The H2 production efficiency varied by a factor of 3.7 for GO dispersions sonicated for various amount of time. Interestingly it was found that too long ultrasound treatment had negative impact on the GO enhancement of hydrogen production which was related to the fragmentation of GO flakes. The photocatalytic system produced the highest amount of H2 when graphene oxide occurs as monolayers and efficiency becomes lower with the decrease of GO sheets size. Our results demonstrate the importance of optimizing the size and the number of layers of the GO flakes prior to preparation of GO-based materials.The present study explored the correlation between the photocatalytic activity toward hydrogen production of the graphene-based materials and graphene oxide (GO) morphology. In this work we applied the technique based on the combination of time-dependent sonication and iterative centrifugation cascades, which were designed to achieve nanosheets size and the number of layers selection. First such obtained GO dispersions were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and optical spectroscopy. Those combined measurements showed that the intensity of the π-π peak at 230 nm seems to be very sensitive to the number of layers of nanosheets. Next, GO dispersions were used to establish influence of the size and the number of layers of GO flakes on the photocatalytic hydrogen production in the photocatalytic system, containing eosin Y as a sensitizer, triethanolamine as a sacrificial electron donor, and CoSO The composites photocatalyst that includes graphene could have improved charge separation which results in better photocatalytic activity. Moreover, graphene due to its unique two-dimensional structure could act as a support that increases the specific surface of the material. However, graphene is a hydrophobic material what limits its application in water splitting process. Therefore, its two hydrophilic derivatives: graphene oxide (GO) and reduced graphene oxide (RGO) were found to be more suitable for application in photocatalytic water splitting system.Visible-light-driven water splitting is a long-standing goal for solar energy conversion. The known systems often suffer from the low photo-to-energy conversion efficiency that is related to poor electron transport between the photosensitizer/semiconductor and the catalyst. Most of the research, that involves systems in which the light harvester and the catalyst are not chemically linked and thus interaction between the components is exclusively controlled by diffusion. A novel strategy to enhance efficient charge separation and transport in photocatalytic H3. In the past 10 years, graphene-based materials were explored by a number of research groups and the concept of incorporating graphene type materials in the photocatalytic systems have been proven valid12. The first example of the use of graphene for photocatalytic hydrogen production was the system composed of eosin Y (EY), RGO with Pt nanoparticles dispersed on its surface13. The largest apparent quantum yield (AQE) of 9.3% was achieved under 520 nm irradiation using triethanolamine (TEOA) as a sacrificial reagent13. More recently, a few graphene-based systems that employ photosensitizers and catalysts  that are both derived from earth-abundant materials were also reported for photocatalytic hydrogen production14. Yuan et al. presented enhanced photocatalytic activity toward hydrogen generation in a non-noble metal system for photocatalytic H2 generation that combined Zn(II)-5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin and RGO decorated with MoS2 as the catalyst14. Enhancement of photocatalytic hydrogen evolution was attributed to the facilitation of charge separation in the presence of graphene. Despite proof-of-concept studies on the application of graphene-based materials in photocatalysis, there are still some challenges to be addressed, which are thought to greatly inhibit practical applications. Surprisingly, despite the fact that it is well accepted that graphene-based materials has the potential to boost the efficiency of photocatalytic hydrogen systems, the link between morphological properties of GO or RGO (flakes size and the number of layers) and photocatalytic performances has not been explored yet.Much effort has been recently applied to design and fabricate multifunctional materials based on GO and RGO19. It was reported previously that many applications require controlled nanosheet properties of materials23. For example, counter electrode in the dye-sensitized solar cell require small nanosheets23 whereas mechanical reinforcement of composites demands large ones21. Lyons et al. presented that smaller flakes result in more junctions and so lower conductivity24. More precisely, graphene can stack into a thick multilayer structure via van der Waals force decreasing significantly the material’s specific surface area and conductivity.However, a polydispersity of graphene flakes has brought scientist attention as it presents important issues in many applications since the properties of graphene are inextricably linked to its structureWe are unaware of any deliberate works in the literature on the control of the morphology parameters of GO that was applied in the hybrid materials for photocatalytic hydrogen production. Since there is no prior study that focuses on the GO size and the number of layers selection it is currently unclear whether those parameters actually matter in photocatalytic hydrogen production. Therefore, a systematic exploration of the effect of the GO size and the number of layers is highly required to provide a reasonable correction for optimizing the photocatalytic hydrogen production efficiency. With this work, we aim at filling this gap. Most graphene-oxide dispersions contain large variations in the number of layers, lateral area, and shape of the graphene oxide flakes. Here we applied the technique based on the combination of time-dependent sonication and iterative centrifugation cascades, which were designed to achieve nanosheets size and the number of layers selection. Such obtained dispersions were used to establish a correlation between the photocatalytic activity of the graphene-based materials and GO morphology. Our results clearly demonstrated that both GO size and the number of layers influences the efficiency of the photocatalytic hydrogen production, while the effect of the number of layers was much more profound.4 was purchased from Alfa Aesar and graphene oxide  was purchased from Abalonyx. All experiments were performed with one batch of GO that ensured constant elemental composition. Solutions were prepared with ultrapure water (18 MΩ cm).Eosin Y and triethanolamine were purchased from Sigma Aldrich, CoSOA total of 90 mg of GO powder were sonicated for various time in 30 mL of ultrapure water using a sonic bath . The dispersion was sonicated under ice-cooling in order to avoid heating effects. The dispersion after various sonication time and dilution were characterized by optical spectroscopy and atomic force microscopy.The dispersion of GO after sonication with the high content of monolayers determined by atomic force microscopy (AFM) referred to as stock dispersion was used for nanosheets size selection. The size selection was obtained by means of controlled centrifugation with sequentially increasing rotation speeds using stock GO dispersion. The dispersion was centrifuged in a MPW-352R centrifuge equipped with a fixed-angle rotor Nalgene 11469 . In the standard primary cascade, stock GO suspension was subjected to centrifugation at 4 krpm (100 min). The sediment was collected while the supernatant was centrifuged at 8 krpm (100 min). Again, the sediment was collected and the supernatant subjected to centrifugation at higher speeds 12 krpm (100 min). The concentration of nanomaterial in each fraction was determined by weighing the sediment after its drying overnight at 60 °C.25. In brief, in a photochemical reaction 3 mL of an aqueous solution of TEOA (0.2 M) and a certain amount of EY, GO and CoSO4 were placed in a quartz 1 cm × 1 cm cuvette. The pH of the solution was 10.8. The mixture was then degassed by bubbling argon through it for 30 min. Then the solution was stirred continuously and exposed to irradiation from a Broadband Halogen Fiber Optic Illuminator . The generated H2 was monitored by a gas chromatograph (GC Agilent 7890B) equipped with a thermal conductivity detector. The experiments were repeated independently four times using each time newly prepared GO suspensions.The photochemical reaction has been performed according to procedure described elsewhere−1 and resonant frequency 150 kHz on the typical scan frequency of 0.2 Hz. For such analysis, the 0.1 mg mL−1 GO water dispersions were highly diluted in the ratio of approximately 50 µL:1 mL, deposited on freshly cleaved mica and left at room temperature for at least 24 h. The morphology of graphene oxide flakes was also examined by scanning electron microscopy (SEM) Quanta 250 FEG, FEI. Four water dispersions of GO (0.01 mg mL−1) with different sonication time were drop casted on silicon wafer substrate and left at room temperature to dry. The measurements were performed in a high vacuum using accelerating voltage 2 kV.Extinction spectra of the GO dispersions were measured using a two-beam spectrometer Cary 100 UV–Vis scanning from 800 to 200 nm with 1 nm step. It should be noted that the extinction (Ext) is a combination of both the absorption (Abs) and scattering (Sca) where Ext(λ) = Abs(λ) + Sca(λ). The morphology of the GO flakes were analyzed using atomic force microscopy (AFM) Agilent 5500. The AFM imaging was realized using soft tapping mode by All-In-One Al cantilever (Budget Sensors) probe C, with a nominal force constant 7.4 N m27.The raw GO sample used in this study were synthesized by a proprietary modified Hummers method and purchased from Abalonyx. The formed GO can stack into thick multilayer structure via van der Waals force decreasing significantly the material’s specific surface area and conductivity which in turn may influence on the photocatalytic performance. A sonochemical approach is widely used as a routine protocol for GO preparation prior to assembly in hybrid materials for photocatalytic hydrogen production. It was reported previously that ultrasonic treatment influences the adsorption properties of GO28 studied the effects of ultrasounds, found that the number of layers in the GO sheets can decrease from more than 30 layers to less than 5 layers after 20 min of the ultrasound treatment. Here the variation in sonication time (1–80 min) of GO dispersion leads to obtain the suspensions, denoted as GO-s1–GO-s13, where GO-s1 was sonicated for just 1 min and GO-s13 was sonicated for 80 min. The resultant ultrasound-selected GO dispersions were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM) .In this work, we investigated the effects of ultrasound sonication on graphite oxide aqueous suspensions by monitoring its structural and optical properties as a function of exposure time to the ultrasonic treatment. Martinez et al.ion time –80 min o31 we verify the apparent height of monolayered GO flakes by the AFM. As the apparent height of monolayered GO flakes remains 1.0 nm after each sonication stage this indicates that the reduction process does not occur upon sonication since for reduced graphene sheet the height of monolayered flake is expected to be reduced by 0.2 ÷ 0.8 nm33.To characterize the morphology of graphene oxide flakes, we used two techniques of imaging: atomic force microscopy (AFM) and scanning electron microscopy (SEM). The AFM is suitable for measuring the number of layers of the flakes but during one scan only a small area of the sample is embraced. If the sample is no homogeneous, drawing conclusions based on limited number of images can be inaccurate. Thus, we used additionally SEM measurements which enabled us to observe the whole area in a reasonable time. The combination of AFM and SEM analysis provided more overall insight into the influence of the GO sonication process onto its delamination and fragmentation. Figure 36. It is known that the lateral size distribution, the mean number of layers per flake and the functional groups on graphene and graphene derivatives are all important factors influencing the extinction coefficient38. As shown in Fig. 37. In our previous study mild reduction of GO by ascorbic acid was accompanied by the significant red-shift of the peak from 230 to 259 nm39. Contrary to peak position that remained unchanged, the intensity of the peak at 230 nm of GO samples increased remarkably with the increase of sonication time due to efficient exfoliation of GO sheets, as observed in AFM measurements, which created more particles in the suspension and absorb more light from the excitation. In addition, the intensity of the extinction spectra of GO dispersion decreased slightly with the increase of sonication time in the region 400–800 nm which might be attributed to the decrease of the light scattering by GO samples that are exfoliated more efficiently.While AFM is a reliable technique for the evaluation of number of layers of nanosheet, such a method is time-consuming and expensive, making it unsuitable for routine batch monitoring. Quantification of the number of layers of graphene oxide nanosheet via optical spectroscopy could facilitate preparation of GO dispersion with high monolayer content. Therefore, we combined AFM with optical spectroscopy in order to establish the link between the GO morphology properties and its extinction coefficients. Optical absorption spectroscopy of graphene flakes suspension produced by high-yield liquid phase exfoliation was extensively employed by Coleman’s group−1). Thus the measured extinction is equal to the GO absorbance. The measured extinction can be converted to an absorbance coefficient, ε, using the Beer-Lambert law, Exc = ε c l, where c is the GO concentration and l is the cell length (1 cm). It was clear from Fig. −1) at 230 nm varied from 700 L g−1 m−1 for GO-s1 suspension that was sonicated for just 1 min to 4830 L g−1 m−1 after 20 min of sonication (GO-s8) , through ε230nm = 2553 L g−1 m−1 for a mixture of monolayered and few-layered flakes (GO-s5) up to ε230nm = 4830 L g−1 m−1 for fully monolayered GO dispersion (GO-s8). From UV–vis spectroscopic studies, it can be inferred that the peak at 230 nm of GO is attributed to the π-π* plasmon peak. It is was reported that the change of UV–vis absorption intensity at 230 nm with the number of GO layers is caused by a conjugative effect related to chromophore aggregation that influences this π-π* plasmon peak40.As demonstrated by combined optical spectroscopy and AFM/SEM measurements, the intensity of the π-π peak at 230 nm seems to be very sensitive to the nanosheet number of layers. Therefore we could correlate the number of layers in GO flakes with absorption coefficient at 230 nm. The number of layers in GO flakes evolve during the progressive sonication from the thick aggregates with hundreds of nanometers in thick down to fully monolayer. Concurrently, the absorption coefficient ε increases during the sonication increases. This implies the proportional correlation between the dispersion excitation coefficient and the amount of GO layers. Thus, the sonication induces the evolution of ε from ε36. Graphene oxide area sorting by means of density gradient centrifugation has been achieved using nano-GO sheets functionalized with polyethyleneglycol with the intention to obtain ultrasmall flakes for cellular imaging and drug delivery41.Another morphology parameter of GO that may influence on photocatalytic activity is sheet size. The randomness of chemical cutting and various accessibilities of carbon on the lateral plane towards oxidants using this method yielded GO sheets with different characteristics. Therefore, to test the impact of the flakes size of GO on photocatalytic activity we have performed the cascade centrifugation in order to get size-selected GO dispersions. It was reported that for controlled centrifugation of graphene dispersions, the average lateral flake size decreases as the centrifugation rate (rpm) is increasedAccording to the Abalonyx product specification, the average lateral size of GO is about 1 µm with a rather wide size distribution. We start with a dispersion of liquid-exfoliated nanosheets, obtained by sonication (20 min) of GO powder in an aqueous solution. Sonication time has been chosen so that number of monolayers in GO dispersion was maximized. This “stock” dispersion contained nanosheets with a broad distribution of sizes. The stock was then centrifuged at a speed (4 krpm) and the sediment collected. This sediment contained the largest nanosheets and we refer to this sample as GO-4 krpm. The supernatant produced during the 4 krpm centrifugation contained all but the largest nanosheets. It can be centrifuged at a higher rate (here 8 krpm) to give a sediment with smaller nanosheets, which we label GO-8 krpm. The associated supernatant was centrifuged again at higher rate 12 krpm. The sediment was labeled GO-12 krpm and the resulting supernatant was referred GO-residual. After each step, the sediment contained smaller and smaller nanosheets, resulting in effective size selection. Size-selected dispersions were prepared by re-dispersing the collected sediments in water after subsequently increasing centrifugation speeds.The whole process is illustrated in Fig. 42.We have characterized the nanosheets collected in each fraction  microscopically using atomic force microscopy (AFM) with typical images displayed in Figs. To identify the GO size-selection of the cascade centrifugation process we performed extended statistical analysis of AFM images based on hundreds of GO flakes from each of fraction. The resulting histogram is shown in Fig. 4 salt was added to serve as a precatalyst in the reaction system, and GO was added to act as an electron mediator. This system was chosen to explore the influence of the GO morphology on photocatalytic efficiency as it was found previously that the addition of GO to these components increases the efficiency of the hydrogen production43. Wang et al. have demonstrated based on TEM, ICP-MS, and XPS measurements that Co2+ ions after irradiation in the presence of GO transform into Co metal nanoparticles surrounded by Co2+ species43. In addition our previous mechanistic studies have shown the existence of a stable charge-separation state after the addition of GO to the system of EY and triethanolamine what explains the role of graphene in the improvement of photocatalytic efficiency in the Eosin Y-based systems25. In the absence of GO, the H2 production from EY and CoSO4 system was 0.84 μmol after 1 h of irradiation. No significant amounts of hydrogen were detected in the absence of either irradiation or the photosensitizer EY suggesting that the visible light activity comes from the EY sensitization. Also no measurable amount of hydrogen was detected for the GO alone. Initially “stock” dispersion of GO-s8 (GO powder after 20 min of bath sonication) was used in order to establish the optimum GO concentration for the H2 production. The results of these experiments are shown in Fig. −1, which is 5.8 times larger than that of the system without GO  of the GO dispersion affects photocatalytic hydrogen production, a simple non-noble metal based system was constructed. The photocatalytic hydrogen evolution was studied under visible irradiation by using triethanolamine (TEOA) as a sacrificial donor and eosin Y (EY) as a photosensitizer. CoSO GO Fig. . Further230nm) is related to the increase of the content of monolayered flakes. The photocatalytic hydrogen generation was explored for eight samples of GO suspensions (GO-s1–GO-s8) with gradually increase of ε230nm (from 700 L g−1 m−1 to 4830 L g−1 m−1) and additionally for five samples of GO (G-s9–GO-s13) with extended sonication process  of GO suspension with ε230nm = 4830 L g−1 m−1  and GO-s8 (ε230nm = 4830 L g−1 m−1) samples. However, more interestingly, the hydrogen production for systems with the addition GO samples that underwent prolong ultrasound treatment decreased. The efficiency of photocatalytic reaction decreased by 18% for the GO-s9 suspension sonicated additional 10 min and by almost 40% for GO-s13 that were sonicated for 60 min longer than time sufficient to achieve maximum absorption coefficient ε230nm = 4830 L g−1 m−1  resolution. Therefore, we conclude that the decrease of the hydrogen production efficiency for the GO samples that underwent long sonication treatment is related to the decrease of the GO flakes size. To avoid negative impact of such fragmentation of GO flakes on the photocatalytic activity it is important to optimize the sonication treatment. Due to various bath sonicators used in the laboratories with different frequency, power or volume there is no unique sonication time that would ensure optimum GO properties for the photocatalytic activity. Thus, the correlation between the UV–Vis absorbance at 230 nm and the amount of H2 is the key, not the specific sonication parameters. Our work has shown that simple measurement of the optical spectra of the GO dispersion is reliable method for the optimization of the monolayer content. In short, the most important practical outcome of our results is that the optimum sonication time of the GO suspension used in photocatalytic H2 production systems should match exactly the time (but no longer) required to achieve the highest absorbance of the GO dispersion at 230 nm.We have performed systematic exploration of the effect of the sonication time of GO on the photocatalytic hydrogen production efficiency since the sonication process is a standard step in preparation GO solutions prior to assemblies of GO in the photocatalytic systems. In all photocatalytic tests that were performed to establish the effect of GO flakes size and number of layers on hydrogen production concentration of all components were kept constant, including GO what ensured identical conditions in all experiments. GO samples treated for various sonication times were firstly prepared and characterized (by UV–vis spectroscopy and AFM) and subsequently tested in photocatalytic hydrogen production systems. As it was shown by AFM the number of layers in GO flakes of the GO sample is correlated with the absorbance at 230 nm. The increase of absorption coefficient at 230 nm (εions GO-s–GO-s8 wi2+ system. The results are summarized in Fig. The investigation of the role of the sonication treatment of the GO suspension on the hydrogen production showed that photocatalytic efficiency is affected not only by the number of layers in GO flakes of the GO sample but as well as by the size of the flakes. To explore this effect in a quantitative manner the size-selected GO dispersion obtained via cascade centrifugation procedure were tested in the photocatalytic hydrogen production systems. The cascade centrifugation was performed four times and collected fractions were used independently for photocatalytic reaction with EY–GO–Co2+ system produces the highest amount of H2 when graphene oxide occurs as monolayers and efficiency becomes lower with decrease of GO sheets size. It is important to choose appropriate conditions of sonication process to obtain GO in optimal form as monolayered flakes with the biggest, possible area. The influence of the GO number layers on the hydrogen production can be related to the change of surface area which subsequently may affect the interaction with precatalyst Co2+. The interaction of GO and EY was found previously to be weak and therefore we do not expect to be dependent on GO morphology25. Therefore, presumably, the change in the size and dispersion of Co nanoparticles on the GO surface is responsible for the observed changes of hydrogen production upon variation of GO morphology. Detailed analysis of the dependence of Co nanoparticles size and dispersion on GO morphology will be the subject of separate paper in the future.The highest efficiency of hydrogen generation was observed for GO-8 krpm which was higher by 22% in comparison to the analogue experiment with the “stock” GO. The small decrease of hydrogen production for GO-4 krpm might be attributed to the presence of graphene oxide aggregates as observed in AFM measurements. For the residual GO the decrease of the hydrogen production by 30% was observed in comparison to GO-8 krpm sample. The residual GO consists mostly of monolayers but with significantly smaller flakes size than fraction GO-8krpm Fig. . The EY–This work provides an important guide for researchers interested in exploiting the application of graphene-based materials in photocatalytic water splitting. We report a systematic and comprehensive study of the influence of the morphology of graphene oxide flakes on their photocatalytic activity toward hydrogen production. This information is absolutely crucial given the interest in using graphene-based materials in photocatalytic water splitting.Therefore our systematic exploration of the effect of the GO size and the number of layers provide a guide for optimizing the photocatalytic hydrogen production efficiency with graphene-based materials. Our results clearly demonstrate that both size and the number of layers of GO flakes do matter for the photocatalytic hydrogen production, but the latter one has more profound impact on the hydrogen production efficiency. Our results demonstrate that sonication of graphene oxide is an important contributor to the variation in efficiency of hydrogen production with GO-based composites. With this concept in mind, it is therefore worthwhile for researchers to control and optimize the sonication time of GO prior to preparation of GO-based materials. Our observed effect of the morphology of GO dispersions on photocatalytic efficiency could be employed for other 2D-material based composites with various potential application.


Open-book examinations (OBEs) will likely become increasingly important assessment tools. We investigated how access to open-book resources affected questions testing factual recall, which might be easy to look-up, versus questions testing higher-order cognitive domains. Few studies have investigated OBEs using modern Internet resources or as summative assessments. We compared performance on an examination conducted as a traditional closed-book exam (CBE) in 2019 (N = 320) and a remote OBE with free access to Internet resources in 2020 (N = 337) due to COVID-19. This summative, end-of-year assessment focused on basic science for second-year medical students. We categorized questions by Bloom’s taxonomy . We predicted higher performance on the OBE, driven by higher performance on ‘Remember’ questions. We used an item-centric analysis by using performance per item over all examinees as the outcome variable in logistic regression, with terms ‘Open-Book, ‘Bloom Category’ and their interaction. Performance was higher on OBE questions than CBE questions , and higher on ‘Remember’ than ‘Understand/Apply’ questions . The difference in performance between ‘Remember’ and ‘Understand/Apply’ questions was greater in the OBE than the CBE . Access to open-book resources had a greater effect on performance on factual recall questions than higher-order questions, though performance was higher in the OBE overall. OBE design must consider how searching for information affects performance, particularly on questions measuring different domains of knowledge. The COVID-19 pandemic has necessitated rapid and unprecedented change in the delivery of medical school examinations  for final-year medical students from a CBE to an OBE. We found that the median mark was the same as the equivalent exam from the previous year .We used data from the single-best-answer (SBA) component of a summative assessment centered around the life cycle and physiological regulation. In 2019, this was conducted as a traditional CBE. In 2020, the assessment for this paper was designed as a CBE but converted to an OBE. The 2019 and 2020 assessments were not the same paper; they had different questions, though 16 were the same. Students were informed of the format change 6 weeks prior to the exam. The 2020 exam was written prior to the COVID-19 pandemic. We compared exam performance over the two years.The exam was performed by second year medical undergraduates. The module comprised mainly basic science with some clinical applications. This was a ‘high-stakes’, summative examination, requiring examinees to pass to progress to the next course year. Each year, the exam contained 128 single best answer (SBA) questions with 4 distractors. The exam was sat as 2 sections with 64 SBAs in each section.Each exam also contained 14 multi-part short answer questions. We opted not to analyze these questions. The inclusion of a different type of question would mean that performance was affected by factors related to item type. Most studies comparing OBEs and CBEs have used multiple-choice questions . This platform was already in use by the medical school for electronic summative and formative written exams since 2017. Using this platform to deliver OBEs online did not incur additional costs. Examinees could perform the OBE on any device with internet access. The platform did not impose any restrictions on other activities such as web browsing.There was no remote proctoring in place during the exam. To minimise the risk of collusion, examinees were each presented with the questions in a different random order. Examinees were reminded that academic misconduct would contradict the medical school code of conduct and the professional standards outlined by the UK General Medical Council.Examinees had 2 h 30 min to complete each of the papers in both years, which equated to around 1 min per SBA question. Instructions to examinees were the same across years. In 2020, examinees were advised that additional resources could be used, although this could add additional time constraints to the completion of all questions. Ebel standard setting was used for both exams and performed by senior faculty at ICSM. The pass mark was the same in both years. Students had no prior experience of OBEs within the medical course.To determine what domain of cognitive knowledge a question was measuring, or the type of information required to answer it , we categorized exam questions using the cognitive domain of Bloom’s taxonomy Bloom, . Bloom’sIt has been suggested that it is harder to measure higher-order processes like evaluation and synthesis using multiple-choice questions  independently categorized each question in the SBA exams according to Bloom’s taxonomy. Both raters had attended a workshop course by ICSM on educational theory that included categorizing questions by Bloom’s taxonomy. Questions that had been categorized differently by each rater were then discussed at a consensus meeting and a final category decided.Due to considerable overlap between boundaries, low numbers of ‘Apply’ questions and no questions from levels 4–6 of Bloom’s taxonomy, we combined ‘Understand’ and ‘Apply’ questions to produce a binary predictor of ‘Remember’ versus ‘Understand/Apply’. We recognize that some evidence suggests no linear relationship between taxonomic level and item difficulty  out of the number of total ‘trials’ (number of students answering the question), which follows a binomial distribution. Our dependent variable was the set of 256 questions , with performance averaged over all examinees. This ‘item-centric’ approach contrasts with a traditional ‘person-centric’ approach, where an examinee’s performance is averaged over all questions.pen-Book (‘open’ vs ‘closed’ book) and Remember (‘Remember’ vs ‘Understand/Apply’). We used model comparison to determine which effects to include in the model. A more complex model can always fit the data better, but inclusion of more complex terms may not significantly improve model performance and could make the model unnecessarily complex. We used Bayesian Information Criterion (BIC), a log-likelihood based model fit index that penalizes complexity, so trading off model fit with parsimony. We compared the following models: null model (no effects), Open-Book main effect alone, Bloom Category main effect alone, and both main effects with interaction. BIC is a relative fit index and lower values indicate better performance. A difference in BIC of > 10 is considered very strong evidence favoring one model over another . Table Table Table Figure Open-Book main effect alone, Bloom Category main effect alone, and both main effects with interaction.We fit the following logistic regression models: null model (no effects), Open-Book and Bloom Category main effects and their interaction. BIC values are shown in Table The best-performing model contained Open-Book. Examinees performed better on ‘Remember’ questions than on ‘Understand/Apply’ questions, shown by the significant main effect of Bloom Category. Importantly, there was a greater difference in performance between ‘Remember’ questions and ‘Understand/Apply’ questions in the OBE than the CBE, shown by a significant Open-Book * Bloom Category interaction.Table To understand the interaction term, we ran two separate post-hoc logistic regressions for the OBE and CBE separately. We used z-tests and Wald confidence intervals as tests of significance, with 99% confidence intervals to adjust for multiple comparisons Table . In eachWe compared a summative, end-of-year assessment performed as an OBE in 2020 to the equivalent examination from the previous academic year, performed as a CBE. In this second-year examination focusing on basic science, we found that performance was better overall in the OBE than the CBE and performance on questions testing recall of facts (‘Remember’ in Bloom’s taxonomy) was better than on questions testing higher-order cognitive domains (‘Understand/Apply’ in Bloom’s taxonomy). The difference between performance on ‘Remember’ and ‘Understand/Apply’ questions was greater in the OBE than the CBE, supporting that the access to open-book resources had a greater effect on questions testing factual recall. However, performance on both types of questions improved in the OBE.We theorized that performance was higher on factual recall questions because answers could more easily be found by accessing information resources, compared to answers to questions at higher levels of Bloom’s taxonomy. Importantly, ours was one of few studies that gave examinees access to modern Internet-based resources in a summative setting  theorized that search complexity is determined by ‘objective’ factors, which are intrinsic to the task and independent of the problem performer, and ‘subjective’ factors of how an individual perceives and interacts with the problem. Objective complexity of a search task depends on its ‘a priori determinability’, which reflects intrinsic uncertainty in the task’s inputs, processes and outputs. We can consider uncertainty in (at least) three domains: (i) what information is required to solve the problem, (ii) how to search for it, and (iii) interpreting the found information  maps mainly to the first domain of objective complexity: the information required to answer a question. We tried to capture this using Bloom’s taxonomy. The gap in performance between questions testing factual recall versus understanding/application was greater in the OBE than the CBE. Open-book resources may therefore confer a particular advantage to fact-based questions, which may be because there is little uncertainty in the information required to answer them, making the search task less complex  in an SBA will be shaped by the information in the question stem and distracters. Uncertainty in how to search has been experimentally manipulated by changing the number and relevance of key terms provided in search problem. Searches have been made more complex by requiring participants to generate their own key words for efficient search queries, rather than providing them  is effectively out of examiner’s control if students are given free Internet access. However, it may be useful during question development to construct searches to answer the question as if one were the examinee, to review what kind of information examinees are likely to find.Subjective factors affecting search complexity will introduce individual differences in when examinees search, how they search, and how they evaluate and use the found information . Secondly, each cohort was fairly large, with over 300 students, making it less likely that there would be systematic differences in ability levels. Thirdly, the course and teaching content were the same for both years. The 2020 cohort had a shift from face-to-face to online learning from March 16th 2020, but this was after most of their timetabled teaching had taken place.The exams taken in each year assessed the same topics but had different questions, raising the possibility that a difference in exam difficulty would confound the results. The two exams were standard-set independently using the Ebel procedure and the pass mark was the same in each year, suggesting they had comparable difficulties. The standard-setting for 2020 was performed prior to the conversion to an OBE and prior to conceptualization of this study. 16 of the items were present in both papers.There is also uncertainty associated with using subjective classification of questions by Bloom’s taxonomy, as well as evidence that students often use different processes than those expected by question-writers Gierl, . We had We have no evidence of academic misconduct in either assessment but also cannot exclude the possibility that differences in exam security could affect our results. We argue this would be unlikely to systematically favour factual recall questions in the OBE and therefore bias our findings.We found that free access to open-book resources, including the Internet, in a summative, end-of-year examination for second-year medical students was associated with higher performance overall, in contrast to our previous findings in final-year students (Sam et al.,


A multi-band analogue electromagnetically induced transparency (A-EIT) metamaterial is proposed. The structure is composed of liquid crystal (LC) layer and a graphene strips layer on both sides of silicon dioxide. The transmission spectrum and electric field distribution of only one graphene strip and two graphene strips have been studied. As a bright mode, the graphene strip is coupled with adjacent graphene strip to realize the A-EIT effect. When multiple graphene strips are coupled with each other, the multi-band A-EIT is obtained due to the electric dipole resonances of the four strips. The results show that the multiband A-EIT effect can be tuned by voltage on LC and graphene layer, respectively. Moreover, changing the incident angle of the electromagnetic wave has had little influence on the transmission window in the low frequency band, it is meaning that the A-EIT effect with insensitive to the incident angle can be obtained. Each transmission window has a high maximum transmittance and figure of merit (FOM). The multi-band A-EIT effect can widen the application on sensor and optical storage devices. Metamaterials are a kind of materials whose subwavelength structures produce properties that natural materials do not have through periodic or aperiodic arrangement. The properties of these materials depend on the artificial structure rather than the material itself ,2. It haAs a kind of anisotropic material, LC has birefringence effect in terahertz (THz) band. LC molecules will reorient under the action of external electric or magnetic field, and the permittivity shows good continuous adjustable ability in a wide range. LC was widely studied as functional devices in the THz band . R. KoweA special physical phenomenon of non-linear effect was produced in the process of interaction between light and matter. After decades of research and development, electromagnetically induced transparency (EIT) has faded its mysterious veil . Neverthλ0. The measurement result was shown a high quality factor transmitted peak [Graphene metamaterials (GMs) have the characteristics of fast response, electrical tunability and ultra thin. GMs have potential applications in electromagnetic lens, electromagnetic absorption and so on. Cheng et al. realized the active and adjustable EIT phenomenon in mid infrared band by using periodic graphene nanoband . A planated peak . Xiang eted peak .A graphene metasurface structureis composed of mono-layer graphenenano-disks coupled with graphene nano-strip exhibited tunable EIT responses at a mid-infrared range . The allIn this paper, a three-layer metamaterial is studied, which is composed of a liquid crystal layer, a substrate layer and a graphene layer. The A-EIT phenomenon of double electric tuning is found by the finite integral time domain method. By analyzing the distribution of electric field and magnetic field, the physical mechanism of A-EIT effect is obtained. Through the coupling of several graphene strips, the multi band A-EIT effect is obtained. The results have potential applications in THz devices.effn is given by Equation (1) to the incident THz wave [n0 and ne represent the ordinary and the extra-ordinary indices, respectively. It is known that the LC is a nonmagnetic material. Thus, effε = effn2. effε and effn varied with bias voltage .The LC is an anisotropic medium, the refractive index of the LC THz wave .(1)neffεG of the graphene be written as [Gσ is the surface conductivity of the graphene, Gt is the thickness of graphene sheet, ε0 is the permittivity in the vacuum.Assuming that the electronic band structure of a graphene sheet is not affected by the neighboring, so the effective permittivity itten as  follows:Gσ whichcan be taken into account in inter-band and intra-band transitionsby the Kubo model of conductivity [ω is radian frequency, ħ is the reduced Planck constant, κB is the Boltzman constant, e is the charge of an electron, T is the temperature, μc denotes the chemical potential, and τ is electron-phonon relaxation time, respectively.For a graphene sheet, the electromagnetic properties are described in terms of the surface conductivity uctivity .(3)σG=σεG can be tuned by the cµ is shown in εG increases with the decrease of cµ. The change of imaginary part of εG is just the opposite.The effective permittivity of graphene z-axis is open. The proposed structureconsists of graphene strip and LC layer patches on the opposite sides of polyimide layer of substrate in l = 18 µm, h = 15 µm, t1 = 1.6 µm, t2 = 0.3 µm, Gd = 1 nm, w = 0.3 µm, l1 = 4.4 µm, l2 = l1 × 1.4 µm, l3 =l1 × 1.42 µm, l4 = l1 × 1.43 µm.The proposed metamaterial structure was calculated by applying the finite integral time domain (FITD) method. The boundary condition ofx and y directionsare unit cell and the εLC. It is demonstrated that the transmission spectrum changing due to effective permittivity of LC can be tuned by the different voltage on the LC. The transmission spectrum has a slight red shift atthe εLC increases from 2.47 to 2.91. The transmission spectrum produces a larger shift when the εLC increases to 3.06.cµ can adjust the transmission spectrum. As shown in cµ from 0.1 eV to 0.8 eV. Increasing the cµ causes the A-EIT window to besignificant blue shifted. The resultsshow that the chemical potential of graphene can modulate the transmission spectrum.It can be concluded that the structure has the double tunableproperty.Changing the chemical potential of graphene To study the A-EIT phenomenon of the designed structure, we study separately the Graphene Strips 1 and 2 and both strips inthe A-EIT structure to clarify the cause of A-EIT effect physical mechanism. Asshown in To further explain the physical mechanism of the A-EIT, l1 on the transmission spectrum. It can be seen that the transmission spectrum has a red shift with the increase of l1. This is due to the length of graphene strip increasing, its resonant frequency will also produce a red shift. This conclusion is consistent with the result in nb, each resonant frequency has a slight red shift. This result has a potential application in refractive index sensing.fa, fc, fe, is smaller and the frequency shift of fg is larger. n of the surrounding environment, which is between 13 and 18.To further discuss the application on refractive index sensing, To compare the results of this work with reported, 2-graphene strips layer. To study the A-EIT effect, we first designedthe structure with only a transmissionwindow of A-EIT, which is composed of two graphene strips and LC on both sides of SiO2 layer. The transmission spectrum and electric field distribution of only one graphene strip and two graphene strips have been studied. As a bright mode, the graphene strip is coupled with adjacent graphene strip to realize the analogue A-EIT effect. The multi band analogue A-EIT is obtained on multiple graphene strips which are coupled with each other. The results showthat the multibandA-EITeffectcan be tuned by voltage on the LC and graphene layers, respectively. The results show that the incident angleinsensitive A-EIT can be obtained. Moreover, each transmission window can achieve high transmittance and FOM on changing the surrounding environment. The multiband A-EIT effectcan be applied on optical storage devices, sensor and large angle devices.In this paper, we studied a metamaterial structure with multi-band A-EIT effect. The structure is consisting of LC-SiO


Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.The opportunistic pathogen  Acinetobacter baumannii towards polymyxins remains largely unexplored. In this study, we established a host-pathogen-antibiotic tripartite in vitro model to examine the complex tripartite molecular interplay from both bacteria and macrophages perspectives, mimicking the physiological infection-treatment condition. This is the first comprehensive proteomics dataset for A. baumannii interacting with host cells, with an extraordinary coverage  of A. baumannii proteome even in infection conditions. For the first time, we report that macrophages and polymyxins utilize complementary mechanisms to disarm several A. baumannii stress tolerance strategies to persist in the macrophages. Our host-pathogen-antibiotic mechanistic study and the discovery of potential druggable targets  represent a significant advance, paving way to antibiotic optimization and drug discovery to tackle multidrug-resistant bacterial infections.Bacterial response towards antibiotics is sensitive to the surrounding environment; however, how the host immune microenvironment affects the response of opportunistic pathogen  Acinetobacter baumannii which causes severe pneumonia, bacteremia and other infections, especially in immunocompromised patients  + PMB) exhibited the most distinct separation from untreated bacterial controls in the first component, followed by the polymyxin B-treated infection group (AB + THP-1-dMs + PMB) and THP-1-dMs infection alone group (AB + THP-1-dMs) . Compared to the untreated bacterial controls, AB + THP-1-dMs + PMB resulted in 607 differentially expressed proteins (DEPs) followed by AB + PMB (549 DEPs) and AB + THP-1-dMs (202 DEPs), indicating that perturbations in the AB5075 proteome were predominantly driven by polymyxin treatment .We examined whether macrophages (THP-1-dMs) alter molecular response of 2FC values of 6.12, 4.12 and 1.23, compared to the untreated controls . Interacting bacteria also upregulated oxidative stress resistance-associated thioredoxin TrxA, glutaredoxins GrxC and GrxD, peptide-methionine (S)-S-oxide reductase MsrA, and an oxidative damage protection protein (ABUW_RS17615) (S1 Table). THP-1-dMs infection alone also led to downregulation in bacterial iron acquisition systems. Several acinetobactin biosynthetic enzymes were significantly downregulated in the AB + THP-1-dMs group, including 2,3-dihydroxybenzoate-AMP ligase EntE, non-ribosomal peptide synthetase BasD, BasC and BasB, TonB-dependent siderophore receptors BauA, TonB-dependent receptor (ABUW_RS14495), FhuE (ABUW_RS08070), MotA/TolQ/ExbB proton channel family protein , and acinetobactin utilization protein BauF . Fig 2A and 2B show the schematics of oxidative stress resistance and iron acquisition pathways, respectively, with up- and down-regulated DEPs.In response to THP-1-dMs, interacting bacteria uniquely upregulated several cold shock proteins namely Csp2 (ABUW_RS12225), Csp1 (ABUW_RS13055) and putative cold shock protein (ABUW_RS15360) with respective log2FC values of 1.46, 1.37, -7.32 and -4.08, respectively . Notably, two thioredoxins (TrxA and TrxC), thioredoxin-disulfide reductase (TrxB), GrxD, and glutathione peroxidases BtuE (ABUW_RS18150) were substantially downregulated  . Furthermore, polymyxin B treatment alone downregulated AB5075 protein quality control systems, including the DsbC family protein (ABUW_RS03275), DsbA family oxidoreductase , thiol:disulfide interchange protein DsbA/DsbL , MsrA and peptide-methionine (R)-S-oxide reductase MsrB .The bacterial proteome signature corresponding to antibiotic treatment alone  includes a unique upregulation of catalases KatE and KatG, and severe downregulation of superoxide dismutases SodB (ABUW_RS05940) and SodC (ABUW_RS01670), with log1Fo ATP synthase subunits AtpA (ABUW_RS18175) and AtpG . In addition, three proteins responsible for energy-mediated uptake of iron complexes, acinetobactin export ABC transporter permease/ATP-binding subunit BarB, energy transducer TonB (ABUW_RS16655) and ExbB, were all mildly upregulated . Conversely, there was a dramatic downregulation in acinetobactin biosynthetic enzymes EntE, BasB, BasF, and utilization protein BauF .Interestingly, we observed a unique upregulation in tricarboxylic acid (TCA) cycle and oxidative phosphorylation proteins with polymyxin B treatment alone. These upregulated proteins included NADP-dependent isocitrate dehydrogenase Idh (ABUW_RS05265), ADP-forming succinate-CoA ligase beta subunit SucC, class II fumarate hydratase FumC, isocitrate lyase AceA (ABUW_RS14030), type I NADH dehydrogenase subunits NuoC, NdhC, and NuoG (involved in shuttling electrons from NADH to quinones in the electron transport chain [ETC]), F2FC, 2.46) and (p)ppGpp synthetase RelA  ; while, intriguingly, a dramatic downregulation of DksA  .Stringent response regulators are critical in bacterial physiological activities promoting survival during stress , and sevFig 1E). Notably, the expression of NdhC changed from a log2FC of 1.35 when exposed only to polymyxin B to a log2 FC of -1.32 in the tripartite condition . Energy-dependent systems that were uniquely upregulated only in response to polymyxin B were also abrogated in the presence of THP-1-dMs. These included multidrug efflux pump membrane proteins , ATP-binding protein MlaF (ABUW_RS01880), methionine ABC transporter ATP-binding protein MetN (ABUW_RS07465), lipoprotein-releasing ABC transporter ATP-binding protein LolD, ATP-dependent Clp protease ATP-binding subunit ClpX, energy transducer TonB, and acinetobactin export associated BarB .To determine whether THP-1-dMs affected the adaptive responses of interacting bacteria towards subsequent polymyxin B treatment, we compared the bacterial proteome signatures from the tripartite condition  with those of the bipartite condition . Despite a high level of similarity in DEPs between the two groups, polymyxin-induced upregulation in energy production and ATP-dependent systems observed in the AB + PMB group was abrogated in the AB + THP-1-dMs + PMB group. For example, upregulation in SucC, NuoG, AtpA and AtpG observed in response to polymyxin B alone was reduced to a level approximating that of the untreated control in the AB + THP-1-dMs + PMB group . However, unique proteome remodeling in the iron acquisition systems of interacting bacteria was also observed in this group, as shown by the tripartite-specific upregulation in TonB-dependent siderophore receptor (ABUW_RS18465) and downregulation in siderophore achromobactin biosynthesis protein AcsC (ABUW_RS10625) (S3 Table). In the interacting bacteria, the tripartite condition also severely downregulated the copper resistance system multicopper oxidase  and abrogated infection-specific upregulation in the heavy-metal associated domain-containing protein CopZ (ABUW_RS13140) observed in the AB + THP-1-dMs group . Interestingly, nickel and cobalt homeostasis associated RcnB family protein (ABUW_RS02965) was upregulated  solely in the tripartite condition (S3 Table). Of note, upregulation in SpoT and RelA observed in the AB + PMB group was both reduced to a level approximating that of the untreated control in the AB + THP-1-dMs + PMB group .Interacting bacteria isolated from the AB + THP-1-dMs + PMB group exhibited insignificant differences in KatE, KatG, GrxC, Csp1 and putative cold shock protein (ABUW_RS15360) compared to the untreated control  perspective . In total, approximately 4,320 mammalian proteins were identified across all samples involving THP-1-dMs. The PCA score plot revealed that the most distinct separation was between the AB + THP-1-dMs group and the untreated THP-1-dMs control . Expression data of THP-1-dMs in the AB + THP-1-dMs + PMB group was closely clustered with those of the AB + THP-1-dMs group; whereas the data of the THP-1-dMs + PMB group were closely clustered with the untreated THP-1-dMs control . Compared with the untreated control group, there were 98, 94 and 2 DEPs with the AB + THP-1-dMs, AB + THP-1-dMs + PMB, and THP-1-dMs + PMB groups, respectively . Collectively, our data show that the THP-1-dMs proteome was predominantly modulated by AB5075 infection, and 1-h exposure to 30 mg/L polymyxin B had little effect on THP-1-dMs whether alone or in the presence of AB5075 infection. Pathway analysis revealed that infected THP-1-dMs from the AB + THP-1-dMs group were enriched in platelet activation and signaling, high density lipoprotein (HDL) remodeling, heme homeostasis and apoptosis .We further examined the proteomics of THP-1-dMs samples to better understand the effects of 2FC, 2.01), heme scavenging associated proteins , and apolipoprotein A-I ) . However, downregulation was observed in heme catabolic enzymes, such as heme oxygenase 1  and biliverdin reductase A  . Notably, host cellular antioxidants were depleted in AB5075-infected THP-1-dMs with downregulation in the modifier subunit of glutamate-cysteine ligase GSH0  and 2-aminoethanethiol dioxygenase AEDO  . Intriguingly, our findings reveal AB5075-induced upregulation of clotting cascade enzymes in infected THP-1-dMs. This was shown by the increased expression of coagulation factor V , prothrombin , fibrinolysis inhibitor alpha-2-antiplasmin , and alpha-2-macroglobulin  in the AB + THP-1-dMs group . Of note, anti-coagulant antithrombin-III (ANT3) was also upregulated in infected THP-1-dMs .Compared to the untreated control, THP-1-dMs in the AB + THP-1-dMs group at 4 h post infection exhibited a significant upregulation of iron-binding proteins, including lactotransferrin  and upregulated intracellular trafficking associated TBC1 domain family member 15 . When the AB + THP-1-dMs + PMB and AB + THP-1-dMs groups were compared, downregulation was revealed in glycolytic enzyme ATP-dependent 6-phosphofructokinase, muscle type , while upregulation in phospholipid phosphatase 6 .In the THP-1-dMs + PMB group, THP-1-dMs downregulated transcriptional regulator SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 . At 1 and 5 h post polymyxin B treatment (4 mg/L), trxC and trxA mutants both showed 4- to 6 log10 CFU/mL additional killing compared to that of the wild-type, with >3 log10 CFU/mL additional killing at 24 h . Enhanced polymyxin B killing was also observed with the katG and katE mutants at 1 h (at least 3 and 4 log10 CFU/mL), the csp1 mutant at 1, 5 and 24 h (at least ~3 log10 CFU/mL), and the grxC mutant at 1 and 5 h (>2 log10 CFU/mL) . Polymyxin B time-kill kinetics of AB5075 mutants with T26 transposon-mutated pseudogenes hisP (ABUW_RS06545) or filB (ABUW_RS15750) mirrored those of the wild-type strain .Next, we investigated the role of identified candidate targets in affecting bacterial killing by polymyxin B. As the wild-type lymyxins , 4 mg/L bauF and exbB mutants, such as at 1 h with additional killing of 6 and 5 log10 CFU/mL, respectively . With the basB mutant, >2 log10 CFU/mL killing was observed at 1 and 5 h, and 1.95 log10 CFU/mL killing with the tonB mutant at 1 h .AB5075 transposon mutants with disrupted iron acquisition genes were more susceptible to the initial killing by polymyxin B, compared to that of the wild-type strain. Much better polymyxin B killing was evident against spoT resulted in the greatest enhancement of killing (6–8 log10 CFU/mL) in the tested mutants across all timepoints . This dramatically enhanced killing matched the observations of our time-lapse imaging study which showed low cell counts of the spoT mutant across 24 h when treated with polymyxin B at 0.25 and 0.5 mg/L . In contrast, at both polymyxin B concentrations growth of the wild-type AB5075 was suppressed only over the first 7–10 h followed by rapid regrowth that approximated the control values by ~14 h . Interestingly, our imaging data revealed that, compared to the wild-type AB5075, spoT mutants exhibited a relatively smaller cell size at 0 h. AB5075 wild-type showed a mean (± SD) cell area of 3.35 ± 0.92 μm2 at 0 h; whereas spoT mutants exhibited a much lower mean (± SD) cell area of 2.58 ± 1.13 μm2 . At 24 h, the mean (± SD) cell area of spoT mutants treated with 0.5 mg/L polymyxin B did not increase (2.98 ± 1.43 μm2); while the mean (± SD) cell area of untreated spoT mutants increased to 4.91 ± 3.42 μm2, comparable to those of wild-type cells regardless of polymyxin B treatment .Notably, with polymyxin B treatment alone, inactivation in the bifunctional (p)ppGpp synthetase/hydrolase dksA also led to enhanced polymyxin B killing, with ~4 log10 CFU/mL additional killing of the dksA mutants at 1 and 5 h and 2 log10 CFU/mL additional killing at 24 h . Additionally, relA mutants exhibited reduced tolerance to initial polymyxin B exposure with additional killing of 2.97 and 4 log10 CFU/mL at 1 and 5 h, respectively . Subsequently, we compared the antibacterial activity of polymyxin B against the three stringent response-associated mutants spoT, dksA, and relA to that of the wild-type in a macrophage infection microenvironment. Herein, 0.25 mg/L polymyxin B  was employed to prevent overkill of interacting bacterial cells and allow comparison of the antibiotic killing dynamics between the interacting wild-type and mutants. Interestingly, during early THP-1-dMs infection in the absence of polymyxin B, all three mutants had significantly fewer interacting bacteria at 4 h post infection, compared to that of the wild-type . At 4 h , the antibiotic treated wild-type group exhibited 2.7 log10 CFU/mL interacting bacteria; however, at this time no interacting bacteria were detected with the dksA mutant while the spoT mutant was reduced by 99.5% compared to that of the wild-type . At 8 h , the viability of the interacting spoT and relA mutants was significantly reduced , compared to that of the wild-type .Inactivation of stringent response regulator in vitro findings, we employed spoT mutants as an example to examine the role of bacterial stringent response in the survivability in an immunocompetent mouse bacteremia model. At 6 h after bacterial inoculation (in the absence of polymyxin B), significantly reduced bacterial load in blood was observed in mice infected by the spoT mutant at both 108 and 109 CFU/mouse inoculum, compared to that of AB5075 wild-type infection control . Using the same mouse bacteremia model with intravenous polymyxin B , the average bacterial count of spoT mutant in blood at 4 h reduced to below the limit of detection of 1.30 log10 CFU/mL, showing at least >1.65 log10 CFU/mL reduction compared to that of the wild-type AB5075 .To validate our A. baumannii, macrophages and an antibiotic using an in vitro host-pathogen-drug model. Using correlative proteomics, we discovered several potential pathogen-directed or host-directed candidate proteins and pathways for future therapeutic targeting. Correlative proteomic profiling of infected macrophages and their interacting bacteria allowed an integrative analysis of the interaction between innate immune cells and pathogen during the infection. A high multiplicity of infection  was employed to achieve a sufficient number of interacting bacterial cells for proteomic analysis. Considering the rapid killing by polymyxins  in A. baumannii), and shares similarity with periplasmic copper oxidase PcoA (BCZ13020.1) in A. baumannii (83.6% identity) which detoxifies oxidizing Cu1+ to less damaging Cu2+ ions ) is a recently characterized MDR isolate collected from a wound of a patient at the Walter Reed Army Medical Center ) or 0.5 mg/L (2×MIC). The treatment slide was incubated on the microscope stage at 37°C. Using 20× magnification, twelve spots per treatment group were marked and traced, and images at all spots were collected every 10 min for 24 h. The time-lapse videos for each treatment group are attached in S6 Videos. For analysis of bacterial cell size changes over time, images of the same spot collected every 1 h for 24 h were examined using ImageJ [Logarithmic-phase cultures of AB5075 wild-type and g ImageJ .spoT mutants and AB5075 wild-type, and polymyxin B efficacy in vivo . Female Swiss mice  were housed with food and water available ad libitum and were used in the immunocompetent mouse bacteremia model . Mice were randomly assigned to different experimental groups. Early logarithmic-phase cultures (100 μL) of AB5075 wild-type or spoT transposon mutants were intravenously administered into each female Swiss mouse to establish bloodstream infection . At 2 h after inoculation, polymyxin B was administered intravenously at 4 mg/kg to the treatment group. Mice were euthanized at 0 h and 4 h post polymyxin B treatment using 100% carbon dioxide inhalation, and blood was collected using cardiac puncture. Mouse blood was serially diluted with 0.9% saline and spirally plated on nutrient agar plates to determine bacterial load in blood (CFU/mL). At least three independent biological replicates per experimental group were examined in this study. All animal experiments were approved by the Monash University Animal Ethics Committee and performed in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.An immunocompetent mouse bacteremia model was employed to examine the survivability of in vitro time-kill assay and THP-1-dMs infection study were quantified using spot assay on nutrient agar plate  and statistically analyzed using two-way ANOVA followed by Tukey’s HSD post-hoc test on GraphPad Prism 9.1.0. In the mouse bacteremia study, bacterial load in blood was quantified by spirally spreading on nutrient agar plates . Multiple unpaired t-tests were performed in GraphPad Prism 9.1.0 to analyze the in vivo bacterial count, with p-value <0.05 defined as statistically significant. The sample size used in each experiment is specified in the methodology sections and figure legends.Bacterial viable count in the S1 Fig(TIF)Click here for additional data file.S2 FigA) PCA score plot of A. baumannii proteomic profiles showing the relatedness of the dataset within and across the experimental groups of untreated AB5075 , polymyxin B treated AB5075 (AB + PMB), interacting AB5075 from infected THP-1-dMs (AB + THP-1-dMs), and interacting AB5075 from polymyxin B treated THP-1-dMs infection (AB + THP-1-dMs + PMB). B) Venn diagram showing common and unique sets of differentially expressed proteins of A. baumannii  between different comparison groups. C) PCA score plot of THP-1-dMs proteomic profiles showing the relatedness of the dataset within and across the experimental groups of untreated THP-1-dMs , AB5075-infected THP-1-dMs (AB + THP-1-dMs), polymyxin B treated THP-1-dMs (THP-1-dMs + PMB), and polymyxin B treated and AB5075-infected THP-1-dMs (AB + THP-1-dMs + PMB). D) Venn diagram showing common and unique sets of differentially expressed proteins of THP-1-dMs  between different comparison groups. Three biological replicates were employed in each experimental group.(TIF)Click here for additional data file.S3 FigVolcano plot generated using WebGestalt showing the enriched KEGG and Reactome pathways (Benjamini-Hochberg FDR <0.05) in THP-1-dMs at 4 h post infection with AB5075 in the absence of polymyxin B.(TIF)Click here for additional data file.S1 TableAcinetobacter baumannii AB5075; PMB, polymyxin B; THP-1-dMs, macrophage-like THP-1 cells.DEPs, differentially expressed proteins; AB, wild-type (XLSX)Click here for additional data file.S2 TableAcinetobacter baumannii AB5075; PMB, polymyxin B; THP-1-dMs, macrophage-like THP-1 cells.DEPs, differentially expressed proteins; AB, wild-type (XLSX)Click here for additional data file.S3 TableAcinetobacter baumannii AB5075; PMB, polymyxin B; THP-1-dMs, macrophage-like THP-1 cells.DEPs, differentially expressed proteins; AB, wild-type (XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.S2 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.S3 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.S4 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.S5 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.S6 Video20× magnification; video speed at 7 frames per second (fps).(AVI)Click here for additional data file.


Amyotrophic lateral sclerosis is a progressive fatal neurodegenerative disease caused by loss of motor neurons and characterized neuropathologically in almost all cases by nuclear depletion and cytoplasmic aggregation of TDP-43, a nuclear RNA-binding protein (RBP). We identified ELAVL3 as one of the most downregulated genes in our transcriptome profiles of laser captured microdissection of motor neurons from sporadic ALS nervous systems and the most dysregulated of all RBPs. Neuropathological characterizations showed ELAVL3 nuclear depletion in a great percentage of remnant motor neurons, sometimes accompanied by cytoplasmic accumulations. These abnormalities were common in sporadic cases with and without intermediate expansions in ATXN2 and familial cases carrying mutations in C9orf72 and SOD1. Depletion of ELAVL3 occurred at both the RNA and protein levels and a short protein isoform was identified, but it is not related to a TDP-43-dependent cryptic exon in intron 3. Strikingly, ELAVL3 abnormalities were more frequent than TDP-43 abnormalities and occurred in motor neurons still with normal nuclear TDP-43 present, but all neurons with abnormal TDP-43 also had abnormal ELAVL3. In a neuron-like cell culture model using SH-SY5Y cells, ELAVL3 mislocalization occurred weeks before TDP-43 abnormalities were seen. We interrogated genetic databases, but did not identify association of ELAVL3 genetic structure with ALS. Taken together, these findings suggest that ELAVL3 is an important RBP in ALS pathogenesis acquired early and the neuropathological data suggest that it is involved by loss of function rather than cytoplasmic toxicity.The online version contains supplementary material available at 10.1007/s00401-021-02374-4. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by adult-onset progressive motor impairments from degeneration of upper and lower motor neurons , 28, 58.While ALS is a disease that specifically affects motor neurons in the central nervous system, many of the altered RBPs involved in ALS are ubiquitously expressed. ELAVL (or Hu) proteins are RBPs highly expressed in neurons and fundamental for the central nervous system development. They were discovered as antigens from patients with paraneoplastic neurological syndromes , 30. FouBecause of the importance of RBPs in ALS pathogenesis, we analyzed RBPs in our two independent studies profiling transcriptional changes in sALS laser captured motor neurons , 48. We Human tissues were obtained from the UCSD ALS tissue repository that was created following HIPAA-compliant informed consent procedures approved by Institutional Review Boards . Spinal cord and brain tissues were acquired using a short-postmortem interval acquisition protocol usually under 6 h. Tissues were immediately dissected in the autopsy suite, placed in labeled cassettes, and fixed in neutral buffered formalin for at least 2 weeks before being dissected and paraffin-embedded for indefinite storage. For this study, we evaluated from 6 control cases, 31 sALS cases, 5 C9orf72 cases, and 4 SOD1 (Supplementary Table 1).Motor neurons were identified by their large size, multipolar cytoplasm, presence of lipofuscin, and large nucleus with a less dense DAPI staining in IF studies. In spinal cord, we examined motor neurons in Rexed lamina IX of anterior horn in cervical, thoracic, and lumbar sections. In the cortex, we examined Betz cells in layer V of the cortex. For quantifications, we chose paraffin blocks that we knew had abundant neurons as revealed by standard H&E light microscopy and previous studies revealing abundant TDP-43 pathology.® HRP Horse Anti-Rabbit IgG Polymer Detection Kit, Peroxidase (MP-7401 Vector) and developed the antibody with Vector® NovaRED® Substrate Kit, Peroxidase (HRP) (SK-4800 Vector) for 1–2 min until the color was noticed.Tissue sections were cut from blocks of formalin-fixed paraffin-embedded ALS tissue, obtained from the UCSD ALS bank collection. 6 µm-thick tissue sections were de-paraffinized through histology grade CitriSolv (two times for 15 min each) and a graded alcohol series . Then, we dipped the slides in de-ionized water, permeabilized for 20 min in 1 × PBS with 0.2% Triton X-100, and performed antigen retrieval with 1% Tris-based (Vector # H-3301) in a pressure cooker at 120 °C for 20 min. Sections were further blocked with 2% Fetal Bovine Serum  and incubated with the primary antibody. Primary antibodies were ELAVL3 (#55047-1-AP from Proteintech (lot number 09000021 was specific for biochemistry) and LS-C408905-50 from LsBio), TDP-43 mouse , TDP-43 Rabbit (#10782-2-AP Rabbit Proteintech), HuR-Antibody , ELAVL4 (#24992-1-AP Proteintech), ELAVL2 (#67097-1-AP Proteintech), and Ubiquitin . The antibodies for ELAVL1 and ELAVL3 were previously validated by KO studies , 59, 62.Sections from blocks of the spinal cord and motor cortex were stained with the antibodies described above. We analyzed two sections of 6 µm per patient. A semiquantitative assessment of RBP nuclear depletion and mislocalization or decreased expression in ALS tissues was performed taking digital photographs using the Nanozoomer at 40X and visualizing under 60 × objective in either Nikon confocal A1/LANA system or Leica SP8 confocal systems at UCSD’s Microscopy Core facility. Depending on the degree of neurodegeneration of each tissue, we counted as many motor neurons as could be identified as described above. Once neurons were identified, motor neurons were scored based on the presence or absence of nuclear ELAVL3 and TDP-43, being either present or depleted. For illustrations, images were taken at a resolution of 1024 × 1024 pixels with a 60 × objective and between 15 and 20 z stack images (0.25–0.50 μm per stack) were collected per frame, and the maximum intensity projection was used to construct the final image series using Fiji software. The .tif files were loaded into Photoshop, where the RGB channels were split. Once the figure was built, each individual channel was inverted to facilitate visualization. The following criteria were used in scoring cytoplasmic accumulations: inclusions were small dot-like, round, diffusely scattered speckles that are relatively uniformly distributed; aggregates were discrete, dense, long, fibrillary, or striae-like (or thread-like) accumulations; and cytoplasmic inclusion bodies were large, round, dense deposits in the cytoplasm present in low number, usually only one per cell.l-glutamine , 100 units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% carbon dioxide at 37 °C. SH-SY5Y TDP-43EGFP cells were plated on 8-well chamber (Ibidi) at 25,000 cells per well. After 24 h, cells were arrested in G1 with Palbociclib (Apexbio) and maintained during the whole length of the experiment. Cell culture media were changed every 3 days. In all the experiments, the SH-SY5Y control cells and the experimental were plated and maintained in the same culture time and media conditions with Palbociclib, without and with rapamycin respectively. 10 nM of Rapamycin was diluted in 100 μl of DMSO to use as stressor. Rapamycin was used at a final concentration of 5 μM in the media to stress the cells. Rapamycin was maintained in the media for 4 weeks. Cells were fixed with 4% PFA in PBS for 10 min at room temperature. After two washes with PBS, cells were permeabilized and blocked with blocking solution  for 1 h at room temperature. Cells were then incubated overnight with the primary antibody in PBS/0.3% Tween 20. The primary antibodies used for staining were: anti-ELAVL3  at day 1 and after 1, 2, and 4 weeks after rapamycin application. After three washes with PBS, the cells were subsequently incubated with fluorescently labeled secondary antibodies diluted at 1:500 in 1 × PBS for 1 h at room temperature. After three washes with 1 × PBS, a secondary antibody (anti-rabbit-555 from Jackson) was applied for 1 h. We washed the cells three times for 5 min each with 1 × PBS and mounted with ProLong Gold Antifade Mountant with DAPI media (Invitrogen) to preserve the cells for imaging. SH-SY5Y cell imaging was performed on a Ti1 Nikon microscope with a C2 confocal camera at 60–100 × magnification.Neuroblastoma SH-SY5Y TDP-43-GFP line was obtained from Cleveland Lab. All cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% FBS , 2 mM ® Fc Imaging System from Li-COR. ImageStudioLite method was used to measure the signal intensity.To characterize ELAV3 expression in spinal cord samples, frozen tissue from donors and patients (Supplementary Table 1) was weighted and homogenized in ice-cold N-PER Neuronal Protein Extraction Reagent (Thermofisher # 87792) with protease and phosphatase inhibitors in 1 ml per 100 mg ratio. The samples were spun down and the supernatants were subjected to Pierce™ BCA Protein Assay (Thermofisher #23225). Samples were boiled with Laemmli sample buffer for 10 min at 90 °C, and to normalize the total amount of proteins among samples, 35 μg were loaded in SDS-PAGE , and transferred into Nitrocellulose membranes (Bio-Rad # 1704159) with Trans-Blot Turbo Transfer System (Bio-Rad# 1704150) . Membranes were blocked using iBind™ Flex Fluorescent Detection (Invitrogen # SLF2019) and were incubated with primary (1:500 ELAVL3 (#55047-1-AP Proteintech),1:5000 GAPDH , 1:500 TDP-43 (12892-1-AP Proteintech), 1:1000 TUJ1 (#801202 Biolegend) and secondary antibodies  using iBind™ Flex Western System (Invitrogen # SLF2000). NewBlot™ Nitro Stripping # 928-40030 LI-COR was use as a stripping buffer between primary antibody incubation when needed. The images were acquired with an Odysseyhttps://www.jax.org/strain/002726), TARDBP tg(tetO-TARDBP) mice and their littermates (C57BL/6J × C3H/HeJ)F1 (https://www.jax.org/strain/016841), and C9ORF72 mice and their littermates (C57BL/6 × C3H) (https://www.jax.org/strain/030581) were studied.SOD1 G93A and their littermates (C57BL/6 × SJL)F1 ; (3) variants with haplotype-based non-random missingness ; (4) variants with an overall missingness rate of ≥ 5·0%; (5) non-autosomal variants ; and (6) variants that significantly departed from Hardy–Weinberg equilibrium in the control cohort .Variant exclusion criteria were: (1) monomorphic or palindromic SNPs; (2) variants that showed non-random missingness between cases and controls (https://imputationserver.sph.umich.edu/). Only variants in ELAVL1,2,3, and 4 genes with imputation quality score (R2) > 0.3 and minor allele frequency > 0.01 were included for association analysis. Covariates that were adjusted for to account for non-disease related cohort stratification were sex, age at disease onset, PC1, PC2, PC3, PC4, PC6, PC7, and PC8. Regional plots for each gene were generated using LocusZoom (http://locuszoom.org/).Imputation was performed against the Haplotype Reference Consortium (HRC 2016) reference panel (hg19) via minimac4 using data phased by Eagle v2.4 provided by Michigan Imputation Server , and for missense and loss-of-function category was 3.60 × 10–6 .Sequence kernel association test-optimized (SKAT-O) analysis was performed on combined missense and loss-of-function mutations present in ELAVL1,2,3, and 4 genes. The analysis collapses all rare variants and tests for difference in the aggregated burden of rare-coding variants between cases and controls. Variants were annotated using ANNOVAR (v.2018–04/16) prior to association testing using RVTESTS (v.2.1.0). Variants were included in the analysis if the minor allele frequency was < 1% and minor allele count was ≥ 3. The Bonferroni threshold for genome-wide significance for missense category was 5.11 × 10t test–non-parametric Mann–Whitney test, and p values less than 0.05 were considered significant. Chi-square tests used an online calculator (https://www.socscistatistics.com/tests/).Data were analyzed using GraphPad Prism version 7.0a and are expressed as mean ± standard error of the mean. Significance was assessed using the Student p = 0.0009) [p = 4.00E−08) [p = 0.02 [p = 0.016 [p = 0.055 [p = 0.001 [p = 0.007) and not TARDBP     and NOL = 0.001 ) . To evaluate ubiquitination, we co-stained those tissues in which ELAVL3 cytoplasmic inclusions were visualized and we did not find colocalization between ELAVL3 and ubiquitin—in these, we did indeed observe ubiquitinated cytoplasmic inclusions, consistent with ubiquitination of TDP-43 .We quantified ELAVL3 abnormalities in 161 motor neurons from 11 sALS nervous systems in the spinal cord using IF. In this, we included only motor neurons that had observable nuclei as identified by DAPI staining —and our preliminary observations in three mice of each model and three respective wild-type littermates support the observation of ELAVL3 nuclear depletion and align with our observations in human samples, although additional studies are needed .We compared ELAVL3 in nervous systems with known genetic contributions. 85% of 59 motor neurons examined in two sALS nervous systems with intermediate length repeat expansions of ATXN2 Fig. d. These To confirm the downregulation of ELAVL3 observed transcriptionally and by immunofluorescence, we measured ELAVL3 protein levels by immunoblotting in sALS, C9, and SOD1 ALS cases Fig. a. Controp = 0.09 using Chi-square analysis and Supplementary Table 4). This supports current consensus that TDP-43 sALS neuropathology has both nuclear depletion and cytoplasmic accumulation and contrasts to ELAVL3, where nuclear depletion is the hallmark neuropathologic change.We evaluated TDP-43 in the same 161 motor neurons from the 11 sALS nervous systems using co-IF. TDP-43 was well visualized in the nuclei of neurons and glia cells in control tissue, and sALS patients’ tissue exhibited the expected mix of TDP-43 nuclear depletion and cytoplasmic accumulations, especially aggregates , ELAVL2 (Ctr = 4 and sALS = 10), and ELAVL4 (Ctr = 6 and sALS = 31). ELAVL1 was expressed in the nuclei and diffusely in the cytoplasm of neurons and glial cells in control nervous systems . At the rare variant level, we performed a gene-based analysis on variants that were predicted to be missense or have a loss-of-function effect using the SKAT-O method and did not find excess presence of rare variants in cases compared to controls. From these results, it does not appear to be that there are rare variants in these genes that drive/modify risk for ALS; at least this is true for this set of imputed variants in the tested cohort.To investigate the contribution of common ELAVL variants to disease risk, we performed association testing on 750 common variants present in ELAVL1,2,3, and 4 genes in 10,599 cases and 42,279 controls . There wBecause of the focal clinical onset of ALS and anatomical propagation of disease over time, the timing of degeneration desynchronizes in motor neurons, and they display different degrees of molecular changes neuropathologically . This alIt is not unreasonable to think ELAVL3 pathobiology in ALS may be different from TDP-43. In TDP-43 pathobiology, the preponderance of evidence indicates that subcellular mislocalization rather than expression is key. Both nuclear depletion and cytoplasmic accumulation of TDP-43 usually appear together neuropathologically, and our neuropathological data indicate both nuclear loss and cytoplasmic accumulation are significant. TDP-43 expression is tightly regulated and much data support that it is not differentially expressed although our own transcriptome data if anything suggest slight upregulation. Unlike TDP-43, ELAVL3 does not have a canonical nuclear localization signal (NLS), or a low complexity glycine-rich prion domain and it is significantly downregulated at the RNA and protein levels. Our data further suggest that ELAVL3 abnormalities are upstream of TDP-43 in the process of motor neuron degeneration. This is supported by three lines of reasoning. First, all neurons with TDP-43 abnormalities displayed ELAVL3 abnormalities, but many neurons with ELAVL3 abnormalities displayed normal nuclear TDP-43. Thus, TDP-43 pathology was a subset of ELAVL3 pathology, not vice versa. Second, ELAVL3 abnormalities were also seen in SOD1 nervous systems, where TDP-43 abnormalities are not present. The role SOD1 plays in sALS is debated. Regardless, it is accurate to say that ELAVL3 pathology is widespread and prevalent in sALS and fALS from the two most common mutations SOD1 and C9orf72. Neither SOD1 nor TDP-43 are as clearly generalizable neuropathologically as ELAVL3 is, although it is possible that TDP-43 or SOD1 are abnormal at a molecular level that are not visible neuropathologically. Third, our cell culture data in which cell cycle arrested non-dividing neuron-like TDP-43-GFP SH-SY5Y cells were artificially stressed clearly showed ELAVL3 abnormalities long before TDP-43 abnormalities, suggesting a more sensitive and prompter response to stress. These results were recently shown in detail in an experimental study reporting uniform downregulation of ELAVL3 in extensive ALS iPSC-MNs models at both RNA and protein levels and, importantly, this decrease was present beginning in early development, suggesting that ELAVL3 is an early modulator of downstream disease . Taken tThe short ELAVL3 isoform was an unexpected finding. Since the changes on immunoblotting mobility are of 3 kDa, the abnormal isoform is expected to be 25–30 amino acids shorter than the normal protein. An isoform of ELAVL3 is known to exist, but it is only six amino acids different (isoform 2 is missing amino acids 251–257). Since we used an antibody to exon 2 epitopes near the N-terminal in both the neuropathological and biochemical studies, it seems reasonable to conclude that the shortening is after exon 2. One possibility is inclusion of a cryptic exon from reduction of TDP-43 such as has been identified in STMN2  and UNC1Previous literature has already implicated ELAVL3 in ALS pathogenesis, although it has received little direct attention and is under recognized. In a systematic survey of 213 proteins harboring RNA recognition motifs in a yeast functional screen, ELAVL3 (but not the other ELAVLs) had a high toxicity score and, as stated, did not harbor a prion domain, unlike TDP-43, FUS, and other RBPs . In humaThe suggestion that ELAVL3 abnormalities are upstream in the process of neurodegeneration led us to question if genetic factors might be related to its misregulation. However, no significant genetic association of ELAVL3 or other ELAVL family members could be identified, indicating that misregulation of ELAVL3 is likely an early acquired abnormality. While common variant association and rare gene-burden analysis at population scale on sporadic ALS cases do not show significant contribution to risk, ultra-rare variants may exist in families that do not carry the common ALS mutations. In summary, we have compelling evidence implicating ELAVL3 upstream in sporadic, C9orf72, and SOD1 ALS pathogenesis that does not appear to be genetically based. The triggers and upstream regulators and downstream mechanisms in the neurodegeneration cascade remain unknown. Further studies will elucidate mechanisms of this important RBP.Supplementary file1 (PDF 317349 KB)Below is the link to the electronic supplementary material.