Natural sediment flocs are fragile, highly irregular, loosely bound aggregates comprising minerogenic and organic material. They contribute a major component of suspended sediment load and are critical for the fate and flux of sediment, carbon and pollutants in aquatic environments. Understanding their behaviour is essential to the sustainable management of waterways, fisheries and marine industries. For several decades, modelling approaches have utilised fractal mathematics and observations of two dimensional (2D) floc size distributions to infer levels of aggregation and predict their behaviour. Whilst this is a computationally simple solution, it is highly unlikely to reflect the complexity of natural sediment flocs and current models predicting fine sediment hydrodynamics are not efficient. Here, we show how new observations of fragile floc structures in three dimensions (3D) demonstrate unequivocally that natural flocs are non-fractal. We propose that floc hierarchy is based on observations of 3D structure and function rather than 2D size distribution. In contrast to fractal theory, our data indicate that flocs possess characteristics of emergent systems including non-linearity and scale-dependent feedbacks. These concepts and new data to quantify floc structures offer the opportunity to explore new emergence-based floc frameworks which better represent natural floc behaviour and could advance our predictive capacity. SPM transport is critical to the fate and flux of sediment, carbon, nutrients, contaminants and pathogens through all natural aquatic environments. Therefore, understanding and predicting floc transport behaviour is essential for the sustainable management of our waterways, fisheries and marine industries.Flocs are fragile, complex, low density aggregates of minerogenic and biogenic material with fluid-filled pore space3. Consequently, critical parameters such as size and shape are frequently measured as 2-dimensional (2D) simplifications of complex 3D structures using e.g., image or laser analysis, whilst density and porosity are estimated indirectly from settling velocity and assuming spherical shape.Floc behaviour is dependent upon the size, shape, density and porosity of floc aggregates, and such data are critical input parameters for the mathematical models that predict fine sediment transport and flocculation. These inherently 3-dimensional (3D) characteristics are challenging to measure. Flocs are fragile, difficult to sample, and range in size across a spectrum from colloidal particles (nanometres) to larger aggregates (1000 s microns) spanning detection and resolution limits of multiple analytical techniques 6. Many studies report that primary particle properties  are the most sensitive parameters that control floc dynamics in a fractal model7. Such properties and the fractal dimension, which is usually derived in 2D4, are relatively simple to measure for mineralogically homogeneous or experimental sediments. A fractal dimension of 1 indicates near spherical, compact flocs, with larger values (up to 3) indicative of ‘looser’, more complex flocs. A single fractal dimension of around 2 is frequently used to solve the fractal analytical flocculation equation8. However, fractal dimension is strongly influenced by sediment composition, and in particular the presence of organic material where extracellular polymeric substances (EPS) can enhance the ‘stickiness’ of flocs9. Therefore, the application of fractal-based models becomes more challenging in compositionally variable natural aquatic environments where sediments have high organic matter, mixed mineralogy and microbial content. In addition, real flocs are multi-component with primary particles of different densities, and measuring real fractal dimensions is problematic.Given these limitations in the availability of observational data for floc characteristics, and to account for floc variability and derive a relationship between density, floc size and dynamic behaviour, flocs are considered to have fractal geometry12. Therefore, the relationship between floc size, density and hence settling velocity vary spatially and temporally13. Empirical formulations also suggest that the fractal dimension varies with floc size14. Unlike Verney et al.13 who utilise a floc diameter parameter, Maggi et al.14 describe floc population based on the number of primary particles in the flocs, which appears to make the incorporation of a variable fractal dimension straightforward. Moreover, Maggi et al.14 adopt a sophisticated collisional efficiency closure that considers the effects of floc size and permeability.Field and laboratory observations confirm that flocculation processes  and the fractal dimension of flocs formed can vary significantly depending on the types of primary particles (sediment composition) and environmental conditions 17 and assign variable fractal dimensions dependent on observations of floc size distribution. These models recognise that multiple levels of floc aggregation (or a floc ‘hierarchy’) must exist based on observations of multi-modal floc size distributions and hence represent flocculation processes using multiple class population balance equations (PBE) and variable fractal dimension, e.g., Eisma18, Manning and Dyer19 and Shen et al.16 simulate the representative sizes and mass fractions using multiple floc aggregation levels—microflocs, macroflocs and megaflocs.In response to this, increasingly many models relax the assumption that the fractal dimension is invariant with floc size 23, and are ‘pseudo-fractal’ at best. Indeed, the fractal nature of natural sediment flocs is widely contested and is largely based on circumstantial evidence and the observed scaling of density (decreasing) and porosity (increasing) with increasing floc diameter. As a result, whilst current fractal-based flocculation models present a mathematically workable approach, they are still not fully robust or efficient24. The development of alternative frameworks is limited by the lack of direct, quantitative, observational data of floc structures and the mechanics of their development, and hence understanding of their behaviour in aquatic environments across the full floc size spectrum.The large variability of floc composition , recognition that fractal dimension varies with size and composition, and the likelihood that different flocculation mechanisms are restricted to specific length scales, collectively all indicates that the structure of natural flocs cannot be self-similar1 nm to 103 µm scale27. Here, we show how these new observations of fragile floc 3D structures and particle interactions across all relevant length scales demonstrate unequivocally that natural flocs are non-fractal. We propose a floc hierarchy that is based on observations of 3D structure and function. We then discuss how our data indicate that flocs possess characteristics of emergent systems including non-linearity and scale-dependent feedbacks. These concepts and new data to quantify floc structures offer the opportunity to improve current approaches to understanding fine sediment behaviour and to explore new emergence-based floc frameworks which better represent natural floc behaviour and could enable the development of innovative mathematical models to predict real floc behaviour.We have previously developed protocols to collect and stabilise flocs, and using correlative tomography, to observe and quantify 3D floc structures and characteristics from 1027. The workflow used a targeted approach, whereby entire flocs were initially characterised at the mm-scale using X-ray computed micro-tomography (X-ray CT) before more focused analysis of sub-micron scale internal composition and structure using 3D focused ion beam nanotomography (FIB-nt). The resulting 3D datasets were transformed into voxel-based (3D pixel) data volumes and composition phases  segmented for quantification and visualisation28. 3D volumetric microscopy was combined with high-resolution  2D scanning transmission electron microscopy (STEM) imagery and energy dispersive spectroscopy (STEM-EDS) to enable the classification of floc components. Most floc components were identified based on their grey-scale value, size and shape, organic structures samples were treated with electron-dense stains and elemental spectra obtained using STEM-EDS to aid compositional identification26. Spatial registration of datasets applied at increasing magnification ensured precise spatial referencing of submerged regions of interest within the floc sample and the ability to correlate floc structures at different length scales from nm to mm, correlating not only internal floc structure but whole floc geometric characteristics.This study utilised a novel sampling and imaging workflow that facilitated the collection and stabilisation of floc samples, and acquisition and correlation of multi-scale, 3D floc datasets. Detailed methodology can be found in Wheatland et al.The floc samples presented here were sampled from natural estuarine cohesive sediment (silty clays) collected from the Thames Estuary, UK.26 was followed. Flocs were first fixed in a buffered solution of 0.15 M sodium cacodylate (pH 7.4) containing 2.5% glutaraldehyde and 2% formaldehyde with 2 mM calcium chloride before being embedded in Durcupan, a hydrophobic resin. Intermediate steps were implemented to improve contrast and replace pore water with resin, achieved by the addition of heavy metal stains  and washing samples in an ethanol series  followed by anhydrous acetone.For the stabilisation of fragile hydrated floc samples the protocol outlined in Wheatland et al.Dark-field STEM imagery was obtained from the ultrathin-sections using an FEI Inspect-F field emission gun (FEG) SEM  operating at 30 kV and fitted with a split detector STEM stage.29 was employed for the steps following sample preparation. Samples were inserted into the stage of the Quanta and raised to the eucentric height (10 mm) where the electron and  ion beams converge. With both beams focused on a coincident point on the sample surface, the stage was tilted to an angle of 52° to bring the trimmed block-face into a position perpendicular to the ion beam. This enabled precision milling and imaging of the exposed subsurface. Prior to serial sectioning a smooth, dense, protective platinum coating with a consistent thickness of ~ 1 µm, was deposited over the selected region of interest to minimise milling fluctuations that can result in morphological defects on the milled cross-section. The target volume was then isolated from the surrounding material by milling trenches on three sides of its perimeter to create a suitably sized cube. An accelerating voltage of 30 kV and a current of 0.5 – 5 nA for the ion beam was selected for milling. Once a cube was prepared the front trench was enlarged to reveal a cross-section for imaging and side trenches eroded to act as repositories into which sputtered material could collect. Serial sectioning was achieved using Auto Slice & View software  and an image sequence acquired using the backscattered signal operating at 3 kV and 4 nA electron beam. Slice thickness was adjusted to match that of the pixel size of the image area to ensure an isotropic voxel resolution (3D pixel). The regular spacing between individual image slices allows entire image sequences to be transformed directly into voxel-based (3D pixel) data volumes suitable for quantitative analysis.Volumes were obtained using an FEI Quanta 3D FEG FIB-SEM . For FIB-nt block preparation and data collection, the protocol of Bushby et al.CT scans were performed using two Nikon Metrology  XT H 225 microtomographs, one configured with a 225 kV tungsten reflection target and the other with a 180 kV tungsten transmission target. Medium-resolution imaging of entire flocs was conducted using the reflection target CT with a focal spot size of ~ 10 µm, whilst high-resolution imaging of smaller sub-regions was undertaken using the transmission target CT which had a spot size of ~ 1 µm. Both CT scanners were fitted with Perkin Elmer  16-bit flat-panel detectors. During data acquisition the source was operated at a voltage of 60–150 kV and a current of 50–160 µA. A 1 mm thick copper filter was used to absorb X-rays in the lower end of the energy spectrum, i.e. ‘soft’ X-ray with energies < 30 kV. The resulting raw X-ray projections represent differences in X-ray energy attenuation, related to material density and the absorbing material’s attenuation coefficient. Raw X-ray projections were reconstructed through tomographic back-projection within CT Pro 3D  to yield 3D volumetric models. During reconstruction data artefacts resulting from beam hardening and the centre of rotation were also addressed.30 (https://imagej.net/Fiji). To address the inherent misalignment between consecutive images within FIB-nt stacks and remove artefacts associated with the drift of the electron beam or sample, an alignment algorithm was applied. Segmentation was performed using a semi-automated segmentation tool capable of machine learning, after which volumes were imported into Avizo v9.0 (Thermo Fisher Scientific) for landmark based registration. Supplementary volumetric renderings were produced within the software package Drishti v2.6.2 (https://github.com/nci/drishti) in which the 2D transfer function editor, in addition to colour and transparency settings, permitted specific properties  to be assigned to segmented materials.All image processing steps were conducted using the imaging software Fiji/ImageJ v226. Once segmented, these image data generate quantitative 3D reconstructions of particle–particle and nm to mm scale (3D FIB-nt and 3D X-ray CT respectively) structural associations and are false coloured to aid visualisation of complex composition. Flocs were characterised into functional groups (FG) according to observations of representative particle–particle interactions and the 3D spatial arrangement and association of floc components.Representative 2D and 3D correlative datasets showing spatially registered images from STEM, FIB-nt and X-ray CT are shown in Fig. 31  and organic matter with a living  and non-living component  Fig. a–d. Iden35. The nature of these inter-particle interactions are dependent on the relative cohesive and adhesive properties of primary particles36 and reflect the environment in which flocs form. Figure Particle interactions within turbulent aquatic environments are inevitable due to fluid hydrodynamics, and organisms actively searching to colonise surfaces for habitat development and food acquisition . These interactions can be observed in 2D and dark-field STEM imagery shows particle–particle associations including simple associations between multiple clay domains, and associations between dissimilar inorganic and organic floc components such as quartz grains, multiple clay domains and bacteria 28 prevents the detection of EPS, but it can be observed in STEM filling the pore spaces between clay particles  is the key interaction operating between chemically similar particles such as clay minerals and is dependent upon clay mineralogy and the ionic strength of the solution1 and 103 nm. Cells become associated with clay particles which provide a source of nutrients and protection from predation38. The radial arrangement of clays around the cell optimises access to these resources, but individual cells can only interact with a finite number of clay domains before becoming isolated inhibiting further aggregation  comprised of densely packed clay minerals and occasional diatom frustules. Figure 33 with weaker electrochemical forces having a secondary effect. Filamentous bacteria  were observed to extend through the FG-3 volumes, and were frequently observed within these units. These units represent heterogeneous associations of primary particles (FG-1) and particle–particle (FG-2) associations confirming previous descriptions of microflocs32, but can be further characterised by highly variable, micron-scale intra-aggregate porosity and aggregation is facilitated by polymer bridging. Whilst, the high resolution and 3D nature of our data also allow the discrimination of inter-aggregate porosity, suggesting the association of multiple microfloc units.Indicative examples of larger structural floc units, characterised by greater complexity and heterogeneity, higher intra-floc porosity and looser structures are shown in both 2D STEM Fig. a and wit2 µm) are a critical contribution to mass settling flux in the environment40. X-ray CT fills a resolution gap between STEM and FIB-nt, and floc cameras and optical/laser techniques providing information on 3D size, shape and internal structures41. Figure 1 µm . A key component of this large floc is the presence of filamentous cyanobacteria .Larger flocs  project beyond the floc periphery and link the FG-4 floc units at nodal ‘anchor’ points to create large FG-5 flocs  and which geometric size ranges most accurately represent floc populations. In particular, there is a focus on defining a static boundary between floc ‘building blocks’ and larger, more fragile flocs, with the boundary ranging from e.g., between 120 and 200 microns46. To confuse matters further, this boundary describes the division between both micro and macroflocs, and macro and megaflocs, depending on the number of size classifications used. Here, we present the evidence defining microflocs (FG-3) structurally and functionally as a collection of primary particles (FG-1) and particle–particle associations (FG-2), with loose µm-scale intra-aggregate porosity, where aggregation mechanisms are dominated by electro-flocculation and polymer bridging. In contrast, macroflocs (FG-4) are defined by the importance of bacterial populations which exert strong control on floc aggregation mechanisms, shape and size of the floc units produced and result in large, tortuous pore channels through floc units and highly irregular floc shapes. Such large pore networks and irregular shapes are likely to have a significant impact on dynamic behaviour and demonstrate the strong influence that the microbiological community could have on fine sediment behaviour.Previous studies have observed multiple modalities within floc size distributions (FSD) and used these observations to develop conceptual, multi-level aggregation models comprising: primary particles, flocculi, microflocs, macroflocs and megaflocs48 The evidence presented here indicates that flocs may possess many of the characteristics of emergent systems, which can be distinguished from other self-assembly behaviour. This includes non-linearity, scale-dependent feedback and interactions and self-regulating, adaptive equilibrium behaviour47. For example, we have evidence for both negative and positive feedbacks across spatial scales and we observe that interactions with bacteria may have both negative and positive feedbacks at different scales .These functional groups operate over multiple, overlapping geometric scales and indicate that the use of modal classes, each with an assigned 2D fractal dimension, will never truly represent or predict the behaviour of natural flocs in the environment. Despite this critical flaw, suitable alternatives remain elusive due to the absence of observations and quantitative data on floc structures and particle–particle interactions. Emergence describes how complex systems arise from their underlying constituent components, exhibiting unexpected patterns, functions and behaviours that cannot be predicted from an understanding of the individual system constituents50 and provides an alternative, potentially more realistic approach for representing real multi-component floc structures with complex non-linear dynamics. Such mathematical process-based models require data which capture quantitative information to describe and explain feedbacks, interactions and spatial relationships (structures) between constituent components at all spatial scales51. This is in stark contrast to fractal-based approaches which are based on static dimensional or geometric data describing bulk floc characteristics . The data generated here provide this quantitative information on floc structures and particle–particle interactions. For example, we have already demonstrated the potential to quantify accurately floc constituents e.g., pore space and bacterial counts26 and it will be possible  to develop algorithms and machine learning approaches to quantify spatial organisation of floc constituents (including pores), and to characterise interactions . Therefore, these new 3D data present the opportunity to inform a new generation of emergent models. Utilising an emergence framework could better account for the spatio-temporal variations observed in natural sediment floc behaviour and provide a level of error checks that are not supported by current fractal approaches.An emergence approach has successfully been used to explain and predict patterns and behaviour in many natural systems41, shape , and 3D fractal dimension, and to semi-quantify density41. In addition, new mathematical approaches can be explored to quantify structural properties of flocs such as the size and connectivity of pore space to inform on the influence of drag and friction as fluid moves through a floc structure, and the spatial arrangement and distribution of individual floc components. Coupling these observations in the laboratory to measures of floc and fine sediment behaviour  could improve our understanding of the controls on floc behaviour and the parameterisation of existing fine sediment transport models. This will be a major advancement as models are currently parameterised using data generated in 2D, or approximations (porosity and density) assuming flocs have spherical shape.The scanning, data processing and visualisation approaches used here to generate fully rendered 3D images are extremely operator- and computer processor-intensive. Therefore, our approach is not suitable for application as part of e.g., a field monitoring campaign. However, whilst only a few indicative 3D images have been presented here, quantitative 3D data for 100–1000 s of individual flocs were generated, particularly for the X-ray CT analysis. These 3D data provide new opportunities to quantify accurately bulk properties of flocs including floc size and porosity (measured as occupied volume)Assumptions of fractal geometry are currently used to aid the mathematical modelling of suspended sediment dynamics. Yet, it is widely recognised that this does not accurately represent natural floc systems.New quantitative 3D data provide unequivocal evidence to demonstrate that natural sediment flocs are non-fractal and that particle composition and inter-particle interactions have a strong influence on aggregate morphology.We propose an alternative five-level hierarchy of floc aggregation based on structure–function relationships rather than observations of geometric floc size distributions.New multi-scale, high resolution 3D data indicate that flocs possess characteristics of emergent phenomena including non-linearity, scale-dependent feedbacks and self-regulation. This offers the opportunity to explore new emergence-based floc frameworks and process-based models based on quantification of particle–particle interactions which better represent natural floc behaviour.Supplementary Information 1.Supplementary Figure 1.Supplementary Video 1.


Nanoparticles of metal–organic frameworks (nanoMOFs) boast superior properties compared to their bulk analogs, yet little is known about how common synthetic parameters dictate particle sizes. Here, we provide experimental evidence for the “seesaw” model of nanoMOF growth. Solution acidity, ligand excess, and reactant concentrations are decoupled and shown to form the key independent determinants of nanoMOF sizes, thereby validating the proposal that nanoMOFs arise from coupled equilibria involving ligand deprotonation and metal–ligand complexation. By achieving the first demonstration of a seesaw relationship between nanoMOF sizes and ligand excess, these results provide further proof of the model, as they required deliberate manipulation of relationships outlined by the model. Exploring the relative impacts of these parameters reveals that ligand excess has the greatest ability to decrease sizes, although low acidity and high concentrations can exhibit similar effects. As a complement to existing models of polymer formation and crystal growth, the seesaw model therefore offers a powerful tool for reliable control over nanoMOF sizes. Nanoparticles of metal–organic frameworks (nanoMOFs) boast superior properties compared to their bulk analogs, yet little is known about how common synthetic parameters dictate particle sizes. Remarkably, MMMs that employ nanoMOFs have been shown to surpass the Robeson limit—an intrinsic trade-off between selectivity and permeability in separation membranes.4,5 While the gas separation performance of nanoMOFs has attracted industrial interest, their improved activities as atomically defined catalysts and drug delivery agents has opened emerging areas of research.6–8 Despite advances in nanoMOF applications, accurate models are still needed to probe fundamental mechanistic details and reliably control particle sizes.Precise size control can yield distinct functional behavior from materials with seemingly similar compositions. Achieving control at the nanoscale, in particular, has uncovered remarkable size-dependent properties, such as the luminescence of quantum dots and the distinct catalytic activities of metal nanoparticles.9–11 Whereas the LaMer model describes distinct stages of a burst nucleation induced by supersaturated monomer concentrations, followed by diffusion-limited particle growth,9 nanoMOFs form at dilute concentrations, and in situ studies reveal continuous nucleation and growth of MOF particles.12 Models based on monomer addition also do not apply to MOFs, as studies suggest MOF formation involves transiently metastable “primary” phases, aggregative growth, and other non-classical events.13–16 Although these existing models can be modified to account for non-classical events, nanoMOF research requires a general model based on the acid–base and coordination chemistry of MOFs to reliably predict and control particle sizes.Several models exist to describe nanocrystal nucleation and growth, including the classic LaMer model, the Watzky–Finke model, and various statistical models, yet recent evidence challenges their applicability to MOF growth.17 This model specifically explained why the use of modulators18—typically monotopic analogs of MOF linkers—causes particle sizes to increase in certain cases, but decrease in others. These trends could be explained by modulators functioning as capping ligands at low concentrations and as acids at high concentrations (2 and UiO-66 nanoparticles20 (2 (ZIF-8) and Cu3BTC2 (HKUST-1). With the results presented here, the seesaw trend appears in at least four compositionally distinct MOF systems, suggesting that the mechanistic model outlined here is universal to all modulated MOF syntheses.Previously, we proposed a novel “seesaw” model of nanoMOF growth based on a metadata analysis of existing literature.trations , as discticles20  could beθ using a copper K-α radiation source. SEM images were collected using a FEI Helios Dualbeam 600i. Samples were prepared by dispersing particle solutions onto Si substrates and drying under N2 flow. Average crystallite sizes were determined by Scherrer analysis, as detailed in the ESI.†All reagents and solvents were obtained from commercial sources and used as received. PXRD patterns were recorded by dispersing solutions on zero-background Si plates using a Bruker D2 Phaser, in the range 6 to 35° 21 using stock solutions of 2-methylimidazole (1.901 M) and zinc nitrate hexahydrate (0.2376 M). For experiments with variable equivalents of excess 2-methylimidazole, metal and linker starting solutions were diluted to equivalent volumes with final linker concentrations of 0.1901 M. For experiments with variable HCl equivalents, HCl (1.0 M) was added to the initial solution of 2-methylimidazole. The metal solution was poured quickly into the linker solution under stirring, then stirring was stopped immediately and the reactions were left undisturbed for 24 h. The resulting white solid was isolated by centrifugation and was washed twice with MeOH, then re-dispersed in a minimal amount of MeOH to store the products as colloids. Full details on reagent concentrations are provided in Tables S17–S19.†Typical syntheses followed a modified literature procedure,2O : EtOH. To achieve the desired stoichiometric ratios of linker and modulator, the appropriate volumes were transferred from each stock solution and combined in a 20 mL vial. The solution was diluted to 9 mL, then the metal ion solution was added. Final concentrations of each reagent for all Cu3BTC2 syntheses are provided in Tables S1–S16.2O : EtOH. Stirring was stopped immediately after complete addition of copper solution and the reaction was left undisturbed for 24 hours. The resulting blue precipitates were isolated by centrifugation and washed twice with 1 : 1 H2O : EtOH and re-dispersed in this same solvent for storage.All syntheses employed stock solutions of 1,3,5-benzenetricarboxylic acid (0.03 M), benzoic acid (0.21 M), and sodium benzoate (0.21 M) in 1 : 1 H21,22 Herein, we simplify the system of equations using assumptions similar to the well-known Initial-Change-Equilibrium (ICE) table method  knowing equilibrium constants for the metal ions and carboxylic acids, (2) knowing the initial reactant concentrations, and (3) by assuming that nanoparticles arise from reaction conditions that develop excess concentrations of deprotonated linkers [Leq and protonated linker species [LH]eq as a function of [H+] donated by the linker and modulator under typical reaction concentrations  as a function of added Hmim ranging from 4 to 14 equivalents per Zn2+, where conditions above 2 equivalents represent linker in excess. Based on analysis of the coupled equilibria, addition of only Hmim without modulator should cause the depletion of [M+], resulting in kinetic trapping of smaller nanoMOF sizes. Indeed, up to a reactant stoichiometry of 16–18 Hmim equivalents per metal, ZIF-8 particle sizes continue to decrease. Above this linker excess, product is simply not observed. These results resemble a previous report by Cravillon et al. that showed excess linker up to eight linker equivalents leads to progressively smaller ZIF-8 nanoparticles,12 but the data presented here show that trend progresses further. We note that particle sizes isolated here are slightly smaller than those reported by Cravillon et al.,14 which we attribute to the different characterization techniques: here, crystallite sizes are reported by Scherrer analysis whereas the previous account reported particle sizes from SEM imaging.With these predictions in hand, we sought experimental evidence for the seesaw model and the independent tunability of nanoMOF sizes through acid and ligand addition. versus number of HCl equivalents with respect to Zn2+ at a fixed linker excess of 14 equivalents. As a non-coordinating species, HCl acts only to increase proton activity. Therefore, HCl addition allowed us to test the hypothesis that nanoMOF sizes increase with higher proportions of protonated linkers incapable of trapping nanoparticles, as indicated in 2+ ion  because many modulated nanoMOF reports have focused on this material. These reports have shown that increasing equivalents of carboxylic acid modulators increases particle sizes, whereas Cu3BTC2 sizes decrease with increased amounts of deprotonated carboxylate, or other basic, modulators.26–29 Hypothesizing that these trends reflect the dual role of modulators acting as acids versus ligands, we investigated the impact of adding benzoate versus benzoic acid to the synthesis of Cu3BTC2. Although literature reports often use SEM or light scattering methods to determine sizes of MOF nanoparticles, sizes in this study are reported from Scherrer analysis because this method gives not particle size, but average size of coherently scattering domains. This metric is particularly useful for identifying the sizes of particles independent of aggregation. SEM images of Cu3BTC2 products in alkaline conditions 2 and trimesic acid at room temperature, whereas particle sizes strictly decreased by adding additional equivalents of sodium benzoate . 3BTC2 nanoparticle sizes versus equivalents of modulator mixtures with benzoic acid (BA) molar contents of 33%, 50%, or 66%, with the remainder comprised of sodium benzoate. Indeed, seesaw curves appear in all three cases with similar qualitative features: a decrease from 7 to ∼10 equivalents, a plateau, and a gradual increase in sizes from 28 to 40 equivalents. When using 1 equivalent of modulator, SEM revealed particle sizes that exceeded 1 μm , equals exp(−t/τ), where τ represents the average time between collisions.51 Similarly, we propose that the probability of MOF nanoparticles not being kinetically trapped by excess ligand decreases exponentially as more modulator enters the reaction mixture. In other words, the probability of particles colliding with capping ligand increases with higher available equivalents of excess ligand. This model helps explain why decreasing slopes in Lastly, we propose that the steep downward slope observed in “region I” of the seesaw curve can be interpreted in terms of classical collision theory. In collision theory, the probability of no collision taking place between particles as a function of time, Ka values and expect that altering the size and acidity of modulators to have a large impact on the particle seesaw dependence. Although the dependence of morphology on solution acidity was unexpected, buffer systems may prove powerful tools for deliberate control over tailoring particle architectures. Taken together, these results demonstrate that the seesaw model offers a fundamental platform for advancing the synthesis and basic understanding of this emerging class of materials.In conclusion, we offer experimental proof of the seesaw model of nanoMOF growth by demonstrating for the first time the existence of a seesaw relationship between nanoMOF sizes and modulator excess through deliberate manipulation of key parameters in the model. Specifically, we show that MOF nanoparticle sizes can be tuned through independent control over solution acidity, ligand excess, and reactant concentrations. Demonstrating that these three parameters control nanoMOF sizes supports the key claim of the model that nanoMOFs result from kinetic trapping of nanoparticles determined by competition between coupled equilibria involving metal–ligand complexation and ligand acid–base chemistry. The relative impact of these parameters on nanoparticle sizes was explored, with ligand excess showing the greatest overall impact. Sizes generally decease with lower acidity, greater ligand excess, and, for dynamic metal–linker bonds, higher concentrations. Importantly, particle sizes were reproducible when approaching similar reaction conditions from different directions on the multi-dimensional reaction space defined by these three parameters. The seesaw model represents a novel perspective for understanding MOF growth in general, and we further show that it complements well-established models of bulk polymer and crystal growth. Despite its clear relation to other models of crystal growth, the competing equilibria should be expanded to account for equilibrium constants associated with events such as oriented attachment, in which multiple metal–ligand bonds form at the same time. We further note that thus far the seesaw curve has been observed by using aromatic carboxylate modulators with similar pThere are no conflicts to declare.SC-011-D0SC04845C-s001


Chronic loss of Augmenter of Liver Regeneration (ALR) results in mitochondrial myopathy with cataracts; however, the mechanism for this disorder remains unclear. Here, we demonstrate that loss of ALR, a principal component of the MIA40/ALR protein import pathway, results in impaired cytosolic Fe/S cluster biogenesis in mammalian cells. Mechanistically, MIA40/ALR facilitates the mitochondrial import of ATP-binding cassette (ABC)-B8, an inner mitochondrial membrane protein required for cytoplasmic Fe/S cluster maturation, through physical interaction with ABCB8. Downregulation of ALR impairs mitochondrial ABCB8 import, reduces cytoplasmic Fe/S cluster maturation, and increases cellular iron through the iron regulatory protein-iron response element system. Our finding thus provides a mechanistic link between MIA40/ALR import machinery and cytosolic Fe/S cluster maturation through the mitochondrial import of ABCB8, and offers a potential explanation for the pathology seen in patients with ALR mutations. ALR have been described for this disorder. Some of these documented mutations result in premature stop codon, while the R194H mutation has been shown to affect the protein stability through increasing dissociation of its cofactor FAD  results in mitochondrial myopathy and cataract, and combined respiratory chain deficiency . Severalctor FAD . Cellulactor FAD . While pErv1-null yeast . Iron destined for Fe/S cluster synthesis is imported into mitochondria by mitoferrin 1/2 and is delivered to ISCU by frataxin. The fully assembled Fe/S clusters are then transferred to Fe/S cluster-containing proteins. Because several cytosolic proteins also require Fe/S clusters for their function, another set of cytosolic proteins, collectively named as the cytosolic iron/sulfur cluster assembly (CIA) machinery, facilitates the incorporation of Fe/S clusters into cytosolic proteins . Howevermyopathy . Conditill death . Previoull death . Additio. brucei .To support adequate Fe/S cluster biogenesis, iron first enters the cells through transferrin receptor-mediated uptake. It can subsequently be stored in the cytoplasm or incorporated into heme and Fe/S clusters in the mitochondria . While aIn yeast, Erv1p, together with Mia40, is part of the oxidative folding machinery required for the import of a subset of mitochondrial proteins. Mia40 oxidizes the cysteine residues within imported proteins, and the reduced Mia40 is re-oxidized by Erv1p, effectively recycling Mia40 for further use . A similIn this paper, we demonstrate that cytoplasmic Fe/S cluster maturation depends on a functional mitochondrial protein import system consisting of ALR and MIA40. Mitochondrial ALR facilitates the mitochondrial import of ABCB8, a protein needed for the maturation of cytosolic Fe/S proteins. Thus, a reduction in ALR and eventual reduction in the cytosolic Fe/S clusters leads to activation of IRP1 and subsequent cellular iron accumulation.Alr in mouse embryonic fibroblasts (MEFs) using pooled siRNA and demonstrated a significant reduction of Alr at the mRNA level  mRNA levels were increased in wild type MEFs with Alr downregulation  knockout (KO) MEFs with Alr siRNA. Alr knockdown in these cells resulted in a significant reduction in ALR protein levels  shRNA in order to examine the effect of Alr downregulation in the absence of IRP1, but we only achieved 40% downregulation of Aco1 at the mRNA level  and achieved a significant decrease in Aco1 transcript levels  using species-specific primer sets confirmed that only endogenous, but not exogenous, Alr was downregulated by siRNA -inducible expression system that allows for monitoring of protein accumulation after DOXY treatment. HEK293 cells stably expressing reverse tetracycline transactivator (rtTA3) were transfected with a tetracycline-responsive bicistronic construct containing GFP and a 6x-His-tagged gene of interest. This system results in mitochondrial protein accumulation in a time-dependent manner in response to DOXY treatment . To corrALR downregulation . Mouse embryonic fibroblasts were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. We have verified that Ireb2 was knockout in the Ireb2 KO MEFs. HEK293 cells were purchased from ATCC and cultured in Minimum Essential Medium  supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 1 mM sodium pyruvate. We have not performed separate verification since receiving the HEK293 cell line from ATCC. The cell lines were regularly using PCR-based mycoplasma detection kit (ABM) and were negative for mycoplasma contamination.Aco1 shRNA constructs were purchased from Openbiosystems and packaged into lentiviral vector as above. The culture supernatant was mixed with polybrene to a final concentration of 15 μg/ml and used to infect MEFs. To select for cells stably expressing Aco1 shRNA, MEFs were maintained in complete media with 6 µg/mL puromycin (Sigma) after infection. pLenti-rtTA3 (Addgene) was packaged into lentiviral vector in HEK293T cells after cotransfection with pSPAX and pMD2.G plasmids. Culture supernatant was used to infect HEK293 as described above, and cells with stable integration of viral genome were selected with 10 µg/mL Blasticidin (Invitrogen).Full-length and cytoplasmic isoforms of ALR were cloned into pHIV-eGFP vector. The resultant vector was packaged into lentiviral vector in HEK293T cells after cotransfection with pSPAX and pMD2.G plasmids. ALR, MIA40, TIM23, and mouse Alr and Aco1 were purchased from Dharmacon. siRNA against 3’UTR sequence of mouse Alr were purchased from Qiagen. siRNAs were transfected into HEK293 or MEF using Dharmafect 1 Transfection Reagent (Dharmacon). Plasmids were transfected into MEF using Lipofectamine 2000 reagent (Invitrogen) and into HEK293 cells using either Lipofectamine 2000 reagent (Invitrogen) or calcium phosphate transfection.Transfection siRNA against human RNA was isolated from cells using RNA-STAT60 (Tel-Test), and reverse transcribed with Taqman Reverse Transcription Reagents (Invitrogen) according to manufacturers’ instruction. Quantification of relative gene expression was done using Fast SYBR Green Master Mix (Applied Biosystems) and run on 7500 Fast Real-Time PCR system (Applied Biosystems). Primer sequences is included in the 55FeCl3 (Perkin Elmer) was first conjugated to nitrilotriacetic acid  before adding transferrin (Tf) in 2:1 molar ratio. The mix was incubated at room temperature for one hour. 55Fe-transferrin was separated from unbound 55Fe by running on PD-10 desalting column . For uptake experiment, cells were incubated with media containing 55 μg/ml 55Fe-Tf for two hours. Residual membrane-bound iron was removed by washing cells with cold 200 μM deferoxamine (DFO) in phosphate buffered saline (PBS). Radioactivity was quantified on a Beckman scintillation counter, and the results were normalized to cellular protein content of the same sample. For steady state cellular 55Fe content, cells were incubated with 280 μM of 55FeNTA for two days. The subcellular fractions were isolated and radioactivity in the fraction was measured as described above.Mitochondria from cells were purified using Mitochondrial Isolation Kit for Cells (Pierce) according to manufacturer’s instruction.Complex I and II activity were measured with Complex I and Complex II Enzyme Activity Microplate Assay Kit (MitoScience), respectively, according to manufacturer’s instruction. Xanthine oxidase activity was measured using AmplexRed Xanthine Oxidase Activity Kit (Invitrogen). For cytosolic aconitase assay, cytosolic fraction was concentrated on Amicon Ultra-0.5 Centrifugal Filter Unit with 10 kDa cutoff (Millipore) and the buffer was exchanged to PBS. The aconitase activity in the concentrated fraction was measured using Aconitase Activity Assay kit (MitoScience).60 hr after transfections, cells were trypsinized and equal number of cells were replated onto Seahorse XF96 cell culture plates in complete media. Cells were allowed to attach for 8 hr, and then washed with PBS twice to remove the complete media before incubating with assay medium . Oxygen consumption was measured using Seahorse Extracellular Flux Analyzer according to manufacturer’s instructions, and mitochondrial oxygen consumption was calculated as baseline oxygen consumption minus non-mitochondrial oxygen consumption (measured in the presence of 50 μM of rotenone and antimycin A).60 hr after siRNA transfection, cells were stained with propidium iodide (PI) and Hoechst 33342. Cell death rate was calculated as the number of PI-positive nuclei over total nuclei number.Twenty-four hours after transfection with Firefly reporter construct and Renilla luciferase construct , cells were solubilized in 1x passive lysis buffer (Promega). The lysates were loaded onto a 96 well plate, and the luciferase activity was determined using Dual Luciferase Assay System (Promega) with Modulus Microplate Reader (Turner Biosystems). The ratio between firefly and renilla luciferase activities was normalized to that of empty vector under corresponding treatments.ATP synthase F1 subunit alpha (ATP5a), cytochrome oxidase subunit 4 (COX4) and α-tubulin (Abcam), FLAG (Sigma), β- actin , GPAT (a generous gift from Dr. Roland Lill [University of Marburg]), or 70 kDa subunit SDH (Invitrogen) overnight, before the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch). The presence of target protein was visualized using Super Surgical Western Pico ECL substrate (Pierce). Quantification of western blotting image was done using ImageJ (NIH).Proteins were resolved on 4–12% Novex Bis-Tris poly-acrylamide gel (Invitrogen) and blotted onto nitrocellulose membrane (Invitrogen). Approximately 20–40 μg of whole cell lysate and approximately 6–10 μg of cytosolic or mitochondrial fractions were loaded onto the gel. Membranes were incubated with primary antibodies for ABCB8 (made in-house), ABCB7, MIA40, ALR, Tim 23, 6x-His, and GFP (ProteinTech), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 36 hr after transfection, cells were treated with 1 ug/ml doxycycline, and harvested after 6 hr for mitochondrial fractionation. For carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment, 2.5 uM CCCP was added at the fourth hour and the cells were incubated for additional 2 hr. The mitochondrial level of protein of interest is normalized to the GFP levels in the cytosolic fraction from the same sample.Mitochondrial pallets were lysed in PBS containing 1% Triton X100 (Sigma), protease inhibitor (G Biosciences) and 10 mM imidazole . The lysate was cleared via centrifugation and loaded to His Spin Trap spin column . The column was sequentially washed with PBC containing 20, 40, and 80 mM imidazole and eluted with PBS containing 200 mM imidazole. 100 mM DTT was included in the lysis buffer when indicated.Data are presented as mean ± standard error mean (SEM). Statistical significance was assessed with ANOVA, with post hoc Tukey’s test for multiple group comparison. All analysis were conducted using GraphPad Prism nine software. A P-value less than 0.05 was considered statistically significant. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This manuscript provides important mechanistic insights into how mutations in the Augmenter of Liver Regeneration (ALR) impacts iron handling and thus cellular iron content. This has potential implications for ALR in regenerative biology in other systems.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Carlos Isales as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Thomas S Weiss (Reviewer #3).Thank you for submitting your article "Augmenter of Liver Regeneration Regulates Cellular Iron by Modulating Mitochondrial Transport of ATP-Binding Cassette B8" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential Revisions:1. While the work is interesting the manuscript suffers from a lack of convincing data with regards to sorting out Alr effects that are driven by the cytoplasmic isoform, versus the mitochondrial isoform. The separation between the different isoforms is shown only from figure 3; therefore, the results shown in figure 1 and 2 may have been interpreted differently if the two isoforms were differentiated. For example, in figure 1 the authors show that downregulating Alr had no significant effect on mitochondrial complex I and II activities, however, since there was no differentiation between mitochondrial and cytoplasmic isoforms, the lack of effect on mitochondrial complexes may imply enrichment of the cytoplasmic isoform. Additional data would be helpful.2. The authors describe a stable MEF line expressing Aco1 shRNA that they created in order to test the effects of Aco1 downregulation, however, they achieved only 40% downregulation of Aco1 at the mRNA level. Therefore, they add siRNA for Aco1 and achieved greater decrease in Aco1 transcript levels. Although this technique proved to be more efficient, it is recommended that the authors discuss why the shRNA technique, which is usually very efficient, did not work in this case, as well as how combining shRNA and siRNA is applicable in studying ALR disease.3. Mitochondrial function studies are missing from the paper. The authors mentioned in the result section that their limitation in performing mitochondrial function experiments was due to a difficulty to attain large amounts of mitochondria from MEF cells, as compared to 293 cells, which were used for this purposes. To my knowledge, MEFs are pretty easy in obtaining mitochondria for mitochondrial studies; furthermore, the authors could use more cells or bigger plates to obtain a high amount of mitochondria. Therefore, to my opinion, this explanation for using 293 cells is not valid.4. In the material and method section as well in the figure caption the authors should present more details about how the experiments are performed enabling scientists to reproduce the data: e.g.How long were the cells treated with siRNA before harvesting and analysis? Does the cells after ALR siRNA treatment show signs of apoptosis or cell stress as reported for murine embryonic stem cells after 72h of ALR-siRNA treatment (PMID: 20147447)?How much protein is loaded on the gel?What are the primer sequences for PCR differentiating endogenous and exogenous ALR (3'-UTR)?5. Endogenous expression and localization of ALR isoforms in MEFs and HEK293 cells should be shown (western blot range from 10 to 30 kDa). Figure S3A: There are no visible bands in EV lane in cytosolic and mitochondria fractions, which would correspond to endogenous expression. Essential Revisions:1. While the work is interesting the manuscript suffers from a lack of convincing data with regards to sorting out Alr effects that are driven by the cytoplasmic isoform, versus the mitochondrial isoform. The separation between the different isoforms is shown only from figure 3; therefore, the results shown in figure 1 and 2 may have been interpreted differently if the two isoforms were differentiated. For example, in figure 1 the authors show that downregulating Alr had no significant effect on mitochondrial complex I and II activities, however, since there was no differentiation between mitochondrial and cytoplasmic isoforms, the lack of effect on mitochondrial complexes may imply enrichment of the cytoplasmic isoform. Additional data would be helpful.We have included a representative western blot demonstrating the localization of various isoforms of ALR in MEF . We utilized a pool of 4 siRNA to downregulate ALR in MEF cells. Three out of the four siRNA target regions shared by the long and the short isoform of ALR. Therefore, we would expect that the long isoform of ALR would be targeted equally  compared to the short isoform of ALR. Although we only showed the change in full length ALR in the main figure, we included an uncropped gel in Figure 1 figure supplement 1B that demonstrated downregulation of the short isoform of ALR after siRNA treatment. We have also revised the manuscript to better highlight the fact that siRNA targets both long and short isoforms of ALR. To further corroborate our model, we demonstrated the defect of mitochondrial ABCB8 import in cells with Mia40 downregulation. Given the similar phenotype between ALR and MIA40 downregulation and the fact that ALR and MIA40 together form one of the mitochondrial protein import systems, we deduce that mitochondrial ALR is critical for ABCB8 import into the mitochondria and therefore regulates cellular iron homeostasis.2. The authors describe a stable MEF line expressing Aco1 shRNA that they created in order to test the effects of Aco1 downregulation, however, they achieved only 40% downregulation of Aco1 at the mRNA level. Therefore, they add siRNA for Aco1 and achieved greater decrease in Aco1 transcript levels. Although this technique proved to be more efficient, it is recommended that the authors discuss why the shRNA technique, which is usually very efficient, did not work in this case, as well as how combining shRNA and siRNA is applicable in studying ALR disease.Aco1 expression, thereby creating an artificial condition to test whether ALR still has an effect on cellular iron homeostasis independent of Aco1. We have added discussion in the text.While shRNAs are generally efficient, knockdown efficiency of the commercially available constructs have not been previously validated. It is well-documented that shRNA efficiency can be influenced by multiple factors, including the level of expression, the secondary structure of the shRNA, the location of the targeted sequences, and the exact sequence and thermodynamic property of the shRNA . The purpose of combining shRNA and siRNA is to utilize a multiple-targeting strategy to suppress 3. Mitochondrial function studies are missing from the paper. The authors mentioned in the result section that their limitation in performing mitochondrial function experiments was due to a difficulty to attain large amounts of mitochondria from MEF cells, as compared to 293 cells, which were used for this purposes. To my knowledge, MEFs are pretty easy in obtaining mitochondria for mitochondrial studies; furthermore, the authors could use more cells or bigger plates to obtain a high amount of mitochondria. Therefore, to my opinion, this explanation for using 293 cells is not valid.We have performed additional mitochondrial functional studies in MEFs and did not observe any changes in baseline oxygen consumption with ALR downregulation. The results are now included in Figure 1 figure supplement 1D.4. In the material and method section as well in the figure caption the authors should present more details about how the experiments are performed enabling scientists to reproduce the data: e.g.How long were the cells treated with siRNA before harvesting and analysis? Does the cells after ALR siRNA treatment show signs of apoptosis or cell stress as reported for murine embryonic stem cells after 72h of ALR-siRNA treatment (PMID: 20147447)?How much protein is loaded on the gel?What are the primer sequences for PCR differentiating endogenous and exogenous ALR (3'-UTR)?Thank you for raising this issue. Cells were harvested 48 hours after siRNA treatment for RNA analysis and 60-72hr after siRNA treatment for protein analysis. About 20-40ug of whole cell lysate and around 10ug of mitochondrial fraction were loaded to the gel. Additional details are also now included in figure legends. We did not observe significant cell death with ALR downregulation, and the results are now shown in Figure 1 figure supplement 1C. This difference likely reflects the distinct roles of ALR in differentiated versus undifferentiated cell types. The exogenous ALR sequence is human origin, and we have designed primers targeting specifically for human versus mouse ALR mRNA. The primer sequences are included in Supplemental table 1.5. Endogenous expression and localization of ALR isoforms in MEFs and HEK293 cells should be shown (western blot range from 10 to 30 kDa). Figure S3A: There are no visible bands in EV lane in cytosolic and mitochondria fractions, which would correspond to endogenous expression.The antibody we used has limited detection of mouse ALR. The lack of signal in Figure S3A was limited by short exposure period and the relatively low protein amount (6ug) per lane loaded onto the gel. We confirmed the localization of ALR in a separate western blot experiment shown in Figure 1 figure supplement 1A.


The increased cancer incidence in patients with glomerular disease can be secondary to an intrinsic immune dysfunction associated with the disease or/and extrinsic factors, especially immunosuppressants. The treatment for paraneoplastic glomerulopathy is different from primary glomerular disease. Immunosuppressive therapy often used for primary glomerulopathy may aggravate concomitant cancers in patients with paraneoplastic glomerulopathy. In membranous nephropathy (MN), measurement of serum circulating autoantibodies against podocyte transmembrane glycoprotein M-type phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A), immunohistochemical staining of kidney tissue for glomerular PLA2R, THSD7A, neural epidermal growth factor-like 1 protein (NELL-1) and specific types of immunoglobulin G (IgG) may be useful adjuncts when screening for underlying malignancies. This review addresses overall cancer risks in individuals with glomerular diseases and employment of biomarkers available for MN. We propose a scheme of screening of cancers frequently reported in the setting of glomerular disease.  Glomerular disease is a leading cause of end stage kidney disease, and it can be idiopathic or result from inherited or acquired disorders, occurring in the setting of systemic autoimmune disease, infections, drugs, or malignancy Observational studies, case reports and case series have supported a link between glomerular disease and cancer. Some glomerular diseases require treatment with long-term immunosuppressants known to have oncogenic adverse effects and they may accelerate oncogenesis and neoplastic progression by direct mutagenesis or disruption in immune surveillance The pathogenesis of glomerular disease secondary to malignancy is poorly understood Intrinsic immune dysfunction associated with glomerular disease may increase the risk of cancer ,26. AnimThe risk of developing a de novo cancer is significantly associated with immunosuppressant use and cumulative exposure ,37. In pThe aim of this review is to determine the risk of paraneoplastic tumours and de novo cancers in patients with glomerular disease and assess recommendations in cancer surveillance. In addition, this review focus on membranous nephropathy which is used as a prototype in understanding the association of glomerular disease and cancer. Several advances have been made in the pathophysiology of membranous nephropathy and the employment of biomarkers may provide more precise differentiation between idiopathic and malignancy-associated nephropathy A population-based study which extracted data from the Danish Kidney Biopsy Registry with a study duration of 30 years (1985-2014) and follow-up period of 6.4 years (± 3.7 years) found that 16% (n=911) of 5,594 patients with glomerular diseases had cancer  ,36. The n=338) developed cancer during a median follow-up of 5.7 years. Approximately one third of cancers were diagnosed within one year of nephrotic syndrome onset. The association of cancer and glomerular disease peaked within a 6-month period following the diagnosis of nephrotic syndrome  and subsequently reduced at 6-12 months  and after a year . The association was most pronounced in younger patients . There was a significant association of nephrotic syndrome and haematological malignancies which included multiple myeloma , Hodgkin's lymphoma , non-Hodgkin's lymphoma . In addition, other solid cancers such as renal cancer  and lung cancer  were noted.Based on the Danish Kidney Biopsy Registry, there was an increase in cancer risk in nephrotic syndrome patients without a prior history of cancer A single-centre retrospective study with 822 patients with primary or secondary glomerulonephritis in Korea found that 5.5% (n=45) of patients developed de novo cancer ≥ 6 months after kidney biopsy during a mean follow-up period of 58.9 ± 44.5 months MN is the most common cause of nephrotic syndrome in Caucasian adults Current literature suggests that MN is closely associated with solid cancers and haematological malignancies ,36,41,42p = 0.003), had lower serum albumin levels  and higher serum C-reactive protein (CRP)  when compared to those without malignancy A meta-analysis  reported a 10% prevalence of cancer in MN p < 0.001)p < 0.001) was reported in a French retrospective study that involved 240 patients with membranous nephropathy There is limited evidence on the prognostic difference between individuals with MN with de novo cancer or those with paraneoplastic glomerulopathy. Regardless of the latter, concurrent MN and cancer is associated with poor kidney and patient survival. In a cohort study with record linkage between the Norwegian Kidney Biopsy Registry and Norwegian Cancer Registry, patients with cancer and MN had a greater mortality rate than patients with MN and without cancer over a mean follow-up period of 6.2 years  was detected in approximately 5-12% of patients with membranous nephrology who are PLA2R-negative ,73. GlomDual positivity of PLA2R and THSD7A was detected in 10% of patients with primary MN p=0.014)Glomerular IgG4 deposition was identified predominantly in primary MN whereas predominant depositions of IgG1/IgG2/IgG3 were often seen in secondary MN including malignancy-related MN. In patieA major technological leap involving laser microdissection of glomeruli followed by mass spectrometry has identified additional antigens which may serve as biomarkers and may identify underlying causes of MN Currently, there is no consensus among clinicians with regards to cancer screening in patients with glomerular disease. In a single-centre study from Italy, full screening {tumour biomarkers , stool for occult blood, chest x-ray, abdominal ultrasound, gastroscopy, total body computed tomography (CT), colonoscopy, mammography, cervical smears and urine cytology} for cancer was performed. In this study, 7.4% (n=12) of malignancies were diagnosed among 163 patients with biopsy proven glomerulonephritis and nephrotic syndrome during a median follow-up of 5.7 years  In general, it is a standard practice to screen for malignancy especially in older patients with newly diagnosed glomerular disease. The Danish Renal Biopsy Registry found the incidence of cancer was rare in patients < 45 years with glomerular diseases but increased significantly beyond 45 years of age Preliminary findings suggested that 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET) may be useful in detecting cancer in patients with MN The Kidney Disease Improving Global Outcomes (KDIGO) guidelines recommended cancer screening in patients with MN though the guidelines did not detail how and when it should be performed ,10. It iA high degree of clinical suspicion for underlying cancer should be maintained and appropriate cancer screening be considered in patients with glomerular disease. This is because delay in diagnosis and introduction of immunosuppressive therapy may be harmful and induce rapid progression of an occult malignancy. It is important that patients with glomerular disease are compliant with standard of care cancer screening programs recommended in the general population. Though recommendations on cancer screening in the general population may vary around the world, we support an approach that adapts consensus cancer screening recommendations from medical societies in America with screening of solid cancers in general population described in Unlike other glomerular diseases, the employment of novel biomarkers can be helpful in distinguishing idiopathic MN from malignancy-related MN. A combination of anti-PLA2R and anti-THSD7A in serum and glomerular PLA2R, THSD7A, and immunoglobulin staining will likely increase diagnostic sensitivity and specificity when screening for underlying malignancies in MN Neoplasia may not manifest itself at the time of kidney biopsy or soon before/after biopsy proven glomerular disease. It is challenging to discern glomerular disease associated with malignancy from de novo cancer. The increased risk of paraneoplastic tumour is mainly limited to a 6–12-month duration before and after kidney biopsy whereas the risk of de novo cancer increases over time following exposure to immunosuppressants. In patients with glomerular disease, the accurate differentiation between idiopathic and paraneoplastic glomerulopathy is important as these conditions have different therapeutic approaches. Along the disease trajectory of glomerular disease, treating clinicians should maintain a high degree of vigilance with regards to malignancy in the context of disease as early diagnosis is likely to influence treatment decisions which can translate to an improved prognosis. Cancer screening should include comprehensive clinical history and systematic physical examination, extrapolation of existing gold-standard cancer screening guidelines followed by targeted tests utilising glomerular immunoglobulin staining and novel biomarkers which may provide a more precise differentiation between idiopathic and malignancy-associated nephropathy. The development and refinement of reliable biomarkers will improve our insight into the pathophysiology of cancers in patients with glomerular disease and help the differentiation between primary and paraneoplastic glomerulopathy.Zaw Thet: Conceptualization, Writing – original draft. Alfred K. Lam: Conceptualization, Writing – review & editing. Dwarakanathan Ranganathan: Writing – original draft. Soe Yu Aung: Writing – original draft. Thin Han: Writing – original draft. Tien K. Khoo: Conceptualization, Writing – review & editing.None.


Currently, there is no clear consensus in cataract surgery if low compared to high energy femto-lasers may enable better capsulotomy quality and induce lower inflammatory response. Therefore, the aim of this study was to compare the intra-operative outcomes achieved with high and low pulse energy femtosecond laser systems for cataract surgery. The charts of 200 eyes of 200 patients aged 68.3 ± 10.3 years who had undergone femtosecond laser-assisted cataract surgery using either group 1 high pulse energy: LenSx (Alcon Laboratories) (N = 100) or group 2 low pulse energy: FEMTO LDV Z8 (Ziemer) (N = 100) laser were reviewed retrospectively. Integrity of anterior capsulotomy, classified as (1) complete (free-floating or with minor microadhesions), (2) incomplete and (3) with capsular tears, intraoperative completeness of the clear corneal incisions , incidences of intraoperative miosis and incidence of subconjunctival hemorrhage were evaluated and compared between the two groups. The proportion of complete capsulotomies was significantly higher in the group 2 than the group 1 . The incidences of intraoperative miosis (0% vs 19%) and subconjunctival hemorrhage (1% vs 63%) were significantly lower in the group 2 than the group 1 (p < 0.001). Completeness of the main incision was comparable  between the two groups. Although not statistically significant, the completeness of side-port incision was slightly better in the group 2 than the group 1 (91% vs 86%). Low energy laser system performed significantly better in terms of completeness of capsulotomy, intraoperative miosis and sub-conjunctival hemorrhage, compared with high energy laser; the CCI outcomes were comparable. Femtosecond laser-assisted cataract surgery (FLACS) has been advocated due to its ability to create a more precise and circular capsulotomy, greater predictability in achieving the desired size and centration of capsulotomy and in turn effective lens position (ELP) as compared to the manual procedure4. Despite the highly precise capsulotomy outcomes, the occurrence of capsular tags, capsule rupture, associated with FLACS created capsulotomy is not uncommon6. In addition, femtosecond laser pre-treatment has been reported to be associated with significant intra-operative pupil miosis (pupil diameter ≤ 6 mm) in up to 32% eyes9.One of the main challenges in phacoemulsification surgery is achieving a continuous curvilinear capsulorhexis10. Unlike most femtosecond laser systems, the FEMTO LDV Z8 femtosecond laser employs low pulse energy (nJ) and a high repetition rate in the MHz range. Previous studies have reported greater incidence of capsular irregularities and collateral damage using high pulse energies11. It has been hypothesized that low energy femtosecond lasers may enable smoother capsulotomy and induce minimal release of inflammatory mediators. Furthermore, femtosecond lasers employ different patient interfaces for the docking step. The use of curved contact interfaces has been found to increase IOP because of corneal compression during the applanation process and create corneal folds during laser cataract surgery leading to incomplete capsulotomy. In contrast, fluid-filled liquid interfaces have been found to prevent corneal folds, improve globe stability, reduce subconjunctival hemorrhage, and lower IOP rise13. Therefore, this study was aimed at comparing the intra-operative outcomes achieved with Ziemer Z8 (a low energy laser equipped with liquid patient interface) and LenSx (a high energy laser equipped with curved contact patient interface) for cataract surgery.Femtosecond lasers employ two different patterns of photodisruption: high-energy pulses (µJ) coupled with low-frequency (kHz) and low-energy pulses (nJ) coupled with high-frequency (MHz). With a high pulse energy, greater spacing between the spots is employed, whereas the low pulse energy laser allows greater number of overlapping smaller sized spotsThis single-center retrospective comparative analysis included 200 eyes of 200 cataract patients that had undergone FLACS between 2015 to 2018 at our facility. Surgeries before January 2017 were performed using LenSx  laser and surgeries after this date were performed using FEMTO LDV Z8  laser platform. Moving backward from this date, consecutive case records of 100 eligible patients were included in the LenSx laser group. For the Ziemer Z8 group, we moved forward from the date Ziemer Z8 surgeries were initiated at our facility and collected consecutive case records of 100 eligible patients that meet the recruitment criteria for the laser platform. One eye per patient was included in the study; if both eyes of a patient met the recruitment criteria, the case record of the first eye that was recruited into the study was analyzed.All surgeries were performed by an experienced surgeon (HYL) at Universal Eye Center, Zhong-Li, Taiwan. The study was conducted in accordance with the tenets of the Declaration of Helsinki for human research and was approved by the Institutional Review Board of Antai Tian-Sheng Memorial Hospital, Taiwan (18-024-B) with a waiver of consent because the data were collected as a part of normal practice care provision.Patients older than 18 years with cataract were eligible to be included in the study. Exclusion criteria included history of glaucoma, retinal detachment, corneal pathology, significant irregular astigmatism, abnormal iris, macular degeneration or retinopathy, lens/zonular instability, previous ocular surgery or trauma, poorly dilating pupil in the operative eye and patients with diabetes mellitus (DM) with uncontrolled fasting blood sugar level (> 200 mg/dl). Patients with complicated or hard cataracts (white or black), which in the experience of investigators could influence study outcomes were also excluded. To account for the initial learning curve, first 50 eyes were excluded for the respective lasers.15.The LenSx femtosecond laser platform uses a wavelength of 1030 nm, pulse duration of 600–800 femtoseconds, maximum pulse energy of 15 μJ and pulse repetition rate of 50 kHz. It employs three-dimensional spectral domain optical coherence tomography (OCT) and video microscope to perform image guided FLACS. This laser system uses curved lens applanating patient interface with vacuum docking17.The FEMTO LDV Z8 femtosecond laser system utilizes the concept of overlapping low-energy near-infrared (1030 nm) femtosecond laser pulses delivering energy in the nano-joule range (< 1 µJ). With a pulse duration of 250 femtoseconds, the laser runs in the MHz range applying up to 1 billion pulses per surgery. The laser system’s integrated three-dimensional high-resolution spectral-domain OCT imaging system enables visualization/identification of the precise location of ocular structures intraoperatively. It uses a non-applanating, fluid filled patient interface with vacuum dockingTo achieve optimal outcomes, default settings recommended by respective manufacturers were used for both the laser systems. Table 2  = 2.89, p = 0.09].All procedures were performed under topical anesthesia. Pupil dilation during the surgery was performed using a combination of phenylephrine and tropicamide eye drops  instilled into the eye 30, 25, 20,15, and 10 min prior to surgery. Ketorolac 0.5% ophthalmic solution  was instilled 4 times/day 3 days before surgery. Femtosecond laser-assisted pre-treatment included lens fragmentation and capsulotomy, clear corneal incision (CCI), and side incisions in both the groups using the femtosecond laser settings enlisted in Table For LenSx laser group, the femtosecond laser portion was performed in a different room adjacent to the operating room. In contrast, the Z8 laser platform allowed the whole surgical procedure  to be performed in the same operating room and under sterile conditions.The videos of all surgeries were retrospectively reviewed, and the following data was collected. Integrity of anterior capsulotomy was recorded as: (1) complete treatment pattern , (2) incomplete treatment pattern (femtosecond laser cut not visible in a certain section of the anterior capsulotomy) and (3) with capsular tears. CCI parameters included completeness of main incision and side ports, evaluated based on resistance experienced by the surgeon when entering the incision. If the incision was easily opened by blunt tip of the viscoelastic cannula , it was defined as ‘complete’ and if the resistance was such that the surgeon had to use a knife to open the incision, it was considered as ‘incomplete’. Additionally, the surgeon also recorded the presence of sub-conjunctival hemorrhage and intraoperative miosis . Cases where the capsulotomy was not visible anymore after the laser pre-treatment, were considered as cases manifesting miosis.20 and Ziemer Z8 laser (average 98.7%)22. Using these reported values for the two lasers, sample size calculations were performed with two-sided alpha of 0.05 and 80% power which revealed a total sample size of 190 (95 in each group). Accordingly, we chose a sample size of 100 in each group.Data analysis was performed using SPSS software for Windows version 17.0 . Categorical data were analyzed using Chi-Square test; however, Fisher’s exact test was used in cases where more than 20% of the cells had expected cell values of less than 5 or if the expected value in any cell of the contingency table was less than 1. In case of continuous variables, normality of the data was checked using Kolmogorov–Smirnov test and quantile–quantile plots. For normally distributed scale data, independent-sample t test was used to compare the means between the two groups. All P-values were two-sided and were considered statistically significant when less than 0.05. The sample size was calculated using literature reported incidence of free-floating capsulotomies achieved with LenSx laser (average 88.4%)A total of 200 eyes of 200 patients  with a mean age of 68.3 ± 10.3 years were included in the study. The mean age of the participants in the LenSx and Z8 group were 64.4 ± 9.9 and 61.7 ± 11 years respectively. The difference between the groups in terms of age was not significant (t(196) = 1.79; p = 0.07).The number of free-floating capsulotomies was significantly higher 100%) in Z8 group as compared to 94% in the LenSx group  = 18.843, p < 0.001, Fig. X2  = 85.501, p < 0.001, Fig. Compared to 19% eyes treated with LenSx laser system, none of the eyes experienced intra-operative miosis in the Z8 laser group  = 0.786, p = 0.375, Fig. Completeness of the main incision was observed to be comparable between the two groups . The outcomes for the completeness of side-port incision were slightly better in the Z8 group (91%) than in the LenSx group (86%), however the difference was not statistically significant  with a high frequency rate to achieve overlapping precisely placed laser spots which has been hypothesized to allow smooth incisions23, capsulotomy4 and minimal inflammatory response24.While numerous studies have provided substantial evidence for the safety and efficacy of FLACS, several complications specific to femtosecond lasers have been documentedThe current study was designed to evaluate if there are any differences in high and low pulse energy femtosecond lasers in terms of integrity of capsulotomy, completeness of CCI, incidence of sub-conjunctival hemorrhage and intraoperative miosis.3. However, several capsular irregularities may still occur with laser-assisted capsulotomies including, capsular microadhesions, incomplete and non-continuous treatment pattern, which may compromise the surgical benefits of femtosecond laser technology17. Variations in intrinsic laser settings, most importantly pulse energy, have been shown to affect the morphology and capsular irregularities associated with femtosecond laser-assisted capsulotomy cutting edges11. A comparison of capsulotomy outcomes with high (15 µJ) and lower pulse energy (7 µJ) revealed more capsular irregularities  with higher pulse energy settings using LenSx Laser System,  or Lensar Laser System, 25. In contrast, cutting edges of capsulotomies performed using lower pulse energy (5 µJ) were found to be smoother and rounder11. The curved contact patient interfaces can create corneal folds that have been associated with incomplete capsulotomies during laser cataract surgery13.Femtosecond laser enables consistently shaped and well-centered capsulotomies which helps maintain accurate IOL positioning within the capsular bag, thus improving postoperative refractive outcomes4. Pisciotta A. et al. reported highly continuous morphology of capsule edges which was attributed to the low energy/high frequency properties of the laser pulse, combined with an overlapped pulse pattern26. Furthermore, Schwarzenbacher et al. have demonstrated that especially low pulse energy lasers do not significantly increase the level of inflammatory cytokines (interleukin), which indicates low damage to the surrounding tissue24. Another article by Verdina T. et al. reported, that low energy capsulotomy group had higher circularity and stability of capsulotomy diameter compared with the standard bimanual microincision cataract surgery technique over the 18-month observation period and was also associated with lower PCO rates27. Also, a report evaluating learning curve with the FLACS found three capsule related complications with the low energy femtosecond laser during initial 60 cases, and none after overcoming the learning curve22.Williams G.P. et al. showed that the low energy FEMTO LDV Z8 created circular, rupture resistant and smooth capsulotomies in both porcine and human globesAs in line with literature, in this study the lower pulse energy settings and the high frequency of Z8 FLACS system and thus the overlapping precisely placed laser spots compared with LenSx showed significantly better capsulotomy outcomes. The same effect was also seen in the lower number capsular tears and incomplete treatment pattern.In the present study, we also investigated the effect of two laser systems on the completeness of main and side port incisions. The results indicated slightly higher percentage of eyes with complete CCIs in Z8 group , compared to LenSx group. It is important to note that the completeness and ease of opening of corneal incisions depend on their proximity to the limbus. While the limbal incisions are desirable with regard to the surgically induced astigmatism, they might be difficult to open as the cornea gets thicker and less transparent towards the periphery. The incisions created by FEMTO LDV Z8 are mostly placed closer to limbus as compared with the LenSx incisions, which are created rather corneal, due to different centration and docking mechanisms. Therefore, the comparable outcomes between two laser systems in terms of completeness of CCI and side-ports must be interpreted considering, that the distance to limbus was not taken into account when the incisions quality was evaluated.9. In an experimental study, liquid optical interface (LOI) performed better in terms of elimination of corneal folds, improved globe stability, reduced subconjunctival hemorrhage and lower IOP elevation, compared to the curved contact lens (CCL) interface system13. Consistent with these findings, the incidence of subconjunctival hemorrhage was found to be significantly higher with contact corneal applanation based LenSx group, compared to Z8 group that employs non-applanation fluid filled patient interface  in this study. Similarly, previous publications also reported significantly lower incidence of sub-conjunctival hemorrhage with liquid interface systems, compared to CCL interface system28. With the availability of advanced presbyopia correcting intraocular lens (IOLs) and changing practice patterns, there is a gradual shift in age of the patients undergoing cataract/refractive lens exchange surgeries. The younger and cosmetically more aware patients are likely to value the lower probability of post-FLACS subconjunctival hemorrhage with low energy Z8 laser, thereby increasing patient satisfaction in such patient population.Subconjunctival hemorrhage after femtosecond laser surgery has been associated with the docking process  and strong suction pressure applied at the patient interface29. Laser pulse energy is thought to play an important role in the degree of inflammatory response generated by the femtosecond laser33. It has been reported that decrease in applied laser pulse energy reduces the damage to surrounding tissues34. Supporting this theory, previous studies have reported a substantially high incidence of intraoperative miosis (as high as 32%) with the use of high energy femtosecond laser systems9. Consistent with the literature, the incidence of intraoperative miosis was found to be significantly higher in LenSx treated eyes, compared to Z8 group  in this study. Apparently, the low energy Z8 laser would induce minimal collateral damage to surrounding tissue, thus ensuing lower amounts of prostaglandin release, thereby reducing the risk of pupillary miosis during the surgery32, compared with high energy laser systems36. In agreement with these findings, a recent study reported no significant changes in pre- and post-laser treated pupil area with low energy Z8 FLACS system37. Conversely, Diakonis et al. revealed significant pupillary miosis with 3 different high energy femtosecond laser platforms38.Intraoperative pupil miosis is the most frequently reported complication during FLACS. It is hypothesized that pupil miosis is linked to the release of prostaglandin and other inflammatory mediators by the ciliary body in response to dissipation of laser energy, leading to increase in temperature, shock waves and vibrations24. Whereas, another publication reported markedly high levels of prostaglandins with the use of high energy laser systems 38.Although, we did not evaluate the levels of prostaglandins released following treatment with the two laser systems in this study, a meeting abstract presented at ARVO 2018 annual meeting reported only a slight increase in prostaglandins with a low energy femtosecond laser system 39. In addition, the entire procedure is carried under sterile conditions of the operating room. As such, housing the femtosecond laser and the operating table in the same room is most suitable40, and must become the only accepted option.Another likely reason for significantly high miotic effect observed with the LenSx laser group could be due to the extended time lapse between femtosecond laser pretreatment and the further surgical steps involved with this laser system. Conventional non-mobile laser systems are usually placed in a different room and after the pre-treatment the patient needs to be transferred to the sterile operating room where phacoemulsification is performed, thus necessitating a transition time for patient transfer and sterile draping. In some settings, the patient may have to move up and the operating room may be a hallway away from the laser room. Whereas, with mobile Z8 laser system (housed in the same room), phacoemulsification can be performed immediately following laser application, thereby causing no delay between femtosecond laser pre-treatment and phacoemulsification41. In this study, preoperative NSAID eyedrops regimen was uniform for the two laser groups, thus eliminating any potential bias due to differences in NSAID regimen responsible for significant difference in intraoperative miosis between the two laser groups. In both groups all eyes received NSAIDs drops combined with the mydriatics prior to surgery. Given the lower incidence of intraoperative miosis in the Z8 group, patients with documented NSAID allergy may benefit from the use of the low energy Z8 laser. The hypothesis is supported by the findings of Mirshahi et al., that did not detect statistically significant changes in the pupil area after laser pre-treatment with the low energy FEMTO LDV Z837.Pre-treatment with topical NSAIDs has been suggested to lower prostaglandin release, thus may have potential benefit of preventing pupil miosis23. The variation in the anterior capsule thickness of a few microns is unlikely to affect the completeness of the capsulotomy, although it cannot be ruled out completely42. Future studies with comparable capsulotomy diameters may help validate the findings of the present study. The two lasers place incisions at different distances to the limbus. Incisions created by LenSx are rather corneal, whereas the incisions created by FEMTO LDV Z8 are mostly placed closer to the limbus. The corneal incisions are expected to be easier to open, and incisions closer to the limbus are expected to be harder due to less transparent cornea in the periphery. Thus, the comparable outcomes between two laser systems in terms of completeness of CCI and side-ports must be interpreted considering that default laser settings were used and the distance to limbus was not taken into account when the incisions quality was evaluated. Another limitation of this study could be that the visualisation of the capsulotomy margin was chosen as criterion of absence of intraoperative miosis, instead of directly measuring the pupil area or diameter.Although patient demographics such as age and sex were comparable between the two laser groups; we did not compare preoperative ocular characteristics such as axial length, nuclear sclerosis grade, co-pathologies and visual outcome which may have influenced the study outcomes and therefore could be a potential limitation of this study. Also, the two lasers were operated using the default settings recommended by the manufacturers in an attempt to achieve optimal outcomes with the respective lasers Table . BesidesTo summarize, low energy Z8 laser system performed significantly better in terms of completeness of capsulotomy, intraoperative miosis and sub-conjunctival hemorrhage, compared with high energy LenSx laser under default laser settings recommended by the manufacturers; however, CCI outcomes were found to be comparable. The authors associate these findings to the low energy laser being gentler on the ocular tissue and causing low inflammation. Future technological innovations and prospective studies in different aspects of low energy femtosecond lasers are expected to further improve the safety and effectiveness of the procedure and allow faster visual rehabilitation and greater patient satisfaction.