesterol synthesis. Statins are reported to reduce PSA levels via enhanced degradation of the androgen receptor protein , 14. Of The trial was conducted at the Beatson West of Scotland Cancer Centre. All patients provided written informed consent. SPECTRE was a single arm Phase II study which used a Simon two‐stage optimal design  to distinguish an ‘ineffective’ PSA response rate of ≤10% from an ‘effective’ PSA response rate of ≥30%  as judged by rising serial PSA measurements, regardless of metastatic status. This was based on a series of at least three readings each taken at least 7 days apart. The third reading was required to be ≥2 ng/mL. Patients who received prior bicalutamide, flutamide or nilutamide had to have proven PSA progression after withdrawal of the drug. Castrate levels of serum testosterone (<1.7 nmoL/L) were required. Patients could not have had therapy with statins or other cholesterol‐lowering drug during a 28‐day period prior to the initiation of trial medication. A screening visit was carried out (Day −28 to Day 1) during which informed consent and medical history were obtained, as well as biochemical tests. Following commencement of treatment, atorvastatin was administered orally, at a dose of 40 mg each day, for a total of 6 weeks. Any adverse effects were recorded, and serious adverse effects immediately reported to the Pharmacovigilance Office, CRUK Clinical Trials Unit.The primary study endpoint was the proportion of patients achieving ≥50% drop from baseline in PSA levels at any time over the 6‐week period of atorvastatin medication (PSA response). PSA levels were measured weekly during this period. The secondary endpoint was maximal percentage change in PSA levels. Additional (exploratory) analyses were performed to study absolute change in serial PSA levels during treatment and PSA velocities before and after statin treatment.Based on serial serum PSA levels obtained during and at the conclusion of the study, the maximum absolute drop in PSA (in ng/mL) was determined as previously reported  and presSerum measurements for the following tests were taken at the beginning of treatment (baseline) and weekly until the end of treatment and performed according to local standard procedures: standard of care clinical assays for PSA, testosterone, lipid profiling, and serum biochemistry. Serum FFA levels were measured using the Free Fatty Acid Assay Kit  as per the manufacturer's instructions. For clinical reasons, serum samples for biochemical analysis were not available for Patient 015, so FFA levels were not measured for this patient.prism 7. Graphs are shown as mean ± standard deviation (sd) with individual patient's data points shown. PSA velocity was calculated using the online STAMPEDE calculation tools [Data plotting and statistical analyses including two‐way ANOVA with Sidak's test, Mann–Whitney and Wilcoxon signed rank test were carried out using GraphPad on tools .Statistical analysis of metabolite levels measured over multiple time points were conducted using the R Statistical Environment, v3.6.3 , the KolA recruited patient was classified as non‐evaluable if: (i) they did not complete 6 weeks of atorvastatin medication and had neither a drop of ≥50% in PSA levels from baseline nor PSA progression, or (ii) only three PSA measurements were available following the start of atorvastatin study medication and none of these corresponded to a ≥50% drop from baseline or PSA progression.It was clarified for final analysis that patients taking at least 80% of the total 6 weeks of treatment were considered to have completed treatment.Eighteen patients consented to participate in the study, four of whom were ineligible and did not start study treatment. Of the 14 patients registered for the study, two had taken less than the required 80% of the study treatment to be deemed evaluable. A total of 12 patients were therefore evaluable . It was performed according to the Research Governance Framework for Health and Community Care  and the Medicines for Human Use  Regulations, 2004 SI 2004:1031 and World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects 1964. All patients provided written informed consent.The SPECTRE trial was designed as a proof‐of‐concept, single‐arm, two‐stage Phase II trial in patients with early evidence of CRPC, testing the impact of combined ADT and atorvastatin on disease progression Fig. . AtorvasAs expected, atorvastatin was well tolerated, with minimal reported adverse effects Table . Consistn = 11) of the 12 evaluable cases recording a decrease in PSA levels. Overall, there was no significant change in PSA from the start to the end of treatment .One of the 12 evaluable patients reported >50% of PSA response . The profile of changes in PSA levels after statin treatment for individual patients is presented as a waterfall plot showing the maximum (percentage and absolute) change during the course of treatment Fig. A, with tent Fig. B. We theent Fig. . Howeverent Fig. . Six of We found no evidence of a difference in means between the two PSA velocity groups for any of our five continuous clinical variables, namely, age (at diagnosis and recruitment), PSA levels, body mass index and weight (see Supplementary Information). There was also no association between Gleason score and PSA velocity group.P values <0.05, with five compounds shared among the 11 evaluable cases (serum samples were not available for Patient 015) and 10 metabolites differentially altered between patients with increasing and decreasing velocities after statin treatment.As statin treatment is thought to result in alterations in the cholesterol and related metabolic pathways, we compared the metabolic profiles of serial blood samples from our patients with diverging PSA velocities. A total of 2551 compounds/features across all samples were detected, with 973 compounds/features passing quality control and taken forward for correlative analysis with patient data. A total of 15 metabolites were identified to have adjusted Six of the 10 observed differentiating metabolites between patients with rising or stabilized PSA velocity after statin treatment were generated by tryptophan Table : tryptophttps://meetings.asco.org/abstracts‐presentations/205381). Consistent with our findings, a recently published window‐of‐opportunity study of preoperative fluvastatin in localized prostate cancer [The SPECTRE trial was the first clinical trial to directly study atorvastatin as a treatment in clinical prostate cancer. Our study is highly timely as there is mounting evidence to suggest evidence of improved patient outcome associated with statin use in advanced prostate cancer, as shown by a recent systematic review and meta‐analysis of cohort studies  were offered for optional patient consent. Unfortunately, not enough prostate biopsy samples were obtained. In accordance with our study design, recruitment was closed following the scheduled interim analysis as the criteria of ≥2 patients achieving >50% PSA drop was not reached. Another limitation of this study was the relative early stage of CRPC among recruited patients, with relatively low serum PSA levels at recruitment. For patients with more significant disease, there was a tendency to proceed with licensed treatment rather than participating in a proof‐of‐concept study. For future study with statins, it would be of interest to consider a combination approach with second‐generation antiandrogen agents or with chemotherapy.AR, TP53 and RB1, and that a biomarker signature combining lipid and genetic abnormalities may be predictive of worse prognosis [Tryptophan metabolism can influence a wide range of pathophysiological conditions including cancer . Consistrognosis .In summary, our proof‐of‐concept study revealed data suggestive of stabilization of CRPC within a 6‐week study period. Given the current treatment options for second‐ and third‐line androgen receptor pathway inhibitors, we found that, for patients with advanced CRPC, both oncologists and patients favoured the commencement of standard of care treatment rather than taking part in the SPECTRE study. Hence, the design of future studies may incorporate the use of statins with one of the androgen receptor pathway inhibitors, requiring the recruitment of large patient numbers with extended duration of statin treatment to fully test the long‐term anti‐cancer effects of statins.The authors declare no disclosures of interest.ADTandrogen deprivation therapyCRPCcastration‐resistant prostate cancerFFAfree fatty acidAppendix S1. Supplementary Information on patients, methods and data analysis, and supplementary.Click here for additional data file.


Tractography is widely used in human studies of connectivity with respect to every brain region, function, and is explored developmentally, in adulthood, ageing, and in disease. However, the core issue of how to systematically threshold, taking into account the inherent differences in connectivity values for different track lengths, and to do this in a comparable way across studies has not been solved. By utilising 54 healthy individuals' diffusion‐weighted image data taken from HCP, this study adopted Monte Carlo derived distance‐dependent distributions (DDDs) to generate distance‐dependent thresholds with various levels of alpha for connections of varying lengths. As a test case, we applied the DDD approach to generate a language connectome. The resulting connectome showed both short‐ and long‐distance structural connectivity in the close and distant regions as expected for the dorsal and ventral language pathways, consistent with the literature. The finding demonstrates that the DDD approach is feasible to generate data‐driven DDDs for common thresholding and can be used for both individual and group thresholding. Critically, it offers a standard method that can be applied to various probabilistic tracking datasets. The resulting connectome showed expected short‐ and long‐distance structural connectivity in the close and distant regions for the dorsal and ventral language pathways, consistent with the literature. In thisThe acquired images were pre‐processed by the HCP and the pipeline is briefly described here. The T1w and T2w images were aligned in native space using FSL's FLIRT and FNIRT functions. A reverse‐phase distortion correction was used to estimate field map distortions and the resulting corrected data were registered to T1w and T2w images using FSL's FLIRT boundary‐based registration (BBR) algorithm. The structural images were subjected to bias field correction  for further processing and whole‐brain probabilistic tracking. Specifically, the diffusion data were first subjected to bias correction using the ANTS flag  of 3D spheres in volume space with a radius of 8 mm that covered the entire left hemisphere in MNI space. For each participant, ROIs were inverted to native diffusion space (using inverse FLIRT and FNIRT transforms) and binarised over the grey and white matter interface (GWI). The GWI‐ROIs were used as a mask to extract the number of streamlines connecting all other ROIs based on the whole brain tractogram. Thus, no streamlines were extracted for those ROIs that were entirely on white or grey matter.A connection score between a pair of ROIs was quantified by computing the proportion of streamlines emerging from the seed and ending at the target ROI with a radial search distance of 4 mm (a default setting in MRtrix3). The connection score did not include streamlines passing through an ROI. As there were two directions of connectivity , we computed the average of the two connection scores generated from each direction of tracking. It was expected that closer ROI pairs would have higher connection scores while distant ROI pairs would have lower scores, reflecting the fact that the number of tractography streamlines and the fraction of true connections  associated with language processing and reading . For concise presentation, here we reported the language connectivity matrix generated by using Euclidean distance. However, the result of the average language connectivity matrix generated by using streamline distance was reported in the Supplementary Figure As we have seen from previous sections that the distance scores generated from the two distance approaches were highly correlated. The resulting patterns of connectivity thresholds also followed a similar trajectory. Furthermore, the resulting patterns of average language connectivity matrices generated from the two distance approaches were highly similar  as in Figure We projected the connection scores (without binarisation) to the brain for an intuitive way to visualise the results Figure  using thWith lenient thresholds , more cross‐region connectivity could be observed, especially for the inferior frontal, temporal and occipital regions. The IFG Orb was connected with the aMTG via the uncinate fasciculus (UF), and the IFG Oper and the IFG Tri were connected to the mFG and pFG via the AF. Collectively, these results demonstrated that the connectivity matrix based on the DDD thresholding approach was able to reconstruct the key white matter tracts that sustain language processing in the language network as previously reported in tractography studies  and (ii) that, as one would expect, some streamline distances through and around the brain's anatomy are longer than the direct Euclidean distance. Figure Our DDD approach dealt with the distance artefact by generating higher thresholds for close regions and lower thresholds for distant regions Figure . As longhttps://www.mrc-cbu.cam.ac.uk/bibliography/opendata/). Despite the computational cost required to generate data‐specific DDDs, a major advance is that one could formally compare results between studies based on the alpha threshold and in the future collated data for formal meta‐analyses. Secondly, the ROIs used to generate DDDs cover the left hemisphere but not the right hemisphere. This was deliberate for two reasons: (a) crossing fibres through the corpus callosum are complex especially for the callosal fibres connecting from the midsagittal slice of the corpus callosum to inferior and lateral brain regions (Chao et al., There are some limitations that are not addressed with the DDD approach. First, the DDDs generated by the present study may not be directly applied to tractography datasets that are acquired using different data acquisition protocols, pre‐processing, tractography algorithms, or different numbers/sizes of ROIs. That is because the sampling distributions of connectivity are data‐specific and generated from the outcome of a specific tractography setting. This means that DDDs would need to be calculated for each study/analysis; however, we have provided detailed methodological descriptions about how to generate appropriate DDDs (downloadable from 5To conclude, the DDD approach was developed as a strategy to formally threshold structural connectivity maps. The approach is in principle applicable to various tractography datasets and allow for comparisons across studies with different levels of alpha. The DDD approach also addressed the distance artefact by providing different thresholds for short‐ and long‐range connections.Ya‐Ning Chang and Matthew A. Lambon Ralph designed and performed research; Ya‐Ning Chang, Ajay D. Halai, and Matthew A. Lambon Ralph analysed data; and Ya‐Ning Chang, Ajay D. Halai, and Matthew A. Lambon Ralph wrote the paper.Data S1. Supporting information.Click here for additional data file.


Arabidopsis thaliana), callose accumulation regulates important biological processes such as phloem and pollen development, cell division, organ formation, responses to pathogenic invasion and to changes in nutrients and toxic metals in the soil. Callose accumulation modifies cell wall properties and restricts plasmodesmata aperture, affecting the transport of signaling proteins and RNA molecules that regulate plant developmental and environmental responses. Although the importance of callose, at and outside plasmodesmata cell walls, is widely recognized, the underlying mechanisms controlling changes in its synthesis and degradation are still unresolved. In this review, we explore the most recent literature addressing callose metabolism with a focus on the molecular factors affecting callose accumulation in response to mutualistic symbionts and pathogenic elicitors. We discuss commonalities in the signaling pathways, identify research gaps and highlight opportunities to target callose in the improvement of plant responses to beneficial versus pathogenic microbes.Cell walls are essential for plant growth and development, providing support and protection from external environments. Callose is a glucan that accumulates in specialized cell wall microdomains including around intercellular pores called plasmodesmata. Despite representing a small percentage of the cell wall (~0.3% in the model plant  In this review, we expose our current understanding of the mechanisms that regulate callose accumulation in response to microbes. The aim is to identify research gaps and highlight opportunities to target callose in the improvement of plant responses to beneficial versus pathogenic microbes. Information was missing on the signaling pathways and interactions controlling the expression and activity of callose metabolic enzymes and their relative contribution to the response to microbial symbionts. Knowledge also lacks on the evolutionary and physicomechanical features that selected callose to function in this process. We also highlight opportunities for stronger plant defenses that emerge from priming callose using non‐pathogenic and mutualistic microbes. Some of these interactions lead to pathogenesis, whereas others mutually benefit the plant and the microbe. Mutualistic symbiosis occurs when plant roots interact with arbuscular mycorrhizal fungi and/or with soil‐borne nitrogen‐fixing bacteria, exemplified by the genus Rhizobium  is finely regulated at the cell wall by the activities of enzymes belonging to the callose synthase (CALS), also known as glucan synthase‐like (GSL) and β‐1,3 glucanase (BG) families Figure . CALSs aLike the widely studied cellulose synthases, CALSs are part of a membrane complex that includes UDP‐glucose transferase, a GTP hydrolase, annexin and a sucrose synthase homolog  that can be grouped depending on phylogenetic relations, structural features and gene expression analysis  but not in mutants in an ER‐ and extracellularly localized stress responsive BG protein (named atbg2)  either by recruiting and stabilizing callose or by physically interacting with the metabolic enzymes. Signaling molecules, including calcium, reactive oxygen species and phytohormones trigger signaling pathways that control the expression and/or activity of CALSs and BGs, modulating callose levels in cell walls. Some of these molecules  play essential roles in coordinating plant responses to microbes. Their role in callose regulation in the context of plant−microbial interactions will be discussed in more detail in the next section.3NONEXPRESSOR OF PATHOGENESIS‐RELATED GENES 1, an interactor of TGACG‐binding transcription factors in the nucleus, which regulates the expression of salicylic acid‐responsive genes encoding pathogenesis‐related proteins. Salicylic acid can be converted into methyl salicylate, which acts as a long‐distance signal to induce transcriptional reprogramming of defense genes in distant tissues/organs . Exposure to these pathogen‐associated molecular patterns induces a burst of nitric oxide and the expression of NbCalS1 and NbCalS12  demonstrate the importance of callose during pattern‐triggered immunity. pmr4 plants are impaired in response to infection with the bacteria Pseudomonas syringae, whereas overexpression of GSL5 restricts penetration of the powdery mildew fungal pathogen  , among other defenses, in response to aphid feeding  cell walls. Enzymatic degradation of fungal beta‐1,3 glucan structures releases fragments or damage‐associated molecular patterns that activate the plant defense response  displayed an increase in callose deposition around infection threads (cytoplasmic invaginations that carry the bacteria into deeper tissues) and, occasionally, around the nodules   and Pseudomonas putida KT2440 (a plant growth‐promoting bacteria) than when treated with any of these microbes alone  showed a delay in callose deposition in comparison to wild type. cngc19 plants displayed a significant increase in fungal colonization to the detriment of plant growth suggesting that the channel activity is vital for maintaining a purely mutualistic symbiosis between P. indica and Arabidopsis. Interestingly, pattern‐triggered immunity responses were also compromised in cngc19 leading to pathogenic invasions.The mechanism regulating callose levels in response to mutualistic symbiosis has not been fully dissected. Jogawat et al.  showed tTo summarize, the initial regulation of callose metabolism is a general response to pathogenic attack and it is associated with the activation of pattern‐ and effector‐triggered immunity. Callose synthesis mechanically reinforces cell walls by interacting with cellulose microfibrils which is important to restrict pathogen invasion. Some BGs are secreted to digest fungal and bacterial cell walls releasing damage‐associated molecular patterns that trigger defense responses. Mutualistic microbes have evolved different strategies to evade plant defenses, some including down‐regulation of the pathways that induce callose. Plants have also adapted by differentially activating genes that regulate callose in response to mutualistic or parasitic symbionts. CALS and BG genes and other key regulatory proteins and factors have been identified to control callose in response to microbial invasions, but we lack a complete mechanistic understanding of these processes. We also do not know how the mechanisms for cell wall reinforcement interact with those that regulate callose to control intercellular transport via plasmodesmata tightly linked to local and systemic responses, a topic that we discuss in the next section.4Plasmodesmata‐manipulation by, and in response to, diverse microbes have been recently reviewed by other authors Figure  Dorokho. The besPenicillum chrysogenum, which activates an abscisic acid‐dependent mechanism that reduces BG expression  and a response dependent on the abscisic acid and salicylic acid signaling pathways. Ectopic expression of REMORIN1.3 in tobacco increases plasmodesmata‐callose and affects virus infection, although this response varies such that the spreading of tobamoviruses  are restricted, but potyviruses  are enhanced , which localizes at plasmodesmata and binds calcium, was induced post flg22 treatment. CML41‐amiRNA lines showed no induction in plasmodesmata callose upon treatment with flg22, whereas a CML41 overexpression line displays higher callose. Callose induction was reduced by a calcium chelator suggesting that calcium signaling is important in this response  and OsGSL2 (a CALS) affects the sugars found in the galls and nematode numbers, indicating that low callose levels are required for proper feeding and reproduction. In support, an effector protein from M. graminicola, MgMO237, was identified to suppress the host immune system and callose deposition   under an infection‐specific promoter leads to defective infection and severe reduction in the number of nodules. PDLPs also seem to play a role in this mechanism as ectopic expression of a PDLP‐like protein identified in M. truncatula, alters callose and improves infection and nodulation in the presence of nitrate  and proteins  are involved in fine‐tuning the differential expression (or activity) of CALS and BG to dynamically control the properties of cell walls (Figure Plants trigger different signaling mechanisms upon perception of beneficial and pathogenic microbes leading to differential callose regulation at and outside plasmodesmata microdomains. Multiple signaling molecules (salicylic acid, reactive oxygen species, Ca,s Figure . Transcrs Figure . Little s Figure . On the s Figure . ResearcAnother area where research is lacking concerns the reactive oxygen species and their specific involvement in determining plasmodesmata and callose regulation in both, mutualistic and parasitic interactions. Oxidative burst is described as one of the first response to pathogenic microbes, but this is also generated during rhizobia infection (Hu et al., Figure Glomus mosseae, which enhanced callose deposition in cell walls surrounding the intercellular hyphae of the pathogenic oomycete Phytophthora parasitica (Cordier et al., R. irregularis induced higher levels of callose and displayed smaller mycelium diameters during infection with the necrotic fungus Botrytis cinerea. More research to identify the factors involved in callose regulation during these microbial interactions would be instrumental in the design of biotechnological approaches to target callose as a tool for crop improvement. When modifying callose we must also take into consideration the potential side effects on plant growth as callose at plasmodesmata regulate the expression of developmental signals, transcription factors, RNAs and metabolites. The newly described role of plasmodesmata in the establishment of an auxin gradient (Band, In spite, opportunities have emerged to exploit interactions between callose regulatory pathways. For example, callose‐priming occurring after infection with mycorrhizal fungi and plant growth‐promoting bacteria has been shown to enhance plant resistance to infections with microbial pathogens. This was seen after infection of tomato roots with the arbuscular mycorrhizal fungi nt Band,  also opeTo conclude, a mechanistic understanding of the molecular components underlying the regulation of callose during papillae formation or that control transport via plasmodesmata is still lacking (Y. Wang et al., All authors were involved in the review writing. Richa Yeshvekar and Liam German generated the first draft and designed the figures. Yoselin Benitez‐Alfonso designed the review, was involved in the manuscript writing and reviewing, integrated comments and contributions from the different authors and reviewers and generated the final submission.The authors declare no conflict of interest.


The use of spinal cord stimulation (SCS) in patients with refractory angina pectoris (RAP) is still under debate. Studies up to date have shown a positive effect with an improvement in quality of life. However, no double blinded randomized controlled trials have been performed.The objective of this trial is to investigate if high density SCS leads to a significant reduction in the amount of myocardial ischemia in patients with RAP. Eligible patients must meet the criteria for RAP, have proven ischemia and a positive transcutaneous electrical nerve stimulator treadmill test. Patients who meet the inclusion criteria will receive an implanted spinal cord stimulator. Patients receive 6 months of high density SCS and 6 months of no stimulation using a cross‐over design. The order of the treatment options is determined using randomization. The primary endpoint is the effect of SCS measured by the change in percentage of myocardial ischemia using myocardial perfusion positron emission tomography scan. Key secondary endpoints are patient related outcome measures, major cardiac adverse events and safety endpoints. The follow‐up period is 1 year for the primary and key secondary endpoints.The SCRAP trial began enrollment on December 21, 2021 and is set to complete the primary assessments in June 2025. To date, January 2, 2023, 18 patients have been enrolled in the study and 3 patients have completed the 1‐year follow‐up.The SCRAP trial is an investigator‐initiated, single‐center, double‐blind, placebo‐controlled, and cross‐over randomized controlled trial investigating the efficacy of SCS in patients with RAP.  Overview of the design of the “Efficacy of Spinal Cord Stimulation in Patients with Refractory Angina Pectoris (SCRAP) trial.” AP = angina pectoris, TENS = transcutaneous electrical nerve stimulation, PET = positron emission tomography, SCS = spinal cord stimulation, SAQ = seattle angina questionnaire, RAND‐36 = RAND 36‐item health survey, 6‐MWT = 6‐min walking test, NRS = numeric rating scale, CCS = Canadian cardiovascular society. Due to advancements in treatment options mortality and morbidity rates in this patient population have steadily declined. However, up to 20% of patients with stable CAD have persisting angina symptoms.Spinal cord stimulation (SCS) is a last resort treatment option for this patient population in which no other treatment options are currently available. SCS is a device with a lead located in the higher thoracic epidural space and an implantable pulse generator (IPG) in the abdomen or buttock that provides stimulation at the level of the myocardial afferent sympathetic neurons (C7–Th1). The potential beneficial effects of SCS are based on four possible mechanisms: reduction of myocardial oxygen demand, reduction of pain perception, and a decrease in sympathetic tone and an improvement in the coronary microcirculatory blood flow.The majority of research into the effect of SCS on RAP has been observational in nature. To date three placebo‐controlled randomized controlled trials have been performed and one study compared SCS with usual care.Although the exact working mechanism of SCS in RAP has not been elucidated studies have repeatedly shown an improvement in the quality of life and a reduction in angina symptoms.The recently published European Society of Cardiology (ESC) guideline “Chronic Coronary Syndromes” describes varying levels of evidence with regard to treatment options in patients with RAP. The ESC guideline concludes that SCS may be considered .The type of SCS traditionally used in patients with RAP is conventional stimulation. With this type of stimulation the patient will feel paresthesia at the target area. For this study a paresthesia free form of stimulation, high density (HD) stimulation, will be applied. Based on previous experience it is a viable treatment modality, it allows adequate blinding of patients because no paresthesia are felt and the patient can be fully informed about the fact that the spinal cord stimulator will be turned off for a period of 6 months.ClinicalTrials.gov with NCT identifier NCT04915157.The aim of this investigator‐initiated, single‐center, placebo‐controlled, double‐blind, cross‐over, randomized controlled trial is to determine if high density spinal cord stimulation, a paresthesia free form of stimulation, leads to a significant reduction in the amount of myocardial ischemia in patients with RAP. The trial has been registered at 22.1The primary objective of this study is to determine the effect of SCS in patients with RAP on the percentage of myocardial ischemia measured using a myocardial perfusion positron emission tomography (PET) scan. Patients with RAP referred to our hospital  will be screened for eligibility to participate in the study. The inclusion criteria must be met, meaning stable angina pectoris CCS class III or IV during at least the previous 3 months, a coronary angiogram (CAG) performed within the last 12 months with significant CAD not suitable for intervention, optimal antianginal medication and proven ischemia using MIBI‐SPECT, perfusion magnetic resonance imaging (MRI), myocardial perfusion PET scan or a fractional flow reserve (FFR) positive lesion (<0.80) with no intervention options. Tables 2.1.1Eligible patients will be invited to the outpatient clinic to perform a standardized transcutaneous electrical nerve stimulation (TENS) treadmill test. The standardized TENS treadmill test is used as a screening tool, as advised by Mannheimer et al in the ESC report, to determine if the patient is eligible for an implanted spinal cord stimulator.(a)Positive: equal to or more than 50% reduction in the time from onset of angina pectoris until resolution of angina pectoris after TENS initiation, compared to the “normal” time it takes for angina pectoris to resolve.(b)Dubious: less than 50% reduction in the time from onset of angina pectoris until resolution of angina pectoris after TENS initiation, compared to the ‘normal’ time it takes for angina pectoris to resolve.(c)Negative: no reduction or increase in the time from onset of angina pectoris until resolution of angina pectoris after TENS initiation, compared to the “normal” time it takes for angina pectoris to resolve.Before the standardized TENS treadmill test the patient will fill out the TENS questionnaire Appendix . The ansPatients with a positive standardized TENS treadmill test are eligible for participation in the study and a myocardial perfusion PET scan is performed to determine the amount of myocardial ischemia present. Patients with a negative standardized TENS treadmill test are excluded from the study. Patients with a dubious standardized TENS treadmill test will receive TENS treatment in an ambulatory setting for a duration of 2 weeks. After this period the effect of TENS will be re‐evaluated by the same team to determine whether the effect was positive or negative (a dubious test result is not possible). If the effect is positive the patient is eligible and a myocardial perfusion PET scan will be performed 1 month after termination of TENS treatment (washout period to prevent possible lingering effects of the TENS treatment)  (PrimeADVANCED™ SureScan® MRI Medtronic), a single lead (Vectris Compact™ SureScan MRI Medtronic) and a patient programmer (myStim Patient Programmer & ADVANCED™ Antenna Medtronic). The spinal cord stimulator implantation will take place in an operating room in accordance with national guidelines concerning aseptic preparations. During the procedure the patient is asked to lie in a prone position to increase the thoracic kyphosis. Moderate sedation is applied using a combination of propofol and remifentanil, secondary field blocks and wound infiltration with local anesthetics will be used to provide analgesia. Sedation levels and vital signs are controlled and monitored by an experienced nurse. A Tuohy needle is used to enter the epidural space at the level of thoracic vertebrae (Th) 7–8. Entrance into the epidural space is gained using loss of resistance and confirmed by advancing a guide wire through the Tuohy needle under fluoroscopic guidance. Next the guide wire is removed and the lead is advanced into the epidural space with the cephalad tip positioned paramedian to the left side at the level of C7‐Th1 using fluoroscopic guidance. To determine if the position of the lead is correct sedation is temporarily lifted and using an external programmer , Samsung Programsystem and Communicator) stimulations are given. The aim is to achieve a paresthesia coverage of the target area (left side of the chest) of at least 80% and subtle modifications of the lead position are performed to reach the optimal coverage based on feedback from the patient during test stimulation. When the optimal position of the lead has been determined the lead is fixated at the thoracodorsal fascia, subcutaneously tunneled and connected to the IPG pocket. The usual subcutaneous position of the IPG is the left buttock. After the implantation has been completed the patient is monitored on the ward and an X‐ray is made to confirm the position of the lead. Depending on the randomization the correct settings will be input by the unblinded study nurse  using myocardial perfusion PET scan at the end of the six‐month period of treatment with SCS compared with baseline.2.2.2An overview of the secondary objectives is given in Table 2.3At baseline the SAQ, RAND‐36, NRS‐scale, and CCS class will be completed when the patient is seen for the initial standardized TENS treadmill test. The baseline 6‐MWT will be performed after the baseline myocardial perfusion PET scan has shown myocardial ischemia. After spinal cord stimulator implantation the follow‐up appointments will take place at the outpatient clinic of the department of anesthesiology. Patients will be seen at the outpatient clinic one week , 1, 3, 6 months, and 1 year after initial spinal cord stimulator implantation.At 6 months the myocardial perfusion PET scan will be repeated before the 6‐month outpatient clinic appointment. The SAQ, RAND‐36, NRS‐scale, CCS class, and 6‐MWT will be completed when the patient is seen for the 6‐month outpatient clinic visit. After confirmation that all data are complete by a member of the research team (excluding the study nurse), the patient has an appointment with the study nurse. At this appointment the study nurse will change the programming according to the randomization, from “SCS ON” to “SCS OFF” or vice versa. The programming of the spinal cord stimulator is performed in the same manner and by the same study nurse as was done at baseline ensuring that the blinding of the patient and the rest of the research team remains intact. At 12 months the myocardial perfusion PET scan will be repeated before the final, 12‐month outpatient clinic appointment. Additionally the SAQ, RAND‐36, NRS‐scale, CCS class, and 6‐MWT will be completed when the patient is seen for the final outpatient clinic appointment by a member of the research team in the same fashion as the 6 month appointment. At this final appointment, after confirmation of complete data, the spinal cord stimulator settings will be set to conventional spinal cord stimulation by the study nurse in the same manner as the 6 month appointment. In addition the patient will be asked during which period of the study, the first or second 6 months, it was thought that the spinal cord stimulator was turned on.For the duration of the study patients are asked to contact our center to inform us if visits to the emergency room, admissions to hospital and/or the undergoing of a PCI and/or CABG have occurred. If complications arise related to the implanted spinal cord stimulator  patients will be asked to contact the outpatient clinic of the department of anesthesiology to determine the appropriate course of action.After completion of the study patients will be invited to the outpatient clinic each year for a total duration of 5 years for long‐term follow‐up. At each outpatient clinic appointment patients will be asked to complete the SAQ, RAND‐36, NRS‐scale, CCS class, and 6‐MWT.2.4Currently, no data with regard to the percentage of myocardial ischemia (as % of the left ventricular myocardium) present in patients with RAP using myocardial perfusion PET scan as imaging modality are available. The data available on this subject is with the imaging modality MIBI‐SPECT. The percentage of myocardial ischemia (as % of the left ventricular myocardium) present in patients with refractory angina pectoris using MIBI‐SPECT ranges from 6.12% (standard deviation of 3.7)T test, α .05 and power .80) a sample size of 32 patients per arm  could detect a relative reduction in percentage of myocardial ischemia of 25%. Assuming a 10% patient drop‐out in the analysis, this means a total of 72 patients should be included in the study.Using these clinical assumptions  and the typical assumptions  consisting of an interventional cardiologist, anesthesiologist and statistician has been established for this study to oversee safety. A blinded interim analysis will be performed after 24 patients have been included and additional interim analysis will be performed when deemed necessary by the DSMB. The DSMB will have access to the unblinded study data  and can give the following recommendations: (i) proceed with the trial according to protocol, (ii) modify the study protocol or study procedures , and (iii) terminate inclusion of new patients into the trial.3Study enrollment started on December 21, 2021 and the estimated completion of the primary assessments is June 2025. To date, January 2, 2023, a total of 18 patients have been enrolled in the study. Three patients have completed the 1‐year follow‐up. The baseline characteristics of the patients currently enrolled are shown in Supporting Information: Table 4The SCRAP trial will be conducted in a large specialized hospital in the Netherlands . In the Netherlands, the “National Health Care Institute” evaluates certain aspects of our health care system to determine if a treatment modality is effective and should be part of insured health care. In November 2019, the National Health Care Institute evaluated the treatment modality neuromodulation in patients with chronic pain, including patients with RAP. The report concluded that, based on the available literature (June 2018), spinal cord stimulation should not be considered as an effective treatment modality in patients with refractory angina pectoris and should therefore no longer be part of insured health care in the Netherlands.The decision to use changes in myocardial ischemia, measured with myocardial perfusion PET scanning, as primary endpoint is based on the afore mentioned report of the National Health Care Institute. An important comment in the report was that evidence of reduction in myocardial ischemia is currently missing, which led to the formulation of the chosen primary endpoint.The choice of 6 months SCS treatment is based on previous research that has shown that changes can be seen in myocardial blood flow (using PET) after 3–6 weeks of SCS therapy.An important concern voiced in previous literature is the risk of adverse events related to the spinal cord stimulator implantation in general. Adverse events range from lead dislocation to device related infections with a reported incidence of up to 40%.5There are a number of limitations with regard to the SCRAP trial. One limitation is the difference in myocardial ischemia on which the sample size calculation is based. The power of the study is based on a relative reduction in myocardial ischemia of 25% after treatment with SCS, in absolute numbers a reduction from 10% to 7.5% of myocardial ischemia. Whilst this is a relatively modest difference, it is deemed clinically relevant. Patients with more than 10% myocardial ischemia have a worse prognosis in comparison to patients with less than 10% myocardial ischemia,6The SCRAP trial is an investigator‐initiated, single‐center, double‐blind, placebo‐controlled, cross‐over randomized controlled trial evaluating the efficacy of spinal cord stimulation on the amount of myocardial ischemia in patients with refractory angina pectoris.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.