However, after adjusting for variables, the type of subtrochanteric femoral fractures were no longer significantly associated with progression to death, whereas older age, higher mean Charlson comorbidity score, and Malay ethnicity were the strongest predictors of death. AFF constitutes a small proportion of hip and femoral fractures with prolonged BP use being the highest risk factor for its development. There is no evidence of increased mortality or morbidity in patients with AFF compared with the typical subtrochanteric fracture. The fear of AFF should not impede treatment of typical osteoporotic fractures in this population. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Bisphosphonates (BP) are the most commonly prescribed effective form of osteoporosis treatment with adverse effects associated with prolonged use such as atypical femoral fractures (AFF). Asians have an elevated risk of AFF at 5 to 6 times those of whites and Hispanics. In this study, we characterize factors associated with AFF and its mortality in a single center in Singapore. We conducted a cohort study of subjects older than 50 years admitted to Changi General Hospital (CGH), Singapore, with fragility subtrochanteric femoral fractures from 2009 to 2015. Using the ASBMR 2014 criteria, fractures are classified into atypical and typical subtrochanteric femoral fractures. CGH uses a nationalized electronic health record that allows review of information on patients' demographics, clinical history and previous investigations. Mortality was assessed as of December 31, 2019. Between 2009 and 2015, there were 3097 hip fractures, of which 393 were subtrochanteric femoral fractures and 69 were classified as AFF by ASBMR 2014 criteria. A total of 52.2% of AFF occurred with BP exposure of median duration 56.5 (28 to 66) months. Multivariate regression showed that BP exposure was associated with the highest risk of AFF (odds ratio [OR] = 6.65 [2.35–18.9]). AFF patients had higher 5‐year survival (0.85 versus 0.62,  BPs inhibit osteoclast‐mediated resorption and remodeling of bone. Many large randomized controlled trials have established the efficacy of BP, showing their ability to increase bone mineral density (BMD) and decrease the risk of hip and vertebral fractures by as much as 40% to 70%. In Singapore, oral BP is still the first line of treatment for osteoporosis for most patients. Its affordable cost and oral route of administration render the drug a viable option to most patients.Osteoporosis is a chronic degenerative disease that causes degradation of normal bone structure. It renders an individual to be at higher risk of major fractures. It is projected that more than 50% of all osteoporotic fractures will occur in Asia by the year 2050.1 Sinc This was followed by larger studies of these fractures  and their relation to BP. The pathogenesis of AFF is currently unclear; BP may alter intrinsic bone properties and healing of microcracks, leading to accumulation of microdamage and stress fracture. Other individual specific risk factors may increase susceptibility, either through alterations in bone geometry or microarchitecture, by interacting with antiresorptives, or by increasing biomechanical stress on the femur. These include ethnicity, younger age, higher body mass index (BMI), genetic factors, comorbidities, and concomitant drugs such as corticosteroid use. A recent large network analysis in California reported that Asian race formed half of their AFF cohorts; risk of AFF in Asians was 4.84 versus whites after adjusting for potential confounding variables. Another recent AFF study in Australia also found that among all Asian patients with AFF, Southeast Asian patients may display the highest risk.Case reports of unusual fragility fractures in the subtrochanteric region and along the femoral diaphysis in BP‐treated patients emerged in the literature about 15 years ago. Singapore was one of the first countries in the world to have reported these fractures.4 This the rate of death associated with AFF has not been well established. A previous study in Sweden found lower mortality in AFF compared with ordinary fractures,) whereas another study in an elderly population in UK found no difference in mortality at 30 days. No previous studies in the Southeast Asian population have been performed. Given the likely higher risk of AFF in this population, we set out to study the incidence of AFF in our Southeast Asian population and its related demographic and clinical risk factors. We also assessed mortality rate of patients who have sustained AFF versus those with typical subtrochanteric femoral fractures.Although increased mortality associated with typical fragility fractures of the femur is known,11 the2 guideline for AFF . All data were collected at time of presentation of the fracture.We conducted a cohort study of all patients admitted to Changi General Hospital (CGH) with acute fragility hip, subtrochanteric, and femur fractures from 2009 to 2015. CGH is a regional hospital serving the eastern population of Singapore estimated at 1.3 million. It employs a nationalized electronic health record that stores demographic information, past biochemical and radiological investigations, and prescription records, which can be accessed from all public health institutions in Singapore. All fragility fractures presenting to CGH between 2009 and 2015 were extracted from the electronic medical records with discharge diagnosis “fracture” and “osteoporosis.” Exclusion criteria include traumatic fractures , and non‐osteoporotic or non‐fragility fracture and age younger than 50 years. We then separated all hip and subtrochanteric femur fractures and further extracted all subtrochanteric femoral fractures for further evaluation. Radiographs were obtained for patients with subtrochanteric or femoral shaft fractures to reclassify them into AFF according to the ASBMR 201414 gui2.1Hemoglobin A1c (HbA1c) level (%) was determined by immunoturbidimetric assay . HbA1C values within 6 months of admitted date were included in this analysis. Serum creatinine (μmol/L) was measured using indirect ion‐specific electrode (Roche Diagnostics) and estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology (CKD‐EPI) Eq. 25‐hydroxyvitamin D (25‐OHD) was measured by radioimmunoassay (Roche Diagnostics), and thyroid‐stimulating hormone (TSH) levels were also measured by immunoassay . Baseline biochemical data were collected at the time of presentation for the fracture.2.22, 2.3% for the femoral neck with a least significant change of 0.041 g/cm2, and 1% for the lumbar spine with a least significant change of 0.022 g/cm2. BMD results analyzed for the study were those that were documented within 6 months of the fracture event.All femoral radiographs performed at the time of patient presentation with acute fractures were retrospectively analyzed by adjudicating the radiographic findings with the reporting radiologist and our study member , who was blinded to patients' identity and clinical characteristics, and classified according to the ASBMR 2014 criteria to ascertain if the radiographic appearances fulfill the definitions of AFF. Periprosthetic fractures were excluded from AFF cases. Healing of fracture was assessed through electronic records to the first documented radiographic report of callus formation or union of fracture during orthopedic clinic follow‐up visit post fracture admission. All BMD scans were performed on a single densitometer . The region of interest (ROI) was set as the total hip (non‐fractured hip site), femoral neck, and first to fourth lumbar vertebrae. We excluded vertebrae with fractures or degeneration causing >1 standard deviation greater areal BMD from the immediately adjacent vertebrae in accordance with the International Society for Clinical Densitometry guidelines for individual vertebrae exclusion. The BMD precision error (percentage of coefficient variation) was 1% for the total hip with a least significant change of 0.034 g/cm2.3p < 0.05) in univariate analysis were included in the multivariate logistics regression to identify the independent factor that was associated with AFF risk.All statistical analysis was performed using STATA 16 . Data were expressed as mean ± standard deviation (SD) for numerical data or frequency (percentage) for categorical data. Binary logistic regression was used to assess univariate and multivariate regression analysis in demographic and clinical variables between patients with AFF and typical subtrochanteric femoral fractures. Variables that showed statistical significance (2.4https://www.singstat.gov.sg/find-data/search-by-theme/population/death-and-life-expectancy/latest-data).Patients were followed up until December 31, 2019, to assess their mortality rate. Date of death was accessed through the electronic health record, and whenever possible, the cause of death was recorded. We calculated Kaplan–Meier curves for death by categories of fracture types at 5 years. We then compared the risk of death between AFF and typical subtrochanteric femoral fractures with potential variables that may affect the progression of death in the follow‐up to assess both unadjusted and adjusted HR for death. This was done using Cox proportional hazard ratio to adjust for age, sex, eGFR status, DM2, race, and bisphosphonate used. To understand the excess mortality rate compared with the age‐standardized mortality of the general population, we compared the rate of death after an AFF and typical subtrochanteric femoral fractures to the rate of death in the general population. National death rates and age‐standardized death rates were accessed on the website from the department of statistics in Singapore  were performed using STDRATE procedure in SAS University Edition . A two‐tailed 3p = 0.087). AFF patients had a lower mean Charlson comorbidity score  and lower prevalence of type 2 diabetes mellitus  compared with typical subtrochanteric femoral fractures. There was a higher proportion of females in patients with AFF , and there were no significant differences in the ethnicity of patients with AFF compared with the typical subtrochanteric femoral fractures. Data for 25‐OHD were only present for 76.4% (n = 353) of the cohort, TSH were recorded in 60% (n = 277), while among DM2 patients (n = 131), 80% had a recently documented HbA1C. There were no statistically significant differences between creatinine, eGFR, TSH, 25‐OHD, HbA1C, prevalence of smokers between patients with AFF, and typical subtrochanteric femoral fractures. There was no documented history of alcohol intake in any patients, and hence this data was excluded from the analysis. However, there was a higher rate of BP use  and higher rate of surgical intervention . There were no significant differences between prevalence of previous fragility fractures in the AFF and typical subtrochanteric femoral fracture group. There was no difference in the time to healing as documented by X‐ray, although there was higher prevalence of prodromal symptoms and bilateral fractures in AFF—this was not statistically significant. There were no differences in ethnicity groups between patients with AFF and typical subtrochanteric femoral fractures. Alendronate and risedronate were the most frequently prescribed BP; a small number of patients in both AFF and typical subtrochanteric groups had sequential treatments of oral BP  and sequential treatment to denosumab. Only one patient was exposed to zoledronic acid. Median duration of BP use was significantly longer in those with AFF . Glucocorticoid use was notably higher in patients with AFF . Table T‐scores in total hip  and femoral neck  in patients with AFF versus those with typical subtrochanteric femoral factures. There were no significant differences in BMD of the lumbar spine.Sixty‐nine cases of AFF were identified by the ASBMR 2014 criteria; 5 cases had bilateral AFFs and 3 had incomplete fractures. AFF made up 2.0% of 3097 total hip, femur, and subtrochanteric fractures between 2009 and 2015. Patients presenting with bilateral AFFs were counted as a single AFF case for the purpose of this analysis. Indeterminate cases were excluded from this analysis. The percentage of AFF within subtrochanteric femur fractures in our population has also remained similar over 2009 to 2015 with the proportion of BP‐related AFF being stable  values observed in repeated regression. Creatinine and eGFR also yielded similar OR with significant degree of collinearity. Hence T‐score total hip and creatinine were selected for the final model. Exposure to bisphosphonate exhibits the strongest association with occurrence of AFF . A higher T‐score at the total hip  was also significantly associated with the incidence of AFF compared with typical subtrochanteric femoral fractures. Denosumab use was not significantly associated with AFF occurrence. A non‐DM2 status was also significantly associated with incidence of AFF compared with typical subtrochanteric femoral fractures.Multivariate regression analysis was performed to assess clinical variables that were significantly associated with AFF Table  in patiep = 0.001)  Fig. . The typp = 0.03) compared with the national standardized age mortality rate, while those with typical subtrochanteric femoral fractures have an excess mortality rate compared with the national standardized age mortality rate . To assess if there are subgroups within AFF and the typical subtrochanteric fractures that may have differing SMR, subgroup analyses were performed. SMR remained lower within subgroups of AFF patients stratified into sex, bisphosphonate use, and age. Within the group of patients with typical subtrochanteric femoral fractures, SMR remained higher in all subgroups except for men and BP users, which showed no differences in their SMR compared with the national standardized age mortality rate  patients.8, 9p = 0.001). Factors associated with higher risk of progression to death were older age, higher Charlson comorbidity score, and Malay ethnicity. Indeed, when hazard ratios for death were adjusted for these variables at 10‐year follow‐up, the differences in mortality between AFF and typical subtrochanteric femoral fractures became non‐significant. This implies that comorbidities in patients presenting with these fractures are more important in predicting their mortality outcomes compared with the fracture type alone. The higher mortality rate in the Malay ethnic group compared with the Chinese is also consistent with observations from a previous hip registry study from Singapore. Other studies looking at mortality post AFF have shown no differences or reduced mortality compared with patients with typical subtrochanteric femoral fractures. A previous study in Sweden found lower mortality in patients with AFF in follow‐up duration of a mean of 4 years. Another study found no differences in mortality in a 30‐day follow‐up. When we compared the mortality rates of patients with AFF and typical subtrochanteric femoral fractures to the national standardized age mortality rate in the population in chronological 5‐year increments, we found that mortality risks for AFF patients were lower compared with the age‐standardized mortality in the general population; however, patients with typical fractures remain at a higher mortality risk compared with the general population. Data on comorbidities in the general population are not included in this comparison and thus the SMR should be interpreted in light of this. To assess the influence of factors such as sex, older age category, and bisphosphonate use, we performed further analysis to ascertain if there are differences in SMR in these subgroups. SMR remains lower in the AFF group compared with the age‐standardized mortality in the general population within the different subgroup analysis. In the typical subtrochanteric femoral fracture group, SMR remains higher compared with the age‐standardized mortality in the general population within the different subgroups except for men and those with BP use, which showed no significant difference in their mortality compared with the general population. This points to the fact that typical osteoporotic fracture such as the typical subtrochanteric femoral fracture remains a very high‐risk event when compared with the age‐standardized mortality rate. Bisphosphonates have been shown to reduce mortality post osteoporotic fracture with increasing evidence of both skeletal and non‐skeletal pathways with benefits observed in cancer and cardiovascular outcomes. This may explain the lack of difference in the SMR in the typical subtrochanteric group compared with the general population in those who were BP users.We also sought to study the morbidity and mortality associated with AFF. Although surgical management in AFF patients is higher and there is a higher rate of bilateral fracture, our study found that time to healing and repeat surgery rates are not significantly different from typical fractures. In our follow‐up, we found that there was a higher probability of 5‐year survival for AFF patients compared with those with typical subtrochanteric femoral fractures  was not significantly associated with progression to death. SMR of AFF is also lower than that of the age‐standardized mortality rate in the general population compared with typical subtrochanteric femoral fractures. There is also no evidence of increased morbidity or need for repeated surgical interventions. These data are reassuring for our population given the much higher risk of AFF that is known in the Asian population. Continued effort to reduce mortality in typical osteoporosis fractures should be pursued with the use of bisphosphonates and other anti‐osteoporotic drugs. The fear of AFF should not impede prevention of typical osteoporotic fractures.The main strength of the study was long duration follow‐up of the patient and the link‐up of the electronic medical record for patients in Singapore, which allows us to accurately document a patient's clinical and medication history. We were able through this linkage to ascertain the date of death to provide accurate mortality data. We were also able to identify AFF by adjudication of the radiograph according to the ASBMR 2014 guideline. Limitations include the observational study design and the potential for residual confounding owing to differences in factors related to frailty, socioeconomic status, BMI, and BMD. There were missing data on 25‐OHD, TSH, and HbA1C, which may not accurately reflect differences present within the study population. There were very small numbers of patients on denosumab, and these patients were all previously exposed to BP. This may imply that we are unable to accurately ascertain the independent association of denosumab with AFF occurrence in this analysis. We also relied on the accuracy of the documented electronic health record for smoking and alcohol intake. There was no documented alcohol history intake in any patients, and we were unable to analyze this as a factor in contribution to the association with AFF and mortality. Ever‐smoker and current smokers were treated similarly in our analysis regardless of their last history of smoking. This may not accurately reflect the effect of smoking on the association of AFF and mortality as recency of exposure may play a significant part in the contribution of smoking as a pathological factor. We were also not fully able to ascertain the cause of death in most patients because the cause of death was not listed. There were also low numbers of patients in the older age groups and as such the interpretation of the results in these older groups should be done with caution. This study is also performed in a Southeast Asian population and as such its results may not be applicable in a different ethnicity setting. Comparison of the SMR with the general population is also limited by data on comorbidities present in the general population that was not adjusted for in the model.In summary, AFF constitutes a small number of fractures in our population, and long‐term bisphosphonate exposure remains the strongest risk factor for its development. A total of 52.2% of AFF occur in BP users, and long‐term bisphosphonate use is the strongest risk factor for the incidence of AFF with median duration use of 56.5 months. Despite higher rates of surgery with AFF, there is no evidence of increased mortality or morbidity in this population compared with the typical subtrochanteric fracture. There was a lower risk of mortality in AFF compared with national age‐ and‐sex standardized mortality rate, but there was a higher mortality rate in the typical subtrochanteric fractures. The fear of AFF should not impede prevention of typical osteoporotic fractures, which carry a high risk of mortality. Further studies would be important to understand risk factors and physiology of the increased risk of AFF in the Asian population.All authors state that they have no conflict of interest.Linsey Gani: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; validation; writing‐original draft; writing‐review & editing. Le Roy Chong: Data curation; investigation; validation; writing‐original draft; writing‐review & editing. Natasha Anthony: Data curation; project administration. Lily Dacay: Data curation; investigation; project administration. Pei Tan: Formal analysis; methodology; validation; writing‐original draft; writing‐review & editing. Thomas King: Conceptualization; formal analysis; investigation; methodology; project administration; supervision; validation; writing‐original draft; writing‐review & editing.5https://publons.com/publon/10.1002/jbm4.10515.The peer review history for this article is available at


Using a new marker of necrosis, pSer46‐MARCKS, which was identified by comprehensive phosphoproteome analysis as a phosphoprotein changed before appearance of extracellular amyloid aggregation, we discovered that neuronal necrosis occurs much earlier in Alzheimer's disease pathology than previously expected. The necrosis is induced by intracellular amyloid accumulation that deprives a critical effector molecule, Yes‐associated protein (YAP), in the Hippo signaling pathway that is essential for cell survival, similarly to TRIAD necrosis observed in transcriptional repression and in other neurodegenerative diseases such as Huntington's disease. The initial TRIAD necrosis due to the intracellular amyloid releases HMGB1 into extracellular space and induces cluster of secondary necrosis around the primary necrotic neurons. Finally, the cluster grows into extracellular amyloid plaque. Inhibition of HMGB1 by anti‐HMGB1 antibody prevents expansion of neurodegeneration. Administration even after onset significantly ameliorates the cognitive decline of Alzheimer's disease model mice. Our results present a new theory of Alzheimer's disease pathology, which can be referred to as the “intracellular amyloid hypothesis". The amyloid hypothesis, which was proposed by Professors Hardy and AllsopUnfortunately, various anti‐Aβ antibodies with different interaction properties for monomers, oligomers, and polymers of Aβ developed by big pharmaceutical companies have not succeeded in improving the symptoms of AD patients, even though, judging from PET studies, most of such anti‐Aβ antibodies have successfully reduced the Aβ burden in patients’ brains.The results of three clinical Phase III studies on aducanumab, the last hope of anti‐Aβ antibodies, are now under review by the Food and Drug Administration (FDA), as of December 2020, while on November 6, 2020, the FDA Peripheral and Central Nervous System Drugs Advisory Committee announced that they did not support evidence of the effectiveness of aducanumab for the treatment of Alzheimer's disease in three clinical studies: Study 302 (EMERGE), Study 301 (ENGAGE), and Study 103 (PRIME) .One explanation for the ineffectiveness of anti‐Aβ antibodies was that the intervention for patients with mild cognitive impairment (MCI) or early‐stage AD in clinical studies was too late. Based on this view, intervention for pre‐onset people who will have AD later should be successful. However, early in 2020, a DIAN study on solanetumab for familial AD patients before the onset of symptoms did not reveal positive results.We have not been interested in whether or not the amyloid hypothesis is true but simply in pursuing an understanding of AD pathogenesis and development of AD therapeutics based on comprehensive analyses that could exclude bias of prejudices. From 2010, we started comprehensive phosphoproteome analyses of brain tissues from model mice and human patients with polyglutamine diseases (Huntington's disease and cerebellar ataxia)Furthermore, we discovered that HMGB1‐TLR4 signal triggers MARCKS phosphorylation at Ser46.Moreover, we analyzed in detail the relationship between the ultra‐early‐stage necrosis and intracellular Aβ accumulation by using time‐lapse imaging of iPSC‐derived neurons and by time‐lapse imaging of living cortical neurons in mouse brains with two‐photon microscopy.Since our group first reported the existence of intracellularly accumulated Aβ in cortical neurons of human familial AD patients with a presenilin 1 (PS1) gene mutation and in AD model mice,Our group performed comprehensive phosphoproteome analyses with four types of AD mouse models  and with postmortem human AD brains. Cerebral cortex tissues were sampled at one, three, and six months of age, and we confirmed with multiple types of anti‐Aβ antibodies that amyloid plaques were not present at one month of age and appeared from three months of age.We next selected four phosphorylation sites of MARCKS from a number of phosphorylation sites under the conditions that they were changed at one month of age and that they were also changed in postmortem human AD brains.At this point, we came to the hypothesis that neuronal necrosis occurs at the site where extracellular amyloid plaque is formed later and that some damage associated molecular patterns (DAMPs) molecules influence neurites connecting or neighboring the necrotic neuron and there induce cell signaling, leading to pSer46‐MARCKS that degenerate neurite and dendritic spines.We examined the hypothesis by adding DAMPs molecules such as Aβ, tau, and HMGB1 and found that HMGB1 induces pSer46‐MARCKS most efficiently. Aggregated Aβ has no effect and Aβ oligomer has a weaker effect than HMGB1 on MARCKS phosphorylation at Ser46.Moreover, we generated a new mouse monoclonal antibody against HMGB1 and revealed that administration of the antibody before the onset ameliorated cognitive impairment at the time point from onset and one month later.We next utilized pSer46‐MARCKS as a marker for necrosis. Neurons under ongoing necrosis accompany reactive degeneration of surrounding neurites, which are stained by pSer46‐MARCKS, and such neurons possess nuclei with weak DAPI signals. Using the criteria, we quantified the frequency of active necrosis in the cerebral cortex tissues of 5xFAD and mutant APP‐KI mice every month from birth and revealed that active necrosis reached peak frequency at two months of age before the appearance of extracellular Aβ aggregates but continued to 18 months of age.Collectively, our analyses of active necrosis in two types of AD mouse models with a new marker pSe46‐MARCKS indicated that accumulation of intracellular amyloid causes neuronal necrosis, leading to extracellular amyloid aggregation. In other words, that extracellular amyloid aggregation is the result of neuronal necrosis caused by intracellular amyloid.in vivo by two‐photon microscopy. Intracellular amyloid accumulation expands ER to induce necrosis and intracellular amyloid remains in the extracellular space after cell death.To examine this new concept, we differentiated neurons from iPS cells carrying APP mutation generated by genome editing and investigated necrosis of the iPSC‐derived neurons by time‐lapse imaging. Consequently, we found that accumulation of intracellular amyloid is followed by abnormal expansion of ER, causing necrotic rupture of cells.Finally, we investigated the molecular mechanism of active necrosis. Active necrosis is morphologically characterized by extreme expansion of ER, and it is identical to the feature of transcriptional repression‐induced atypical cell death (TRIAD) related to YAP that we reported in transcriptional repressionTherefore, we examined the therapeutic effect of adeno‐associated virus (AAV)–YAP rescuing the impairment of YAP function and revealed that AAV–YAP not only rescued cognitive decline significantly but also decreased extracellular amyloid aggregation of AD model mice remarkably.Our data‐driven research, which is independent of the existing AD hypothesis, presented an obviously distinct view for AD pathology. Extracellular amyloid aggregation is the result of TRIAD necrosis caused by intracellular amyloid accumulation. Neuronal degeneration spreads to surrounding neurons due to DAMPs such as HMGB1 being released from original necrosis , which accelerates the AD pathology. The phenomenon at a glance seems homologous to prionoid transmission of degenerative proteins, while it is clearly distinguished because DAMPs mediate the expansion of degeneration. Inhibition of the degeneration expansion by anti‐HMGB1 antibody and suppression of necrosis by AAV‐YAP both have therapeutic effects, and they are complementary and synergic as therapeutics targeting sequential phases in the ultra‐early phase pathology of AD Fig. .


Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane‐embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high‐resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state‐of‐the‐art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design. Recent single‐molecule FRET studies on members of various transporter families reveal the potential of this technique to advance the mechanistic understanding of this protein class. HeA particular challenge has always been the investigation of large proteins and oligomeric transporters. However, recent advances show that the strategic labeling of oligomeric transporters can even be an advantage.[Ph transporter. Here, each subunit was labeled with either a donor, an acceptor, or a surface‐binding tag.Ph transporters in liposomes. Only molecules of the desired orientation, i. e., with the surface‐binding tag on the outside of the liposomes were immobilized whereas molecules that were inserted in the opposite direction were washed off the surface.A particularly successful example is the homo‐trimeric Glt−/proton exchanger CLC‐ec1 from E. coli, where the dynamics of the individual subunits was explored.via point mutations in the gating motifs. After the self‐assembly of the monomers into dimers, only CLC‐ec1 molecules with at least one inactive and surface‐binding monomer were immobilized and only those dimers with a labeled second monomer were detectable in the smFRET experiments. Thus, all dimers contained one active and one inactive monomer, which allowed the authors to selectively monitor the dynamics of only one monomer in the assembly.Another example of smFRET experiments with oligomeric transporters is described in the recent study of the dimeric ClPh.via biotinylated lipids and the subunits of the transporter were labeled with donor and acceptor dyes in a 1 : 2 or 2 : 1 ratio. Afterwards, the different species were separated on the basis of the expected dye‐stoichiometries, using the Alternating‐Laser‐Excitation (ALEX) method.A distinct approach for smFRET experiments with an oligomeric protein was used in another study on Gltet al. The combination with ALEXA proof‐of‐concept study on the novel concept of “caging chromophores”2.2An important factor in the study of transporters is the membrane environment, which is often difficult to realize experimentally.[Ph, dynamics in micelles do not differ significantly from those found in a lipid bilayer.[via liposomes or nanodiscs/nanoparticles.For certain transporters such as GltHowever, creating liposomes of complex lipid composition3Ph[SmFRET has already been used to study transporters from various families. Representatives for each family and their respective substrates are illustrated in Figure 3.1ATP binding cassette (ABC) transporters are primary active transporters.In addition, importers possess PBPs that bind the substrate and deliver it to the transmembrane transporter.[E. coli and OpuA from Lactococcus lactis) and eukaryotic (PgP from Mus musculus and MRP1 from Bos Taurus) ABC transporters.[SmFRET studies were carried out on prokaryotic .[−1 or longer in nanodiscs, the protein was more dynamic in liposomes.A careful comparison showed that the data for McjD are overall consistent between detergent and a lipid environment with only minor differences. The opening of the TMDs in the presence of substrate is slightly wider and the closure of the NBDs with ATP is less tight in liposomes compared to detergent.In contrast to exporters, no spontaneous fluctuations of the NBDs and TMDs were observed for the importer BtuCD. Yet, the time‐resolution in these experiments was 50 ms and faster dynamics may have been missed.Finally, we would like to note that homology modeling had been successfully used to design labeling positions as shown for OpuA.3.22+‐ATPase ortholog LCMA1 from Listeria monocytogenes2+, are rate limiting. In addition, smFRET allowed to monitor so‐called de‐occluded conformations that couple ion transport and ATP hydrolysis. Unfortunately, these states have not yet been described by x‐ray crystallography or other imaging techniques to date.Another well‐studied family of primary active transporters are Phosphorylation‐type (P‐type) ATPase transporters. These membrane proteins are found in all kingdoms of life where they pump cations against their concentration gradients utilizing ATP hydrolysis, which preserves the electrochemical potential across cell membranes.3.3Bacillus cereuss.et al. crystallized the inward‐open conformer in a cross‐linked form and utilized smFRET to verify that this form is indeed similar to the native conformation.Membrane embedded Enzymes IIC (EIIC) are essential components of the phosphoenolpyruvate‐dependent phosphotransferase system that are responsible for sugar uptake. In complex with EIIC‐transporters, sugar is phosphorylated and dissociates.Further smFRET experiments on the native transporter without the crosslinker, identified two conformations in detergent solution out of which the outward‐open conformation was thermodynamically favored, which explains the crystallization bias towards this conformer.3.4The Major Facilitator Superfamily (MFS) is a superfamily of secondary active transporters that is ubiquitous in all kingdoms of life.via an alternating access mechanism.[All MFS transporters share a common fold. It is characterized by 12 transmembrane helices (TMs) that are grouped into two bundles of six consecutive helices each. The bundles are referred to as the N‐terminal and C‐terminal bundle and enclose the substrate binding site.E. coliThe first smFRET experiment on an MFS transporter was carried out on OxlT, an Oxalate:formate antiporter from Subsequent smFRET studies focused on elucidating the conformational states of MFS transporters during the transport cycle, the timescale of transition between those states, and the influence of substrate and pH.[E. coli and the multidrug efflux pump LmrP from Lactococcus lactis adopt inward‐open and outward‐open states in detergent solution.[E. coli on the contrary almost exclusively populate inward‐open states under these conditions.[In its apo state, the multidrug efflux protein MdfA from −1 compared to<10 s−1 for LmrP.[Interestingly, MFS transporters operate at different timescales. In the absence of substrate, MdfA switches conformations with a rate of 0.2 sMost importantly, the studies on DtpA and LmrP revealed that the periplasmic and cytoplasmic side of the transporters are decoupled in detergent,[Finally, it should be mentioned that the conformational state adopted by LmrP and MdfA is influenced by pH. Both proteins preferentially adopt the inward‐open state at low pH[In summary, smFRET studies revealed that MFS transporters are highly flexible proteins and our simplistic model of rigid body movements do not fully describe the transport cycle. Moreover, whereas general transport principles are shared among different transporters, dynamics, conformations, and the response to pH changes or substrate additions can differ substantially.3.5Besides the MFS superfamily, the amino‐acid‐polyamine‐organocation (APC) superfamily is another large superfamily of secondary active transporters.Since the study of BetP (BCCT family) focused mainly on developing smFRET methodology using “caged chromophores”Secondary active NSSAquifex aeolicus, identified conformational variety with opening‐closing dynamics of ∼60 s. The residence times in the outward‐open state were longer in the presence of substrates, thus stabilizing this state and slowing the transport cycle. In addition, Zhao et al. unraveled the molecular basis of the specific TM1a helix and its role in the intracellular gating mechanism using various mutations followed by smFRET measurements. Further smFRET studies+ ion in the second binding site of the transporter. The different substrates and ion modulation in solution from Na+ to Li+ ions, affected the kinetic rates in the transport cycle.Several smFRET studies on LeuT,[+‐binding to the extracellular side of the transporter did not only serve as driving force for transport, but it also provided the required selectivity for a substrate, closed allosterically the intracellular gate, and prevented the premature diffusion of Na+ to the intracellular side. Furthermore, the authors showed that open states on both sides of the membrane exist simultaneously, which deviates from the paradigm of the alternating access model.Another smFRET study of LeuTet al. outline the specific amino acids F259 and I359 in the binding pocket and their action as a “volumetric sensor” that dictates different transport rates according to the chemical identity of the substrates . The study revealed that the substrate size affects the rotamer dynamics of a phenylalanine residue (F259) and is associated with the occupancy of the intermediate conformational state (IO2), which in turn controls the intracellular release of Na+ that was already proven to be pivotal in the rate determining step.The most recent study of LeuTBacillus halodurans with a LeuT‐like structure−1. In the case of the substrate leucine, the second half of the transport cycle, i. e., the return from the apo form to the outward‐open conformation, was found to be the rate‐limiting step with a rate constant of about 1 s−1. But in contrast to the current transporter model for NSS transporter dynamics, the second half‐cycle depended on the transported substrate of the first half of the reaction cycle, suggesting the presence of an extracellular allosteric substrate binding site.The amino acid transporter MhsT is an NSS homolog from + ions increased the transport rate in MhsT while Na+ lowered it.Surprisingly, unlike in LeuT, H3.6Similarly to the NSS family, members of the Excitatory‐Amino‐Acid‐Transporters (EAAT) family are also secondary active sodium‐dependent transporters.Ph from Pyrococcus horikoshi is a suitable model for smFRET experiments because high resolution structures are available for several conformations.[The homolog Glt276,395‐GltPh). Ensemble experiments with this variant but also with the human transporter showed faster dynamics compared to the wild type protein.Ph, were absent in the H276,395−GltPh variant. In addition, smFRET histograms showed that the locked configurations, which are responsible for the quiescent periods, were disrupted in H276,395−GltPh. The inward‐open states of the variant showed an increased donor‐acceptor distance compared to wild type, suggesting an enlargement of the binding pocket in the inward‐open state, which was confirmed using x‐ray crystallography and molecular dynamics simulations.Initial smFRET studiesPh studied the synchronicity between the movements of the homo subunits of the transporter in a vesicle environment. Erkens et al. explored the dynamics that create the symmetric outward‐facing statePh monitored aspartate‐uptake in liposomes using an L‐aspartate‐binding FRET sensor. Surprisingly, uptake rates for single molecules were found to be preserved over multiple uptake cycles, thus exhibiting “molecular memory”, i. e., transporters with slow rates and transporters with fast rates preserve their speed over successive uptake cycles.Another smFRET investigation3.7E. coli. This transporter functions as an antiporter that removes drug molecules from the cell via a coupling to the import of protons.et al. were able to show that EmrE is organized as an antiparallel dimer in which both subunits transport the substrate at the same time but in opposite directions.Another family of secondary active transporters studied by smFRET is the small multidrug resistance (SMR) family. Members of this family are found in prokaryotes where they utilize an electrochemical proton gradient to expel toxic substances such as quaternary ammonium compounds from the cell.[3.8E. coli− ions and simultaneously exports H+ to the environmentThe chloride‐carrier‐channel (CLC) family consists of ion channels and transporters. SmFRET studies of a CLC transporter  family do not require an additional energy source for transport. SWEETs facilitate the transport of mono‐ and disaccharides along the concentration gradient.[A. thaliana provided new insights.A recent x‐ray study combined with smFRET measurements and functional characterizations of AtSWEET13 from 4With the recent advances of high‐resolution techniques,[SmFRET has been shown to complement such structural data with information on alternative conformers and their associated timescales of interconversion. This is a key advantage, not only compared to x‐ray crystallography or cryo‐EM, but also to other ensemble biophysical measurements.To understand transport mechanisms on a molecular basis, data interpretation of smFRET experiments can highly benefit from available high‐resolution structures, combined with MD simulations, mass spectrometry, SAXS and NMR studies. The power of combining smFRET studies with NMR spectroscopy has already been demonstrated on model systems such as EmrE and members of the glutamate transporter family.[In this mini‐review, we discussed recent smFRET results on membrane transporters and highlighted their contributions to advance the mechanistic understanding of this protein class. Technical developments in the field of smFRET spectroscopy, including available commercial setups and the fact that multiple protocols and tools for labeling proteins with fluorescent dyes are available, suggest that this method will evolve to a standard approach for investigating membrane proteins in the future. We expect that it will be possible to record full transport cycles of diverse transporters at a single‐molecule level and to deduce their kinetic characteristics. Such setups not only open the door to understand the impact of different lipids on transport rates and conformational dynamics but they will also be essential in elucidating the function of transporter binding proteins, which have recently been discovered.[The authors declare no conflict of interest.Kim Bartels studied chemistry and biochemistry at the Ludwig Maximilian University Munich (Germany). During her studies she worked on the structure determination of bacterial transmembrane proteins under the supervision of Dr. K. Beis . After receiving her MSc degree in biochemistry, she joined the group of Christian Löw at EMBL Hamburg as a PhD student in 2016. Here, she was using single molecule FRET to study the structural dynamics of proton‐dependent oligopeptide transporters.Tanya (Tatyana) Lasitza Male studied B.Sc. in Chemistry in Tel‐Aviv University (Israel). Afterwards she continued to M.Sc. in the Department of Physical Chemistry (Tel‐Aviv University). Her M.Sc project was guided by Dr. Eli Stern and Prof. Dan Huppert She is currently studying for her Ph.D. in the Department of Chemical and Structural Biology of the Weizmann Institute of Science (Israel), under the supervision of Dr. Hagen Hofmann. She is specializing in single‐molecule fluorescence spectroscopy and dynamics of membrane proteins.Hagen Hofmann (born 1978) studied biochemistry and received his PhD from the Martin‐Luther University Halle‐Wittenberg (Germany). He did his post‐doctoral research in single‐molecule biophysics at the University of Zurich (Switzerland). He then joined the Department of Chemical and Structural Biology of the Weizmann Institute as group leader, where he is working on the dynamics of intrinsically disordered proteins and protein complexes, especially using single‐molecule fluorescence spectroscopy.Christian Löw (born 1979) studied biochemistry at the University of Bayreuth (German) and received his PhD from the Martin‐Luther University Halle‐Wittenberg (Germany). He did his post‐doctoral research in membrane protein structural biology at the Karolinska Institute in Stockholm (Sweden). He then moved as group leader to the European Molecular Biology Laboratory (EMBL) in Hamburg and joined the Centre for Structural Systems Biology (CSSB), where he is studying integral membrane nutrient transporters, mainly using structural and biochemical approaches.


Processes of the patient and public involvement (PPI) in health research shifted quickly during 2020. Faced with large‐scale issues, such as the COVID‐19 pandemic, the need to adapt processes of PPI to uphold commitments to nurturing the practice of ‘nothing about us without us’ in research has been urgent and profound. We describe how processes of PPI in research on patient‐oriented methods of knowledge translation and implementation science were adapted by four teams in a Canadian setting.As part of an ongoing quality improvement self‐study to enhance PPI within these teams, team members shared their experiences of PPI in the context of this pivotal year during interviews and facilitated discussions. Drawing on these experiences, we outline challenges and reflections for adapting processes of PPI in health research on methods in times of urgency, conflict and fast‐moving change.Our reflections offer insight into common issues encountered across teams that may be amplified during times of rapid change, including handling change and uncertainty, sustaining relationship‐building and hearing differing perspectives in processes of PPI.These learnings present an opportunity to help others active in or planning patient‐oriented methods research to reflect on the changing nature of PPI and how to adapt PPI processes in response to turbulent situations in the future. The key reflections presented draw heavily from perspectives shared by eight patient and public partners in interviews and facilitated discussions . PPI involves doing research with, or by, the public rather than to, about, or for the public.The full impact of COVID‐19 on practices of PPI is not yet known. Academic institutions and community organizations face challenges  in operating within the rapidly altered landscape, which has the potential to jeopardize advances in PPI.In this viewpoint, we contribute to a small but growing literature that aims to support research teams to uphold commitments to nurturing PPI across all communities through times of fast‐moving change.2https://bcsupportunit.ca/about/methods-clusters/knowledge-translation-and-implementation-science). Each project team is diverse in its composition and process of PPI. Each team also has a separate focus, including studying consensus methods in integrated KT to promote patient‐oriented research, a hermeneutic approach to IS, the creation of an online set of systems‐thinking tools for community groups, the development of an online portal for citizen science, and using documentary as a method of KT to reach the ‘sandwich generation’. All are led by senior researchers with long histories of PPI and involve at least one patient partner.Across Canada, a Canadian Institutes of Health Research's national initiative, Strategy for Patient‐Oriented Research (SPOR), funds 11 provincial SUPPORT (SUpport for People and Patient‐Oriented Research) Units that aim to support partnerships in patient‐oriented research between academics and others, including patients and the public.https://albertainnovates.ca/programs/arecci/), it was determined the self‐study was a low‐risk quality improvement study that did not need research ethics board review. At the design stage of the self‐study, we did not anticipate COVID‐19 and its impact, nor that the self‐study would come to offer insight into how to adapt PPI during times of turbulent social change. Figure In 2019, we began a quality improvement self‐study with the project teams to gain a better understanding of what it means for patients and the public to partner in research on methods. We conceptualized self‐study as a methodology for studying professional practice that was self‐initiated, collaborative and aimed towards improvement through learning from experience.As part of the self‐study, 18 members of four project teams  took part in one‐to‐one interviews between January and April 2020. One project team chose not to participate in the interviews due to team priorities before and during the COVID‐19 pandemic; however, the team's project lead provided regular updates on their activities and contributed to overall self‐study reflections. Each interviewer  had over 10 years of experience in conducting qualitative interviews and neither were members of project teams at the BC SUPPORT Unit. Interview participants were asked to describe their experiences of engagement, including any successes, surprises or tensions. Twenty‐five project team members  then took part in a reflective discussion informed by the interviews in September–October 2020. Two meetings took place in November–December 2020 among authors with a purpose to discuss further theme development in light of perspectives shared during facilitated discussions. During these meetings, authors reflected on how project teams adapted their PPI over the turbulent year. These reflections were informed by ongoing analysis of interview data and perspectives shared during the facilitated discussions, as well as first‐hand experiences of L. L., M. M. and N. O. as project team members. Themes from the ongoing self‐study will be published separately. Reflections from the debrief meetings are presented here to offer early empirical insight for others to consider when aiming to optimize PPI through times of turbulence.33.1Given the little evidence available to guide PPI in studies of KT‐IS methods, many team members identified that they had been feeling out of their comfort zone at various stages of their involvement before the COVID‐19 pandemic was announced. Although many team members had extensive experience of PPI in research, few had been involved in PPI in research specifically on KT‐IS methods before. At the beginning of the COVID‐19 pandemic, projects in the Methods Cluster halted immediately. Faced with this unexpected interruption, team members relied on approaches they used prepandemic to attend to change and uncertainty COVID‐19 brought about.One approach identified to attend to change and uncertainty was remaining flexible. Before COVID‐19, plans had often been adapted in response to ‘uncontrollable’ aspects involved in exploring how to do partnered research in KT‐IS methods. Adapting to change and uncertainty with kindness, empathy and patience for other team members as well as themselves was articulated as critical among project team members, to ease feelings of frustration, confusion or guilt that could arise. The role of the project lead was also identified as crucial to providing ‘good orientation’ during times of uncertainty and change. This involved timely check‐in conversations with team members to ask open‐ended questions about expectations and discuss changes to project aims and/or designs due to external contextual demands on team members.Similar approaches are also supported in the emerging literature. For example, the value of placing a renewed focus on empathy, humanism, honesty and humility is highlighted by Carson et al.3.2Many project team members had already built relationships with each other before joining their respective projects, with some having research partnerships that had developed over years. The benefits of forming the project teams with established relationships were recognized among team members before COVID‐19, particularly because easier social connections and trust that had been built over time helped to mitigate feelings of unease during times of uncertainty and change. Having established these trusting relationships was a particular advantage to sustaining relationship‐building after the outbreak of COVID‐19 because they allowed team members to be more open and responsive to each other's needs in the fast‐changing context.Before COVID‐19, sustaining relationships had involved taking opportunities to engage in conversation or ‘chit‐chat’ that was non‐project‐related with other team members. The project lead created these opportunities during regular in‐person team meetings or by making themselves available outside of these meetings. A research staff member also described meeting a patient/public partner over coffee to connect on a more informal level while discussing their project. During the COVID‐19 pandemic, the need to evolve these usual methods for sustaining relationships was immediate, especially with physical distancing measures and no in‐person activities. Leveraging online communication platforms was welcomed, particularly by a patient partner who had used technology regularly before COVID‐19 to join team meetings. He emphasized too, however, there were sometimes technical glitches, or had difficulty sharing documents or ideas when brainstorming with other attendees.Others also reworked quickly with large teams to continue PPI during the COVID‐19 pandemic, supported by long‐standing relationships.More can also be learned about how online communication could provide opportunities to build new relationships with additional partners who previously would not have been able to travel to meetings in person. Hausmann et al., for example, describe how, in a few short months, social media platforms enabled researchers and patient and public partners (including over 100 patient support organizations) to partner during the COVID‐19 pandemic as part of the COVID‐19 Global Rheumatology Alliance.3.3Before the outbreak of COVID‐19, project team members identified hearing differing perspectives as a vital component of successful PPI. The importance of hearing others' perspectives in the process of establishing shared goals and reaching mutual benefit was commonly highlighted. A key benefit of hearing differing perspectives shared by patient and public partners was that it kept discussions focused or ‘grounded’ in the real‐world significance of the studies, which was particularly important given the seemingly more abstract nature of research on methods.In Plain Sight, was released in November 2020 describing widespread systemic racism and discrimination against Indigenous peoples in the British Columbia health care system, perpetuating the existing inequities.During facilitated discussions of the self‐study, project team members revisited conversations on what it meant to hear differing perspectives in PPI in 2020. These discussions were particularly prompted by ongoing issues of social justice that gained increased attention through the Black Lives Matter (BLM) movement and efforts of Truth and Reconciliation with Indigenous Peoples. The COVID‐19 pandemic had intensified the spotlight on existing inequalities and the value of engaging with patient and public partners most at‐risk, including Indigenous, Black, Latino and other Minority Ethnic partners.Others have highlighted that PPI has been criticized for not sufficiently including patient and public partners from Indigenous, Black, Latino and other Minority Ethnic communities.4Our reflections contribute to the growing literature that can support meaningful PPI practices to adapt and persist in turbulent times of fast‐moving change. Recent historic events disrupted many aspects of PPI. It is imperative researchers take advantage of the opportunities this disruption has brought to improve PPI practices so that all patient and public partners are meaningfully involved in shaping the future of their health through involvement in research if they choose.The authors declare that there are no conflict of interests.All authors contributed to the conceptualization of this manuscript. Jenny Leese led the initial draft with substantial contributions from Martha MacLeod. All authors commented and contributed to the revision of the final version. Jenny Leese and Leana Garraway are research associates with experience in qualitative research and patient and public involvement in research.


A 51‐year‐old man had hematochezia, anemia, and an intraabdominal mass. Gastroscopy and colonoscopy showed no significant lesions with intraluminal bleeding, while radiological examinations showed bulky swelling of the lymph nodes around the abdominal aorta and a tumor in the left ischial bone. Small intestine endoscopy detected a dark purpuric protruding tumor of the jejunum and its biopsy specimen brought a definitive diagnosis of primary jejunal epithelioid angiosarcoma from positive staining of AE1/AE3, CD31, and erythroblast transformation specific related gene in immunohistochemical studies. The patient underwent surgical resection with adjuvant chemotherapy but died of progression of metastases 7 months after the diagnosis. Epithelioid angiosarcoma of the gastrointestinal tract is an extremely rare malignancy with poor prognosis and it is challenging to distinguish from undifferentiated carcinoma or melanoma. Immunohistochemistry is necessary for a definitive diagnosis. Sufficient biopsy specimen may aid a prompt diagnosis of this disease of the small intestine, which may present as obscure gastrointestinal bleeding. We present a case of successful diagnosis of small intestinal angiosarcoma settled by endoscopic biopsy of the jejunum. Angiosarcoma is a rare malignant entity, accounting for just 1–2% of all types of sarcomas; it generally occurs in the skin and the subcutaneous tissue.A 51‐year‐old man visited a clinic with tarry stool, loss of body weight, and severe anemia for a month. The patient was a government official and not an occupationally exposed person. Ultrasound scan showed bulky swelling of the lymph nodes around the superior mesenteric artery (SMA). He was presented to our hospital with a temporal diagnosis of malignant lymphoma for a detailed treatment. He was febrile and had fatigue, and laboratory tests showed anemia  and hypoalbuminemia . Other significant laboratory data were serum iron level 10  and serum soluble interleukin 2 receptor (sIL2R) level 775 . Computed tomography (CT) showed enlarged lymph nodes around the SMA to the abdominal aorta and segmental wall thickness of the jejunum Figure . F‐18 flWe performed laparoscopic partial resection of the jejunum as the patient's recovery from anemia was difficult, despite repeated blood transfusion. A protruding tumor of 10 × 12 mm in size was found in resected tissue of the jejunum Figure  and it hPostoperatively, he received weekly administration of paclitaxel as a first line chemotherapy. His therapeutic regimen was then changed to doxorubicin because of allergic reaction. Rapid disease progression with cancer pain and cancerous cachexia resulted in his death 7 months after the diagnosis.Angiosarcoma is a rare malignancy of endothelial origin, which ranges from well‐differentiated tumor to high‐grade spindle cell type and a unique morphologic subtype of angiosarcoma that shows a predominantly epithelioid appearance with sheet‐like growth. This atypical epithelioid subtype has been defined as epithelioid angiosarcoma,The etiology of angiosarcoma is still unclear, although several risk factors  relate to its pathogenesis.Definitive diagnosis of epithelioid angiosarcoma of gastrointestinal tract is quite difficult because of its various symptoms, insufficient specimen sampling and histopathological similarity of poorly differentiated carcinoma.Treatment modalities for gastrointestinal epithelioid angiosarcoma include surgical resection, radiation therapy, and chemotherapy, which can be performed alone or in combination depending on the progression of each case.In conclusion, our patient with jejunal epithelioid angiosarcoma may be the first case to be successfully diagnosed by biopsy specimen from small intestine endoscopy. Gastroenterologists should consider this rare disease as a differential diagnosis for obscure gastrointestinal bleeding. We recommend immunohistochemical staining of CD34 and ERG positively and S‐100 and EMA negatively.The authors declare that there is no conflict of interest.The authors report the details of the patient's case in accordance with the ethical standards of the Declaration of Helsinki.None.


The authors have no potential conflicts of interest to disclose.Conceptualization,Writing ‐ original draft,Writing ‐ review & editing: Fenglin Jiang.Dear Editor,We have read with great interest the article “Electrocardiographic markers of increased risk of sudden cardiac death in patients with COVID‐19 pneumonia” by Alareedh et al., .E. coli culture? ACE2 mediates the destruction of endothelial cells, and thrombosis may be the mechanism. Where is prethrombotic state and hypercoagulable state? It is just an academic discussion of assumptions. The ECG of pulmonary embolism is the camouflager of the heart world. It has various manifestations. Autopsy seems to have proved everything and myocardial infarction.The difference between respiratory system virus, circulatory system virus, pulmonary hemorrhagic fever virus, and enterovirus is not very important. Is there still less virus in Acute cardiac injury incidence in COVID‐19 is about 13 times higher in the intensive care unit /severely ill than in less critical patients. The cytokine release with complement and iNO dysregulation are established mechanisms potentially leading to sepsis‐related cardiomyopathy, making sepsis per se one of the potential mechanism leading to acute cardiac injury in COVID‐19 patients. Moreover, the hyper‐inflammation with endothelial dysfunction is likely be responsible of both pulmonary in situ platelet aggregation and deep thrombosis potentially leading to severe pulmonary embolism and right ventricular failure(Tavazzi et al.,


Markets are increasingly being incorporated into many aspects of daily life and are becoming an important part of the conservation solution space. Although market‐based solutions to environmental problems can result in improvements to conservation, a body of social science research highlights how markets may also have unforeseen consequences by crowding out or displacing 3 key types of behaviors potentially relevant to conservation, including people's willingness to engage in collective action and civic duty; tolerance for inflicting harm on others ; and desire for equity. Better understanding of the contexts and mechanisms through which this crowding out occurs and whether specific market‐based instruments are more prone to different types of crowding out will be crucial to developing novel conservation initiatives that can reduce or prevent crowding out. Article impact statement: Markets are important to conservation‐solution space but can crowd out 3 aspects of human behavior relevant for conservation. Markets play a critical role in the mediation of people's relationship with nature through their influence on social  actually manifests as equality, as assumed in many economic games (Starmans et al. A key question that remains is whether and how conservation initiatives can prevent the potential displacement of collective action, equity, and intolerance for externalities by markets? We suggest that answering this question will require a novel research agenda with three key foci. The first is testing the mechanisms underlying crowding out. A range of psychological mechanisms that can result in crowding out have been suggested, such as frame shifting, release from moral responsibility, reduced internal satisfaction, and “control aversion,” whereby a reduced sense of agency motivates resistance (Rode et al. Further, existing research also suggests that the degree of crowding out can vary depending on the types of motivations people have for engaging in prosocial or pro‐environmental behavior (Ariely et al. The second key avenue for future research is identifying the contexts under which crowding out of conservation‐relevant behavior may be more or less likely. For example, crowding out has been shown to be more likely when external implementing agencies are perceived as controlling rather than supportive and when existing norms of reciprocity and cooperation are strong Vollan . In regaFinally, testing whether conservation initiatives can be coupled with countermeasures to prevent or reduce crowding out is a further fruitful area for future research. Examples include coupling conservation initiatives with measures that foster intrinsic motivations or reinforce people's moral responsibility, recognizing multiple stakeholders may hold heterogenous motivations. For example, research on early childhood education suggests that intrinsic motivation can be fostered through supporting autonomy or agency, strong social bonds or capital, self‐evaluation, and limited external rewards (Carlton & Winsler Economic orthodoxy suggests that properly functioning markets provide incentives for the efficient use of natural resources Stavins . Example


For nearly 2 decades, regulators have adopted a harmonized approach to drug development, which has succeeded in bringing new pharmaceuticals to market without significant cardiac liability. Ushered in by technological advancements and better understanding of cellular electrophysiology, the initial paradigm detailed in the 2005 International Conference for Harmonization E14 and S7B documents has undergone evolutionary changes designed to streamline drug development and improve regulatory decision‐making and product labeling. The intent of this review is to summarize the new US Food and Drug Administration (FDA) Question and Answer update from August 2020 and key messaging from a subsequent FDA webinar describing best practices for preclinical and clinical data integration into a QT risk prediction model. In August 2020 the International Conference for Harmonization (ICH) Implementation Working Group (IWG) developed a draft guideline document titled “Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential: Questions and Answers.” This was followed by a US Food and Drug Administration (FDA) webinar in October 2020 addressing and clarifying some of the key issues enumerated in the IWG guidance and discussing new approaches for an integrated nonclinical‐clinical QT proarrhythmic risk assessment. The ICH has followed a 5‐step approach regarding updating the original ICH E14/S7B guidance to ensure safe and cost‐effective drugs are developed. These steps are building consensus among experts, adopting a draft guideline, soliciting public input and instituting revisions to the document, formulating a new harmonized guideline, and implementing the guideline, which is currently targeted for release around 2022. The webinar represented the third step in this process, with public feedback having been requested through November 2020.A number of excellent reviews concerning proarrhythmic risk have been published whose focus was to describe the individual preclinical assays, clinical studies, and analysis schemes involved in assessing a compound's effect on ventricular repolarization.The QT interval on the electrocardiogram (ECG) has garnered the attention of clinicians for more than 60 years beginning in 1957 with descriptions of sudden death in children who had associated deafness (Jervell and Lange‐Nielsen syndrome).l‐type calcium current producing early afterdepolarizations that can provide the substrate to trigger TdP. Since TdP is a very rare event in drug development, a surrogate end point, the QT interval, became the focus of the E14 guidance and formed the basis for the recommendation that a dedicated TQT study be performed on all new pharmaceuticals that have systemic exposure to assess QT liability. Monoclonal antibodies and large proteins that have low likelihood of affecting cardiac ion channels are typically exempted from this requirement, as would many dermatologic preparations that do not generate systemic exposures and do not cross cardiac cell membranes. Oncologic agents for patients with advanced cancer, per ICH S9, would similarly not be required to undergo a stand‐alone cardiovascular safety studyThe in vitro electrophysiologic substrate for TdP is typically block of the human ether‐à‐go‐go related gene (hERG) channel, which governs movement of the predominant outward delayed rectifier potassium current known as Ikr and results in altered ventricular repolarization. Inhibition of this channel results in an increase in action potential duration, which manifests in vivo on the ECG as QT prolongation. In addition, hERG block may increase the probability of That same month in 2005, the ICH also published S7B, which refers to “The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization  by Human Pharmaceuticals.” The hallmarks of this publication included evaluating potential block of the hERG channel coupled with in vivo nonrodent mammalian animal studies to assess their effects on ventricular repolarization. In addition, action potential duration in Purkinje fibers or ventricular wedge preparations was also suggested. Unfortunately, QT prolongation and hERG block are both imperfect predictive markers for TdP risk, and these preclinical assays have routinely been marginalized and interpreted independent of clinical trial findings. They have been primarily used to sanction the safety of proceeding with the test article in human subjects, whereas clinical studies were the principal drivers of QT liability and proarrhythmic assessment.The seminal ICH E14 document has been the subject of multiple question‐and‐answer (Q&A) commentaries published in 2008, 2012 (Q&A R1), 2014 (Q&A R2), and 2015 (Q&A R3). The latter document reviewed the role of CRA in regulatory decision‐making (question 5.1) and alternative study designs when a conventional TQT study could not be undertaken (question 6.1). Most recently, the IWG Q&A document from August 2020 revised initial responses to questions 5.1 and 6.1 from 2015 to provide more clarity and address ambiguities while adding a new section on questions and answers pertaining to S7B preclinical assays.Approximately 19% of recent drug studies reviewed by the FDA have demonstrated QT prolongation, as noted in Figure Obtain information on multiple ion channels beyond hERG/Ikr.Develop in silico proarrhythmic risk models based on the data gleaned from the ion channel assays employing human ventricular action potential reconstructions.Utilize human‐induced pluripotent stem cell cardiac myocytes (hiPSC‐CMs) to assess arrhythmogenicity of the new pharmaceutical.+ and l‐type Ca++ channel currents, which may mitigate risk of TdP in the setting of a prolonged QT interval.Measure the biomarker J‐T peak interval on clinically acquired ECGs to assess late inward NaIn an effort to lessen premature drug discontinuation and reduce the high number of costly and resource‐intensive TQT studies that have been conducted in recent years, several initiatives have been introduced facilitated by our understanding of cellular electrophysiology in conjunction with advances in laboratory technology. In 2013, the Comprehensive In Vitro Proarrhythmic Assay (CiPA) was promulgated by a public‐private collaboration that described a suite of 4 predominantly nonclinical assays as a way to mechanistically profile a compound's proarrhythmic risk and shift focus away from the QT interval.In 2014, a pilot study involving the FDA and Cardiac Safety Research Consortium demonstrated that it was feasible to use CRA to detect a slight QT effect in a small group of individuals who participated in a dose‐escalation protocol.The CiPA paradigm was a beneficial addition to the S7B guidance and reflected a shift away from a traditional QT‐centric focus to a pathophysiologic paradigm of arrhythmogenesis, whereas CRA was viewed as an important alternative to the original E14 primary statistical analysis tool known as the Intersection Union Test (IUT). CiPA was also noteworthy in that it provided a more scientifically rigorous schema that underscored the need to more fully link and integrate preclinical data with human studies into a working proarrhythmic risk model. The culmination of this need was the IWG August 2020 question‐and‐answer publication followed by the FDA webinar in October and most recently, the report from the 35 members of the ICH E14/S7B Industry Support Group in 2021.QUESTION #5.1: What is the role of concentration‐response analysis in QTc assessment, and what are the design pathways in which a positive control could be waived in early‐phase studies?ANSWER #5.1: CONCENTRATION‐RESPONSE ANALYSIS. CRA has been deployed for many years as a secondary analysis tool in conventional TQT studies, where the by‐time point or IUT was the primary analysis modality to assess QT risk. However, CRA is now being used and increasingly accepted as an alternative to the IUT to evaluate the presence of a QTc safety signal and can be part of the body of evidence regarding arrhythmia risk prediction and decision‐making. This enhanced role has been profiled in reviews by Grenier et alWhen CRA is used to estimate a drug's risk, the upper bound of the 2‐sided 90% confidence interval around the estimated maximal effect on ΔQTc should be less than 10 milliseconds at the highest clinically relevant exposure to conclude that the investigative product is low risk and expanded ECG collection in later‐phase studies is not essential. The growing popularity of CRA as the primary analysis tool for QT risk assessment during the past 5 years is evidenced by a reduction in submitted TQT studies from 62% to 34% with a corresponding increase in first‐in‐human protocols from 10% to 42% .max and/or steady state that exceeds the maximal therapeutic dose that is essential for discerning a small QT effect. The acquisition of high‐quality ECGs is also necessary for this type of analysis, whereas pooling of data across studies is acceptable assuming there is no heterogeneity that would introduce bias in the interpretation of results. Evaluating the time course and magnitude of QT prolongation as well as categorical outliers are additional elements that need to be incorporated into proarrhythmic risk assessment. Furthermore, the model and methods used for analysis need to be specified along with tests for hysteresis and goodness of fit.CRA is most often undertaken in first‐in‐human single‐ and/or multiple‐dose escalation studies aiming for an exposure at either CA potential shortcoming of CRA is that on occasion the model may not fully explain the observed QT effects. Changes in ventricular repolarization may be independent of drug exposure secondary to protein trafficking and ion channel gating, abnormal protein synthesis, nonionic mechanisms, or autonomic changes. In addition, delayed QT effects because of hysteresis of the parent compound or metabolites and alterations in glycemia homeostasis may also contribute to QT changes not identified with CRA.ANSWER #5.1: WAIVING A POSITIVE CONTROL. There are 2 main pathways presented for conducting studies without a positive control using CRA, and to understand these scenarios, it is incumbent to define the relevant dose and exposure terms  accounting for both intrinsic  and extrinsic factors . This circumstance has been previously described in the 2018 white paper by Garnett et al.both of these assays are deemed negative for altered ventricular repolarization and QT prolongation, respectively, the so‐called “double‐negative”, a positive control would not be required if only the high clinical exposure scenario is fully covered.The second pathway to avoid a positive control involves the performance of best practice‐designed preclinical in vitro hERG channel testing and in vivo nonrodent telemeterized animal studies. In the event that new initiative that supports lower clinical exposures than previously recommended and is a departure from typical FDA recommendations of performing a TQT study when sufficiently high exposures cannot be attained because of tolerability, safety, or saturation absorption concerns. The potential impact of this initiative in reducing the number of dedicated TQT studies is supported by data from Strauss and colleagues, who found that between 2016 and 2020, only 42% of submitted QT studies to the FDA covered twice the high clinical exposure scenario and would not have necessitated further QT study if the “double‐negative” criteria had been met.This latter pathway represents a There is a robust hERG safety margin comparing the test compound with a library of compounds with defined TdP risk, all tested under the same experimental conditions.The in vivo animal study being of “sufficient sensitivity conducted at exposures of parent compound and human‐specific major metabolites that exceed clinical exposures” does not reveal QTc prolongation of a magnitude that would be seen in traditional QT studies.To fulfill the “double‐negative” requirement and to elicit confidence in these preclinical assays conferring “low likelihood” of risk, the following criteria have been proposed:QUESTION #6.1: What adaptive study designs are acceptable to profile a compound's QT risk and proarrhythmic potential when a traditional TQT with a supratherapeutic dose, placebo, and positive control cannot be implemented or safety concerns preclude its administration to healthy individuals ?ANSWER #6.1: There are 3 basic criteria required to proclaim that a drug has a “low likelihood of proarrhythmic effects” when integrating clinical and nonclinical data for drugs that cannot undergo a dedicated TQT study:The hERG patch clamp assay utilizing best practices demonstrates a safety margin of the test compound that exceeds the known TdP threshold of reference drugs employing the same assay for both data sets AND,The animal in vivo QT study being sufficiently powered to identify QTc prolongation to the same degree as would be seen in a conventional TQT trial and employing best practices shows no QTc prolongation in which both the parent and any relevant metabolites cover the worst‐case clinical exposure scenario.Last, to declare that the compound has low proarrhythmic risk, high‐quality ECGs should be obtained in patients in which that the upper bound of the 2‐sided 90% confidence interval around the estimated maximal mean effect on ΔQTc is less than 10 milliseconds without any significant imbalance in subjects exceeding outlier values. In addition, the prevalence of adverse events in the cardiac safety database for that drug does not indicate a propensity for arrhythmia events.new initiative that may be applicable in a subset of QT studies submitted to the FDA and will likely have an important impact on the development strategy for both oncologic and other investigational products, as approximately 25% of QT studies submitted to the FDA since 2016 would fall in this category.The first 2 criteria constitute a “double‐negative” nonclinical assessment and support alternative study designs for proarrhythmic risk assessment in lieu of a formal TQT study. This is a There are a number of caveats regarding the “double‐negative” assessment, and the guidance does not offer sufficient prescriptive recommendations for each assay to facilitate decision‐making. For example, what is considered an acceptable hERG safety margin, and what are the essential design and analysis elements to ensure that an in vivo study is adequately powered and is truly negative? As such, the criteria for excluding a false‐negative assay need to be delineated so that sponsors can have assurance that they have fulfilled the “double‐negative” scenario and can safely move forward with their compound's development program.As a correlate, interpreting results in a binary manner as either positive or negative is simplistic, as indeterminate or low‐risk findings may be present. In these cases, it is unclear which, if any additional preclinical assays should be undertaken or whether optional ECG biomarker evaluation  would be helpful. Moreover, low proarrhythmic risk does not translate into no risk, and it remains to be defined what constitutes a drug having “low likelihood proarrhythmic risk” and how this impacts the development strategy including acquisition of ECGs in later‐phase studies and product labeling. Finally, there is a paucity of data concerning when, how often, and which of the S7B assays should be undertaken and how each is weighted in the totality of evidence supporting drug submission. In this regard, it is of interest that recent commentary by Vargas et alA final issue is that there is mention of “confounding” heart rate effects as part of the integrated risk evaluation and a threshold value of >20 beats per minute (bpm) was quoted, which may require subject‐specific correction factors or other methods to inform risk. However, a recent publication cites a threshold of ±10 bpm as significant where fixed heart rate QTc corrections can be “problematic” and individual heart rate corrections should be considered along with assessment of QT/RR hysteresis.There were 4 major topics covered in the S7B document pertaining to the role of preclinical assays in assessing ventricular repolarization, requisite elements to standardize the in vitro and in vivo tests, proper formatting of results, and how best to integrate this information with clinical data into a proarrhythmic risk prediction model Figure .+ current, metabolites that interfere with ion channels, and nonionic mechanisms have all been identified and linked to TdP. As such, the need for best practice in vivo animal data is critical to complement the patch clamp studies and ascertain whether a “double‐negative” is present, which would inform the development scheme going forward. In cases in which the “double‐negative” is not present in core assays because of either hERG block or QT prolongation, then the totality of evidence from optional supplemental preclinical assays needs to be integrated with human studies to profile a compound's proarrhythmic potential.In recent years our knowledge about cellular electrophysiology and the substrate for ventricular arrhythmias and TdP has increased considerably. For example, drugs such as verapamil and amiodarone that inhibit the hERG channel are not viewed as proarrhythmic, whereas other compounds such as pentamidine and arsenic trioxide that do not directly block hERG but increase the QTc interval can lead to dangerous arrhythmias.As stated in the Q&A, the best‐practice considerations are designed to be used by sponsors when an integrated risk assessment with clinical data is desired as outlined in Q&A 5.1 and 6.1. Otherwise, if the preclinical data are aimed at informing first‐in‐human drug design, screening for signals that would influence go‐no‐go decisions or aiding in commercialization, then the best‐practice considerations need not be undertaken.Defined end point(s);Defined scope and limitations of the model including the experimental protocols utilized;Prespecified analysis plan;Detailed algorithm of how experimental data will be incorporated into proarrhythmic risk;Variability in model input should be quantified, which might influence predicted risk;Mechanistic interpretation of the model about how this would translate into arrhythmia prediction.Although model input may vary between models, the output should be similar between them and therefore be beneficial in drafting a compound's predisposition to QT prolongation and TdP. Moreover, the magnitude of risk should be independent of which model is employed, acknowledging that human arrhythmia risk may not be precisely predicted from preclinical experimental data. The following are the general principles required for all proarrhythmic risk prediction models and should be integrated with clinical information.When referring to in vitro studies, the IKr/hERG patch clamp assay is the gold standard to determine if hERG channel block poses a risk of delaying or altering ventricular repolarization. There have been a variety of protocols and practices leading to significant data variability, and former guidance lacked enough critical detail to permit consistent and uniform performance of the assay. Based on recent improvements in modeling and better understanding of the molecular mechanisms involved in hERG blockade, there is now the opportunity to develop a systematic approach in executing patch clamp studies to assess the parent compound and any relevant metabolite's effects on IKr. To this end, careful attention to detail is essential when performing the hERG study, as there are a number of experimental conditions that can affect the results of the assay Table .50), and this can be compared with the estimated clinically relevant exposure to calculate a safety margin. This safety margin can then be benchmarked against drugs with known TdP risk to provide a human safety margin estimate. A robust margin of >30 was previously proposed by RedfernhERG block potency is the amount of free drug in the steady state, which inhibits the potassium current by 50%  and what alternative study designs in special cases (Q&A 6.1) would be acceptable when a traditional TQT study cannot safely be performed. Most importantly, the Q&A paper serves to link often marginalized and independently analyzed preclinical information with clinical data into an integrated proarrhythmic risk prediction model. Moreover, it provides important specifics regarding the conduct and reporting of preclinical assays so as to harmonize methodology and reduce variability. Last, it broadens the circumstances and provides insights about how to structure and analyze studies using CRA that would suffice as a substitute for a conventional TQT study.How does a sponsor ascertain that the preclinical package is adequate to fulfill regulatory requirements, and, conversely, how do they determine if it is not acceptable?There needs to be continued pivoting away from a QT‐centric focus given its lack of positive predictive value for TdP and malignant ventricular arrhythmias in concert with avoiding the binary interpretation of assay information and adoption of a graded continuum of risk based on the totality of preclinical and clinical data.The guidance gives significant weight to hERG and in vivo studies as part of the “double‐negative” assessment, although other nonrodent in vitro and in vivo models and clinical assays may be helpful to characterize proarrhythmic risk assuming they are executed using standardized best practices. For example, these additional assays might include ventricular wedge preparations, methoxamine‐sensitized rabbit models for TdP,It would be of interest for regulators to provide metrics concerning the number of cases in which the “double‐negative” scenario has been accepted or rejected. In cases in which the “double‐negative” is not fulfilled or preclinical assay results are discordant, subsequent recommendations by the agency would be enlightening; acknowledging this would presumably be done on a case‐by‐case basis.Despite these advances, there are still a number of unanswered questions and gaps in information that will undoubtedly be addressed as regulators refine the ICH documents and construct a comprehensive database of results and metrics from completed protocols to better inform all stakeholders. Chief among these is:As there is a desire to reduce the number of dedicated TQT studies, how often and under what circumstances is a stand‐alone study still advisable and recommended by regulators? The guidance leaves open the question of how to identify and define drugs with “low likelihood of proarrhythmic risk” and what additional evaluation, if any, needs to be undertaken prior to approval to assess the drug's arrhythmia liability. In addition, how do sponsors determine which clinical research organizations are optimally positioned to perform preclinical assays and what steps are necessary to ensure that best practices are followed and at what cost? What is the optimal approach to assess chimeric moieties for their proarrhythmic potential?Finally, the recent review by Vargas et alThe most recent guidance is an important step in the evolutionary process of drug development by providing a more robust harmonized and integrated road map for all stakeholders to profile a compound's proarrhythmic potential while informing regulatory decision‐making and restrictive labeling. It remains to be determined whether the impact of this guidance will succeed in continued reduction in the number of stand‐alone TQT studies while conserving resources, accelerating development of novel drug candidates, and building on the ICH E14/S7B mission of preventing unsafe drugs from coming to market. Lastly, the ongoing efforts of the IWG to develop additional guidance regarding preclinical‐clinical data integration intended to inform ECG collection in later‐phase trials to clarify appropriate labeling of compounds with a positive QT signal and to address whether certain small molecules with limited bioavailability can circumvent undergoing a QT clinical study, are most appreciated.The author is a Celerion employee.


It is a grand challenge to ensure the food security for a predicted world population of exceeding 9.7 billion by 2050, especially in an era of global climate change, land degradation and biodiversity loss. Current agricultural productions are mainly relying on synthetic chemical fertilisers to boost plant productivity but have undesirable effects on the environment and soil biodiversity. A promising direction in sustainable agriculture is to harness naturally occurring processes of beneficial plant‐associated microbiomes to ensure sustained crop production and global food security. Despite the significant progress made in the development of beneficial microbes as inoculants to enhance plant performance, challenges remain with the translation of knowledge of plant and soil microbiomes to successful microbial products in the agricultural sector. Here, we highlight how fertilizer technology should be renovated by harnessing microbiome‐based innovations to promote plant productivity and contribute to the end of hunger. Conceptual diagrams to harness the plant‐microbe interactions for the development of new microbial products to improve plant performance. We propose to identify the core group of crop‐specific plant signalling molecules under various environmental stresses, and to extract the selected signalling molecules with the capacity to promote activities of beneficial microbes in the rhizosphere. The high‐throughput isolation approach can be used to isolate plant beneficial microbes in a high‐throughput manner, and the isolated bacterial cultures can be used to create various synthetic communities to test their effects on plant performance in controlled and field experiments. These beneficial microbes have positive effects on plant growth through enhancing nutrient availability, biosynthesis of phytohormones, stimulation of root system architecture, and protecting host plants from biotic or abiotic stresses  enriches Flavobacterium spp. in the rhizosphere to suppress pathogens (Kwak et al., The plant−microbe interaction is a two‐way communication process, mediated by a large number of diverse chemical and molecular signalling molecules such as sugars, organic acids, proteins, peptides, lipids, phytohormones and secondary metabolites in plant rhizosphere soils (Venturi and Keel, et al., et al., et al., et al., Under nitrogen‐limited conditions, plants can sense and respond to the distribution and availability of external nutrient pools (Gent and Forde, et al., et al., et al., In order to improve the diversity of microbial inoculants for a better field performance, a promising direction to innovate the design of new biofertilizers is to generate a tailor‐made core microbiome transfer therapy for different crops under specific environmental and agricultural settings (Gopal The increasing emphasis on sustainable agriculture has opened up a wealth of opportunities to integrate microbiome‐based products into modern agricultural management programmes for long‐term sustained benefits. We envision that embracing a holistic view of plant−microbiome interactions for developing new microbial products that are tailored and integrative in diverse agricultural systems will result in a sustainable agriculture of important crops while increasing food production at the same time. These multidisciplinary innovations in microbial products will lead to better integrated efforts to develop appropriate strategies to address the challenges of achieving food security in more efficient and more profitable ways.None declared.