Sargassum ilicifolium, provides shelter and food for numerous coral reef species, but it can also be detrimental at high abundances where it outcompetes other benthic organisms for light and space. Here, we investigate the microbial communities associated with S. ilicifolium in Singapore, where it is an abundant and important member of coral reef communities. We collected eight complete S. ilicifolium thalli from eight island locations along an approximate 14 km east-to-west transect. Each thallus was dissected into three separate parts: holdfast, vesicles, and leaves. We then characterized the bacterial communities associated with each part via polymerase chain reaction (PCR) amplification of the 16S rRNA gene V4 region. We then inferred predicted metagenome functions using METAGENassist. Despite the comparatively short distances between sample sites, we show significant differences in microbial community composition, with communities further differentiated by part sampled. Holdfast, vesicles and leaves all harbor distinct microbial communities. Functional predictions reveal some separation between holdfast and leaf communities, with higher representation of sulphur cycling taxa in the holdfast and higher representation of nitrogen cycling taxa in the leaves. This study provides valuable baseline data that can be used to monitor microbial change, and helps lay the foundation upon which we can begin to understand the complexities of reef-associated microbial communities and the roles they play in the functioning and diversity of marine ecosystems.The large canopy-forming macroalga,  Sargassum ilicifolium (Phaeophyceae: Sargassaceae), is a large and abundant canopy-forming brown seaweed found throughout the coral reefs of Singapore. It provides habitat and food for numerous reef-associated species [The macroalga,  species ,2, and a species . Microbi species ,6,7,8,9. species . Bacteri species ,12,13.Sargassum ilicifolium dominates many reef flats in Singapore where it comes into frequent contact with corals [h corals ,14. Thish corals ,16, and h corals ,18,19. Th corals , which ih corals ,22,23,24h corals ,26, and h corals ,28.Sargassum spp., with differences in photosynthetic rates [Sargassum ilicifolium .Research examining the macrophyte-associated microbiome shows that microbial communities associated with different tissues or plant structures contain different communities, likely a consequence of the different roles different parts play in promoting host survival ,30. Thesic rates , phlorotic rates  and C:N ic rates  being reic rates . SimilarSargassum ilicifolium in the waters of Singapore, the acknowledged influence the microbiome has on host survival, and the growing interest in how species will interact and change under the projected climate change scenarios, characterizing microbial communities in marine environments is important. It provides a baseline on which we can monitor change and, importantly, it provides a framework on which we can build to understand the complex interactions that constitute the marine microbiome as a whole.There is rapidly increasing interest in the marine microbiome, particularly in how coral reefs and other coastal organisms such as mangroves and seagrasses interact with one another, and more broadly regarding how the entire marine microbiome can be influenced by the species present along with terrestrial and atmospheric inputs and interactions . ConsideSargassum ilicifolium thalli were collected over two days in January 2018 from each of the eight islands  at 8 m/s for 2 min, and DNA was extracted with a Qiagen DNeasy Powersoil kit following the manufacturer’s instructions. PCR amplification targeting the V4 region of the 16S rRNA gene was performed using the bacterial and archaeal primers 515F and 806R . Forward and reverse primers were modified to include Illumina adaptors, a linker and a unique barcode . Each rePCR products were visualized on a 1% TBE buffer agarose gel. The normalization and cleaning of PCR products were performed in SequalPrep normalization plates  and submitted for sequencing on the Illumina MiSeq platform  with a 30% PhiX spike (Macrogen Korea).https://benjjneb.github.io/dada2/ for a detailed explanation of filtering parameters, accessed on 22 April 2021).Sequences were demultiplexed, and barcodes and adaptors removed with Cutadapt . Reads wThe DADA2 algorithm was used to estimate error rates from all quality-filtered reads and to merge forward and reverse reads and infer amplicon sequence variants (ASVs). Chimeras were removed with de novo detection. Sequenced extraction negatives were used to identify and remove any possible contaminants using the prevalence method implemented in the decontam R package , and remAny ASVs assigned to mitochondrial or chloroplast genomes and those not present in at least 5% of samples were removed. Rarefaction curves were produced using the rarecurve function implemented in the vegan R package version 2.5.6 . SequencThe filtered ASV table was used to predict metagenome functions using METAGENassist . ASVs wiAll sequences associated with this work have been deposited at the National Center for Biotechnology Information under BioProject ID: PRJNA681720.R2 = 0.30, p < 0.001) and between island locations   . There wp > 0.05), while the holdfast is significantly different from the leaves and vesicles (p < 0.01) and contains the lowest diversity. Diversity at all islands is similar and not significantly different between any pairwise comparisons (p > 0.05) , with holdfasts appearing to have the most distinct community composition. These findings are in agreement with other work from the region that shows microbial community divergence at small spatial scales in a diverse range of marine taxa such as seagrasses [In this study, we use high-throughput amplicon sequencing to characterize the microbial communities associated with the commonly encountered reef macroalga, agrasses , mangrovagrasses  and coraagrasses .There are no obvious barriers to dispersal between the islands in this study, and the prevailing water currents run from east to west with semi-diurnal tidal oscillations . These oS. ilicifolium from different locations are not a consequence of seasonality, as all collections were made over a two-day period in January 2018. Rather, we suggest, much like P. acuta [The differences seen in the microbial communities associated with P. acuta , the sigP. acuta . It is aLocalized rainfall-created runoff does not affect the submerged reefs in this study; while salinity will be reduced in the short term in the waters over these reefs, it will quickly return to the same salinity as the surrounding water with tidal mixing. Conversely, precipitation that falls on islands requires time to reach the reef; while this means that drops in salinity are likely more gradual and less dramatic, they probably occur over a longer duration and take on the chemical characteristics of the substrate they travel over or through  ,66. Our S. ilicifolium structures have also been shown to harbor distinct fungal communities in Singapore [Our analysis shows a differentiation in the microbial communities associated with the structures sampled, with the holdfast harboring the most distinct community. This is in line with studies performed on other marine organisms from the region, which have shown microbial community divergence over small spatial scales in seagrasses, mangroves and corals ,33,34; tingapore . Like otingapore .Sargassum horneri collected in the Yellow Sea indicating that bacteria likely play important roles in nutrient cycling in Sargassum species [Sargassum communities, particularly under nutrient limitation [Sargassum-associated microbial communities in nutrient cycling on coral reefs.Metagenome functional predictions suggest that leaf-associated communities contain more taxa with the potential to perform nitrogen cycle functions, while the holdfast-associated communities contain more taxa with potential sulphur cycling roles, and these predictions are consistent with results from  species . Nitrogemitation ,71, and mitation . Sulphurmitation ,74, and Sargassum ilicifolium, in the highly urbanized marine environment of Singapore. Characterizing the microbes present in marine environments, living within hosts, in the reef matrix, or water column, is an important first step in understanding how the marine microbiome is structured and could respond to change, and this is especially crucial for some of the most commonly encountered species such as S. ilicifolium. Work such as this provides foundational information that can be further advanced with the development and testing of explicit hypotheses that facilitate a greater understanding of how species and their associated microorganisms interact and evolve as environments change.Our study characterizes the microbial communities associated with the reef macroalga,


Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease and similar neurodegenerative disorders take their toll on patients, caregivers and society. A common denominator for these disorders is the accumulation of aggregated proteins in nerve cells, yet the triggers for these aggregation processes are currently unknown. In ALS, protein aggregation has been described for the SOD1, C9orf72, FUS and TDP-43 proteins. The latter is a nuclear protein normally binding to both DNA and RNA, contributing to gene expression and mRNA life cycle regulation. TDP-43 seems to have a specific role in ALS pathogenesis, and ubiquitinated and hyperphosphorylated cytoplasmic inclusions of aggregated TDP-43 are present in nerve cells in almost all sporadic ALS cases. ALS pathology appears to include metal imbalances, and environmental metal exposure is a known risk factor in ALS. However, studies on metal-to-TDP-43 interactions are scarce, even though this protein seems to have the capacity to bind to metals. This review discusses the possible role of metals in TDP-43 aggregation, with respect to ALS pathology. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by a specific and progressive demise of upper and lower motor neurons in the motor cortex, brainstem and spinal cord ,2,3. DeaC9orf72 hexanucleotide repeat extension, develop frontotemporal lobar degeneration (FTLD) and progressive cognitive decline with behavioural changes subsequently leading to clinical frontotemporal dementia (FTD) 9+ with an approximate mass of 20,098 Da [Zn is so far also the only metal for which the binding to TDP-43 has been characterised. In a study by Garnier et al. , the RRM0,098 Da . In furt0,098 Da . Simulta0,098 Da . The oli0,098 Da ; further0,098 Da . Later, 0,098 Da .bis(thiosemicarbazonato complexes inhibited TDP-43 aggregation, likely via the inhibition of phosphorylation, suggesting a neuroprotective function of the compound.Fe and Cu have important roles in intracellular redox reactions ,131,132.In a recent case report study, Mitani et al.  describeElevated Mn concentrations have been observed in CSF from ALS patients  as well Aggregation of TDP-43 has been observed in the majority of all studied ALS cases  9,31,32,3231,32.Zn is the only metal where binding to TDP-43 has been characterised at the molecular level . RRM1/2 Full-length TDP-43 aggregates are predominantly detected in the spinal cord , and somIn this review, six metals have been discussed in relation to TDP-43, yet several other metals may correlate with ALS and/or other TDP-43 proteinopathies, such as Cd and Al . ApplyinThe roles of metal imbalances and metal exposure in neurodegenerative disorders remain to be clarified. Many of these disorders have been proposed to be metallopathies , as metaMetal imbalance and/or environmental exposure to neurotoxic metals may contribute to the harmful aggregation of the protein TDP-43 as seen in amyotrophic lateral sclerosis.


Graphene is a two-dimensional material, with exceptional mechanical, electrical, and thermal properties. Graphene-based materials are, therefore, excellent candidates for use in nanocomposites. We investigated reduced graphene oxide (rGO), which is produced easily by oxidizing and exfoliating graphite in calcium silicate hydrate (CSHs) composites, for use in cementitious materials. The density functional theory was used to study the binding of moieties, on the rGO surface , to CSH units, such as silicate tetrahedra, calcium ions, and OH groups. The simulations indicate complex interactions between OH/rGO and silicate tetrahedra, involving condensation reactions and selective repairing of the rGO lattice to reform pristine graphene. The condensation reactions even occurred in the presence of calcium ions and hydroxyl groups. In contrast, rGO/CSH interactions remained close to the initial structural models of the epoxy rGO surface. The simulations indicate that specific CSHs, containing rGO with different interfacial topologies, can be manufactured using coatings of either epoxide or hydroxyl groups. The results fill a knowledge gap, by establishing a connection between the chemical compositions of CSH units and rGO, and confirm that a wet chemical method can be used to produce pristine graphene by removing hydroxyl defects from rGO. The che devices ,22. TherThe most widely used technique for preparing graphite oxide by oxidizing graphite is known as Hummer’s method ,23,24,253 treatments with active solvated electrons as a strong reducing agent [1s X-ray photoelectron spectroscopy spectra. By considering Fourier transform infrared spectra, they found a marked decrease in the number of hydroxyl and epoxide groups. Pei et al. [2H4·H2O solution, and a 50 mM NaBH4 aqueous solution at room temperature. They found that after reducing the GO films with HI, most of the oxygen-containing groups from the GO film had been removed, causing the C–C bonds to become dominant, giving a C/O atom ratio ≥ 12 and an electrical conductivity of up to 298 S/cm. Much lower C/O atom ratios and electrical conductivities were found for higher GO films that were reduced using N2H4·H2O and NaBH4.Many recent studies have focused on manufacturing very pure rGO sheets  from GO . Feng etng agent . Liao etng agent  producedi et al.  fabricat9 t and it is currently increasing by 5% per year, and contributes 5–10% to global anthropogenic carbon dioxide emissions [Concrete- and cement-based materials are only second to water, in terms of their use around the world ,34,35,36missions ,38. The missions ,40. Thismissions ,41. CSH missions ,42,43, imissions ,45, a glmissions ,47,48, amissions ,50,51. Tmissions ,39,52, wmissions ,54,55,563H) groups, with a 10% ultra-confined coverage with the calcium silicate hydrate gel (CSH), as shown in The current main challenge in the field area lies in improving our understanding of the mechanisms involved in the interactions between the chemical components of cementitious CSH gel moieties  and prisTo our best knowledge, the mechanism involved in the interactions between rGO and monomers in CSH gels during the fabrication of cementitious composite materials c have noThe interactions between rGO and CSHs are studied by performing DFT electronic structure calculations . The Vie4 monomers, because these moieties occur in solution when the calcium silicates in cementitious clinkers are hydrated. SiO(OH)3− units that were produced in small amounts during the hydration process, causing the pH of cement, were also studied. The contributions of van der Waals interactions were not considered in all the configurations, because the differences in van der Waals potential energies were found to be small when calculated in some tests. The ΔE was calculated from the difference between the energies of the relaxed configuration and the ground-state structure. Further, rGO structures with oxygen-to-carbon reduction ratios (O:C) of 1:50 were used in the simulation models.  is high . Thus, sads) relates to interactions between the sorbent and substrate, and was calculated using Equation (1), as follows:Total is the total energy of the composite systems, ESub is the energy of the graphene or rGO plane, and EAb is the energy of the absorbed moiety [The adsorption energy 4 in two different positions relative to the hydroxyl/rGO sheets, as input geometric structures, were therefore first prepared. Two dissimilar Si(OH)4 unit configurations, with respect to the distance to the hydroxyl/rGO sheet, were found. There was a stable configuration in which the starting geometry did not change much after optimization, as shown in 3 unit, as shown in 4 deposited slightly further from the hydroxyl/rGO sheet than in the expected configuration.Feasible configurations of hydroxyl- and epoxide-reduced graphene with CSH gel moieties were calculated to allow the investigation of the mechanisms involved in the interactions between the different moieties. We were interested in investigating the mechanism involved in the interaction between Si(OH)3 and Si(OH)4 configurations were −1.683 eV and −0.094 eV , respectively. Therefore, the SiO(OH)3 unit in the ground-state structure had a lower adsorption energy than the other configurations, indicating that the system containing SiO(OH)3 with a water molecule next to a graphene plane was the most favorable configuration. Importantly, a condensation reaction occurs when a hydroxyl group at the GO interface becomes dissociated and combines with a hydrogen atom released by Si(OH)4.The calculated adsorption energies for the SiO(OH)3 unit on graphene were considered, to determine the optimum location of the water molecule. The geometric structure consisting of a system containing SiO(OH)3, a water molecule, and graphene, shown in 3, and, most importantly, it is near the graphene surface, implying an interaction with the graphene surface and an increase in the bonding energy.Then, several configurations of water molecules in a system with a SiO(OH)3 unit on hydroxyl/rGO, in a singly negatively charged system, was also considered. Perhaps surprisingly, we found that in the ground-state structure, hydrogen is dissociated from the hydroxyl/rGO surface and is being transferred to the SiO(OH)3 unit to saturate the dangling oxygen atom, as shown in 3 on hydroxyl/rGO in a neutral system was placed as another initial simulation model, and we also found that the hydrogen becomes dissociated from the hydroxyl/rGO surface and is transferred to the SiO(OH)3 unit. This indicates that a hydrogen atom becomes dissociated from the hydroxyl/rGO surface regardless of whether the system is charged or neutral. A dangling oxygen bond therefore points towards the graphene plane and is almost fully occupied through charge transfer from the graphene sheet.A SiO(OH)3 unit on the graphene surface. The unit on the graphene plane was placed at several distances in four different configurations, with the system being either neutral or singly negatively charged. All four configurations were optimized, and the ground-state structure was found for the configuration associated with the unit that was physisorbed to the graphene sheet. The configurations for the neutral cases are shown in 3 physisorbed to the graphene sheet, which has a lower energy state (−0.23 eV) than the chemisorbed configuration. When the system is singly negatively charged, both relaxed structures have the SiO(OH)3 unit physisorbed to the graphene sheet.Next, we considered the adsorption properties of a SiO(OH)3, for the physisorbed ground-state structures and the next chemisorbed configuration for the neutral system, are −1.59 and −1.36 eV, respectively, as shown in 3 to form SiO (OH)3− for the total charged system, so the initial energy state must change by the difference between the neutral and charged silicate units. We expect that the adsorption of SiO(OH)3 to the doped graphene sheet in the neutral and singly negatively charged state is almost the same, because the doping electron must be shared between a large number of carbon atoms. The corrected adsorption energy for the SiO (OH)3− unit, typical in a basic solution, was found to be exothermic by −1.4 eV, because charge fills the oxygen levels. It seems that the adsorption of silicate hydrate units onto rGO would remain favorable at basic pH values and with negatively charged units.The calculated adsorption energies for SiO(OH)4 and hydroxyl/rGO sheets ending in SiO(OH)3 were performed. Previously studied structures, including Si(OH)4 and SiO(OH)3 units with a hydroxyl/rGO substrate, were used, but in the presence of Ca ions. Two different Si(OH)4 and hydroxyl/rGO configurations are used with a Ca ion at different positions, one with the Ca ion close to Si(OH)4 and the other with the Ca ion far from Si(OH)4, as the initial geometric structures. The structures were optimized, and the ground-state energy was found to be 0.495 eV lower for the configuration with a Ca ion close to Si(OH)4 3. Therefore, in a system containing a Ca ion, the Ca ion interacts directly with SiO(OH)3. Some of the charge on the Ca ion is transferred to the 2p orbital of an oxygen atom in SiO(OH)3, to occupy the orbital fully, and the remaining charge is transferred to the graphene sheet. The structure of Si(OH)4 on a hydroxyl/rGO substrate was also optimized in the presence of a Ca ion, in a doubly positively charged system. The hydroxyl group dissociates from the hydroxyl/rGO substrate and a water molecule is formed, and the Ca ion also interacts with SiO(OH)3. The distance between the Ca ion and the oxygen atom in the ground-state structure is ~2.075 Å, as shown in Comprehensive investigations of systems containing Si(OH) Si(OH)4 a than fo3 in the four different configurations, with a water molecule on the graphene surface, vary between −1.43 and −1.69 eV; the latter is given as the ground state indicated in 3 model are slightly higher than the adsorption energies of −1.36 to −1.59 eV, when no water molecule is present on the graphene surface  surface . The enea Ca ion a,b. In o3 on the hydroxyl/rGO sheet in the initial simulation model, the hydroxyl group is found to not dissociate from the hydroxyl/rGO sheet. A lower energy, −1.93 eV, is found for the ground-state structure than in other configurations. However, the ground-state structure in the initial simulation model relates to the configuration with the Ca ion near SiO(OH)3. Optimization for the ground-state structure indicates that the hydroxyl group becomes dissociated from the hydroxyl/rGO, and the Ca ion is involved in bonding, so charge transfer from the Ca ion to two neighboring oxygen atoms occurs, to cause a nearly full occupation of the oxygen orbitals (When a Ca ion is far from SiO(OH)orbitals b.4 and SiO(OH)3, and the hydroxyl/rGO sheet, in the presence of a hydroxyl group and a Ca ion. The results indicate that the ground-state structure has the same configuration as the structure with a Ca ion close to the silicate monomer, which causes the hydroxyl group to dissociate from the hydroxyl/rGO sheet and the charge to be compensated by charge transfer from the Ca ion to the two neighboring oxygen atoms on two hydroxyl groups, as shown in 4 and SiO(OH)3 have energies that are almost 3 and 0.4 eV lower, respectively, than the next lowest energy configurations. In contrast, for the configuration with a Ca ion that is far from the silicate, the hydroxyl group is not dissociated from the sheet. The adsorption energy for SiO(OH)3 in the ground-state structure, in the presence of a hydroxyl group and a Ca ion on the graphene plane, is −2.63 eV, which indicates that stronger adsorption occurred than for the removal of only hydroxyl, as shown in 4 remain strongly bonded to the Si atom.We also studied the interactions between Si(OH)4 is on the epoxide/rGO surface, and the ground-state structure remains similar to the initial structure, with Eads~0.127 eV . The mechanism involved in the interaction between SiO(OH)3 and the surface of the epoxide/rGO sheet was investigated, and the results are shown in 3 on the epoxide/rGO surface of −0.853 eV, which is lower than the adsorption energy for a neutral Si(OH)4 silicate hydrate moiety. The SiO(OH)3 silicate unit moves away from the epoxide groups, as shown by the physisorption model described above. The nearest distance between the dangling oxygen atom and the epoxide/rGO sheet is 2.931 Å. Even longer distances were found in several previous studies. For example, Gao et al. [2S and CH4 on intrinsic graphene, which were −0.038 and −0.022 eV, respectively, and distances of 3.813 and 3.865 Å, which are larger than the distances included in 3 and the epoxide/rGO sheet was considered to be neutral in the presence of a Ca ion and two hydroxyl groups, as shown in 3, in the presence of a Ca ion and hydroxyl functional groups of hydroxyl, is therefore −1.52 eV lower 3 on the graphene plane, for the neutral and singly negatively charged systems, were assessed. The Bader charge analysis method indicates that the charge transfers between the graphene and SiO(OH)3 were similar to the charge transfers in the ground state on the rGO, as shown in The electronic properties of the ground-state structures given by the physisorption model of SiO(OH)3 on the graphene plane in the neutral system, with respect to the graphene sheet (200 electrons) and the SiO(OH)3 unit (31 electrons), are shown in 3 unit. The adsorption energy for SiO(OH)3 on graphene was calculated with respect to the negative unit, and was in the range of a few electron volts. This is typical for Coulomb interactions over a few angstroms distance, caused by charge transfer. Water molecules near the graphene increase the adsorption energy because they form hydrogen bonds with SiO(OH)3, and because the oxygen atom in a water molecule becomes more stable by interacting with the depleted positive charge in the graphene layer.We constructed the 3D charge density difference plots to determine the spatial distribution of the charge. The charge density differences for the ground-state structure of SiO(OH)We assessed the changes in the hydroxyl/rGO electronic properties, caused by adding CSH. The structures with the most favorable adsorption energies were analyzed. The DOS and Bader charge distributions are shown in 3 on the graphene plane in the neutral system are shown in 3 unit resembles the bond in the basic bonding model described in 3 occurs in the neutral system, to give an almost fully occupied oxygen 2p orbital.The charge density distribution of the neutral systems, determined by Bader charge analysis, and the charge density differences for the molecular orbital isosurfaces, are shown in A DFT method was used to study the mechanism involved in the interactions between hydroxyl or epoxide rGO and the CSH moieties, such as CSH gel in cement. The DFT calculations for silicate tetrahedra, Ca ions, and hydroxyl groups improve our understanding of the bonds between rGO and primary CSH moieties. The results led to the following conclusions. The interactions between hydroxyl/rGO and silicate tetrahedra can repair hydroxyl defects selectively in the rGO lattice, and cause graphene to re-form. The dissociation of defects in the graphene plane, and the formation of water, even occurs in the presence of Ca ions and hydroxyl groups. In fact, the main interactions between the graphene plane and CSH gel are Coulomb interactions, caused by charge transfer. In contrast, the ground-state structure remains similar to the initial structure model for interactions between epoxide/rGO and CSH gel. Consideration of the strong interactions in this way could allow improvements to be made in the design of composite materials.


TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative  Increasing genetic evidence, neuropathological and clinical observations have identified a substantial overlap between these two disorders2. Several genes have been identified where mutations can cause either FTD or ALS (FTD-ALS genes) including C9orf72, VCP, OPTN, SQSTM1, TBK13 and, most recently, CYLD4. In addition, FTD and ALS share neuropathological similarities: ~ 95% of ALS and ~ 50% of FTD patients show cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in the brain6, while ~ 5% of ALS and ~ 10% of FTD patients show cytoplasmic inclusions of fused in sarcoma protein (FUS)8. In healthy cells, TDP-43 and FUS are largely expressed within the nucleus. In FTD and ALS, affected neurons can display a redistribution of TDP-43 or FUS to the cytoplasm, as well as insoluble TDP-43 or FUS aggregates10. The importance of TDP-43 and FUS is further demonstrated by the fact that mutation of their encoding genes TARDBP and FUS is sufficient to cause ALS12 or, rarely, FTD13.Frontotemporal dementia (FTD) is one of the most common forms of presenile dementia and involves the progressive degeneration of the frontal and temporal lobes of the brain. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects the upper and lower motor neurons leading to muscle weakness and paralysis15; however, for the remainder of patients the origin of the disease is uncertain. Despite TDP-43 being a prominent feature in the majority of FTD and ALS cases, including all those carrying FTD-ALS gene mutations13, this fact has not been applied to the screening of FTD and ALS candidate gene variants on a large scale. In previous studies, TDP-43 and FUS mislocalisation has largely been assessed by subcellular fractionation, immunohistochemistry and manual analysis of confocal microscopy24 or high-content imaging27. Whilst studies utilising these techniques have been informative, these assays are labour intensive and low-throughput or, expensive and in some cases produce qualitative data. In addition, some microscopy-based techniques could be subject to bias due to manual selection of the cells to be analysed.Identified genetic mutations cause 20% of FTD and 5-10% of ALS cases28. We validated this technique using known genetic drivers of TDP-43 or FUS mislocalisation. We were able to detect increased cytoplasmic localisation of exogenously expressed FUS and TARDBP mutations relative to wild-type (WT) sequence. We also observed mislocalisation of endogenous FUS upon treatment with the apoptosis inducer staurosporine. Importantly, we could also detect more subtle changes in the cellular distribution of endogenous TDP-43, arising from mutation in a secondary gene (CYLD p.M719V). This validated methodology can now be utilised for rapidly prioritising newly discovered FTD and ALS gene variants by quantifying their effect on TDP-43 and/or FUS localisation.Here we report the development of a rapid, cheap and unbiased technique for the in vitro study of TDP-43 and FUS cytoplasmic mislocalisation. TDP-43 or FUS staining in nuclear and cytoplasmic subcellular compartments was measured in thousands of cells from confocal microscope images, using the freely available analysis software CellProfilerFUS mutations that drastically alter FUS subcellular localisation29. Expression of green fluorescent protein (GFP)-tagged FUS protein with missense mutation FUSR521C or truncation mutant FUSR495X in the neuronal-like neuroblastoma cell line, SH-SY5Y, led to a significant increase in the fluorescence intensity of cytoplasmic FUS  and FUSR495X , respectively, relative to FUSWT  and FUSR495X  mutants in human embryonic kidney (HEK293) cells , ImageJ36 and Huygens (RRID:SCR_014237). We chose CellProfiler based on its ease of use, the ability to perform batch analysis of images, cost and accessibility.Previous studies examining TDP-43 and FUS have quantified cytoplasmic mislocalisation in different ways. Many studies have reported the proportion of the cell population that display cytoplasmic TDP-43 or FUS expressionAnother general observation in our experiments was that SH-SY5Y cells often displayed a greater degree of cytoplasmic mislocalisation than that in HEK293 cells under the same conditions. This may be due to SH-SY5Y cells being a neuronal cell line and thus more disease relevant, or due to the longer transfection time required to achieve optimal transfection efficiency in SH-SY5Y cells , causing additional stress on the cells. It should also be noted that transfection efficiency also dictated the number of images required to reach the desired cell number per replicate (300 cells). SH-SY5Y cells, which had a lower transfection efficiency, required more images to achieve the same number of cells for quantification.FUS mutations has been widely established12. This pathology has been recapitulated by expression of FUS mutations in mammalian cells29. In this study, exogenous expression of the severe truncation mutation FUSR495X caused the majority of FUS to be mislocalised to the cytoplasm as previously demonstrated by Bosco et al.29 using live-cell confocal microscopy and 3D images (11–28 cells). Our quick and simple methodology was able to duplicate these results using 2D images and a larger number of cells . While we recognise the merits of quantifying the entire cell volume, measuring such a small number of selected cells is not representative of the population and may not be able to detect more subtle or variable effects in protein localisation.The cytoplasmic mislocalisation and/or aggregation of FUS in neurons and glia of post-mortem tissue from patients with ALS caused by R521C missense mutation and treatment with staurosporine (1–10 μM) also reproduced previous qualitative microscopy and subcellular fractionation results37 showing a significant increase in the FUS cytoplasmic/nuclear ratio. Confirmation of these results demonstrates the effectiveness of this new methodology in providing quantitative data where previously qualitative or labour-intensive experiments were required. Our ability to quantify FUS cytoplasmic mislocalisation following staurosporine treatment also demonstrates the possibility of this technique for use in testing the ability of novel FTD and ALS drug treatments to modulate FUS or TDP-43 mislocalisation.Expression of the FUSTARDBP are absent6. Mutations in TARDBP have been associated with both FTD and ALS and lead to TDP-43 cytoplasmic mislocalisation13. Unlike FUS, the expression of TARDBP mutations in primary cells and cell lines does not reproduce the dramatic cytoplasmic mislocalisation seen in post-mortem tissue40. In addition, overexpression of exogenous TDP-43 in vitro is sufficient to induce mislocalisation even for WT sequence23. Any differences between WT and mutant TDP-43 for this characteristic are therefore harder to discern and represent a significant limitation for all TDP-43 exogenous expression assays, including the one described here, and may be responsible for differing results being reported for the same mutations. Our technique was able to detect a significant increase in the TDP-43 cytoplasmic/nuclear ratio in TDP-43A315T and TDP-43A382T cells when compared to TDP-43WT. This confirms previous results for TDP-43A315T19 and TDP-43A382T42. The TDP-43M337V variant, which displayed a significant decrease in cytoplasmic TDP-43 in SH-SY5Y cells, has previously been shown to increase cytoplasmic TDP-43 in some24, but not all studies22. We note that TDP-43M337V has previously been shown to aggregate into nuclear puncta in SH-SY5Y cells21, which we observed in our experiments. As these puncta fluoresce at a higher intensity than diffuse TDP-43 expression, this may lead to an overall decrease in cytoplasmic/nuclear fluorescence ratio relative to TDP-43WT. Thus, in some cases quantification of nuclear and cytoplasmic puncta may be a useful adjunct to assess the pathogenicity of a given mutation. However, the disease relevance of nuclear inclusions in in vitro studies of TDP-43M337V is unclear: although both intranuclear and cytoplasmic inclusions were reported in a transgenic TDP-43M337V mouse model43 and it is reminiscent of intranuclear inclusions observed in post-mortem tissue from sporadic FTD patients9, such inclusions are rare in human disease. In the only neuropathological report to our knowledge that concerns a patient with a TARDBP M337V mutation, no mention was made of intranuclear inclusions44. We also note that mislocalisation of TDP-43 is not the only mechanism by which TARDBP mutations lead to disease: for example, changes in protein stability or interaction partners have also been described31. However, we envisage that the mislocalisation assay as implemented here would be a useful tool in a battery of assays to screen novel TARDBP variants.The majority (~ 95%) of ALS and ~ 50% of FTD cases are characterised by the abnormal accumulation of TDP-43 in the cytoplasm of neurons and glia, even though in most cases mutations in 4. In comparison to the other experiments used to validate this quantification method, the degree of change in the endogenous TDP-43 cytoplasmic/nuclear ratio between the empty vector, the CYLDWT and the CYLD mutations was very small: e.g., absolute increase in ratio of ~ 0.03–0.04 between CYLDWT and CYLDM719V 46, and TDP-43 forms homodimers via its N-terminus48. By tagging the protein at the opposite end to its NLS or dimer-binding region we hoped to minimise any effect on the normal localisation pattern of TDP-43 and FUS. Additionally, aggregates of TDP-43 contain C-terminal fragments49, which may have been undetectable had we used a N-terminal GFP tag. We note that the cytoplasmic/nuclear ratio of exogenous GFP-FUSWT  at the C-terminus aa497-526)645. Conv9 contribute to its mislocalisation and aggregation49. Antibodies raised against the extreme N-terminus of TDP-43 would therefore not be suitable for a mislocalisation assay, as they would not detect these cleavage products. In contrast, the anti-TDP-43 antibody we used  recognises all major truncated TDP-43 forms9.A parameter of equivalent importance for detection of endogenous FUS and TDP-43 mislocalisation is selection of an appropriate detection antibody. This is particularly important for TDP-43, where C-terminal TDP-43 fragments observed in FTD and ALS patientsIn summary, this study demonstrates a simple methodology for unbiased quantification of TDP-43 and FUS cytoplasmic mislocalisation in vitro. This technique can easily be added to studies utilising qualitative data to strengthen clearly noticeable phenotypes in an unbiased manner, as well as to highlight subtle changes that may have not been previously identified. Utilisation of this fast and cost-effective technique to aid in assessing pathogenicity of gene variants observed in patients with FTD or ALS will help to prioritise these variants for more intensive research efforts into how they cause disease.CYLD, TARDBP and FUS mutations were introduced into the pCMV6-CYLD, pCMV6-TARDBP and pCMV6-FUS constructs (Origene) by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). Mutated CYLD and TARDBP cDNA sequences were then subcloned, using AsiSI and MluI restriction sites, into the pCMV6-AC-GFP vector (Origene) for expression of C-terminal GFP-tagged CYLD and TDP-43 proteins, respectively. Mutated FUS cDNA sequences were subcloned similarly into the pCMV6-AN-GFP vector (Origene) for expression of N-terminal GFP-tagged FUS protein, to avoid interference of the GFP tag with the C-terminal NLS of the FUS protein45. All clones were verified by restriction digestion and sequence analysis.4 cells/well and HEK293 cells were seeded at 8 × 104 cells/well. After 24 h, cells were transfected with GFP constructs (250 ng/well) for 24 (HEK293) or 48 (SH-SY5Y) hours using Lipofectamine 3000  as per the manufacturer’s protocol. For the staurosporine experiment, cells were incubated for 48 h after seeding, then treated for 5 h with 0.1, 1 or 10 μM staurosporine, to induce apoptosis. Staurosporine dilutions were prepared from a 1 mM staurosporine stock solution dissolved in dimethyl sulfoxide . All vehicle and staurosporine treatment groups were adjusted to a final concentration of 1% DMSO.HEK293 and SH-SY5Y were purchased from ATCC and authenticated by STR analysis prior to use in this study. HEK293 cells were maintained in Eagle’s Minimum Essential Medium  and SH-SY5Y cells in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12  each containing 10% heat-inactivated fetal calf serum (Sigma-Aldrich). For immunocytochemistry, 8-well chamber slides (Ibidi) were coated with 0.005% poly-L-lysine (Sigma-Aldrich) for 1 h and then washed twice with Dulbecco’s phosphate-buffered saline . SH-SY5Y cells were seeded at 5–7.5 × 10Transfected or drug-treated cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min in the dark. For visualisation of exogenous TDP-43 and FUS, cells were then washed twice with DPBS and mounted using DAKO fluorescent mounting medium containing 4′,6-diamidino-2-phenylindole . For visualisation of endogenous TDP-43 and FUS, non-specific background staining was blocked after cell fixation using 1% bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated overnight at 4 °C with rabbit anti-TDP-43  or rabbit anti-FUS  antibodies. The next day, cells underwent 2 × 5-min washes with DPBS, incubation with rabbit Alexa Fluor-647 secondary antibody  for 1 h, followed by 2 × 5-min washes and mounting as above. Cells were imaged for fluorescence intensity quantification with a 40 × objective on a Nikon A1R confocal microscope. Images of representative cells were obtained with a 100 × objective for figure preparation. Laser intensity, gain and offset settings were kept constant and at least five random fields of view were imaged for each experimental replicate.28. To prepare images for analysis, ND2 microscope files were converted to TIFFs, blinded and split into individual wavelength channel images using ImageJ 36. In CellProfiler, the DAPI channel was analysed first using the Identify Primary Objects module and a global, three-class Otsu thresholding method. This allowed for identification and separation of each nucleus within an image. Strict filtering for cell diameter (30–60 pixels) eliminated any irregular or overlapped nuclei that were present. TDP-43- or FUS-positive cells were also identified using the Identify Primary Objects module. Cell diameter filtering for this step had a much larger range and was dependent on experimental conditions and cell type (20–150 pixels). The Relate and Filter modules were used to link filtered nuclei to TDP-43- or FUS-positive cells. Centroid distance filtering, which limits the distance between the centre of the nucleus and the centre of the cytoplasm (maximum 40 pixels) was also applied to remove any debris or abnormal cells that may be fluorescing in the TDP-43/FUS channel. For the experiments quantifying endogenous TDP-43 in cells expressing GFP-tagged CYLD constructs, additional steps in the analysis were required. GFP-positive CYLD cells were identified in the same way as TDP-43- or FUS-positive cells using the Identify Primary Objects module, described above. However, prior to this, TDP-43-positive cells were identified by association with their corresponding nuclei, using the Identify Secondary Objects module. The watershed-image method was used with global, three-class Otsu thresholding. Size filtering was also applied (maximum 3000 pixels) to remove any clumped cells. The Relate and Filter modules were used to select only TDP-43-positive cells that were also positive for GFP. TDP-43- and GFP-positive cells were then linked to their corresponding nuclei using the Relate and Filter modules described above. For all experiments, in each TDP-43/FUS-positive cell, the nucleus and cytoplasm were separated into individual objects using the Identify Tertiary Objects module. Integrated  fluorescence intensity for both cellular compartments were then measured from the TDP-43/FUS channel image using the Measure Object Intensity module. Using these measurements, the cytoplasmic to nuclear ratio of TDP-43 or FUS was calculated for 300 cells per group, for each of the six replicates of the experiment. All filtering and thresholding steps in each experimental pipeline were kept consistent throughout all experimental replicates. Filtering values were specific to each experiment and require adjustment for different cell types and microscope parameters.Quantification of changes in the cellular localisation of TDP-43 or FUS between the nucleus and the cytoplasm was done by utilising CellProfiler 3.1.9 software Data are presented as mean ± standard error of the mean (SEM) from at least six independent experiments of n = 300 cells. Mean fluorescent intensity of TDP-43 or FUS across six biological replicates was in most cases compared by repeated measures one-way ANOVA and Dunnett’s multiple comparisons test. The effect of CYLD on endogenous TDP-43 was analysed using repeated measures one-way ANOVA and Sidak’s multiple comparisons test. All statistical analyses were performed using GraphPad Prism 9. Significance for all tests was set at p < 0.05.Supplementary Figures.


Macrotermes species: termites preferentially deposit soil on the mound surface at the boundary of the high-humidity region characteristic of the mound interior, implying a coordination mechanism through environmental feedback where addition of wet soil influences the humidity profile and vice versa. Here we test this potential mechanism physically using a robotic system. Local humidity measurements provide a cue for material deposition. As the analogue of the termite's deposition of wet soil and corresponding local increase in humidity, the robot drips water onto an absorbent substrate as it moves. Results show that the robot extends a semi-enclosed area outward when air is undisturbed, but closes it off when air is disturbed by an external fan, consistent with termite building activity in still vs. windy conditions. This result demonstrates an example of adaptive construction patterns arising from the proposed coordination mechanism, and supports the hypothesis that such a mechanism operates in termites.Many species of termites build large, structurally complex mounds, and the mechanisms behind this coordinated construction have been a longstanding topic of investigation. Recent work has suggested that humidity may play a key role in the mound expansion of savannah-dwelling  Mound-building termites of several different genera are known for their prowess in collective construction: colonies of millions of insects construct mounds that can be several meters tall, with elaborate outer features and complex networks of internal tunnels . These eThe classic explanation for how termites coordinate their building activity is based on a putative cement pheromone: a hypothetical chemical added to pellets of soil deposited by workers, which attracts other workers to the same site and triggers further deposition Grassé, . HoweverMacrotermes michaelseni mounds in Namibia  and determine with certainty that their actions are solely due to humidity levels. To explore whether the humidity template mechanism on its own can produce the kinds of building patterns seen with the insects, in this work we test an artificial agent-based construction system in which humidity alone is the defining template characteristic. Simulations can indicate the feasibility of the basic mechanism , but simWe constructed an autonomous mobile robot equipped with humidity sensors Carey, , and an Initially, a pair of parallel walls provide a short tunnel; tiles between the walls are saturated, while those beyond the tunnel's end are left dry. A humidity bubble, like that observed during active building on the termite mounds  The external air is still, as is typical for termites building on the surface of their mound at night, or in protected areas on calm days. (2) A fan blows air across the arena outside the tunnel, corresponding to wind, typical especially during the day. (3) The robot's water reservoir is left empty, corresponding to conditions experienced by termites in the dry season.We performed experiments under three conditions, analogous to those experienced by These methods are described in detail in the t-test demonstrates a significant difference in deposition trigger location between the still high-humidity experiments vs. the disrupted and dry trials  from the mound interior and freshly deposited material, or  from the wet tiles. The second is the evolving physical structure itself, which provides the substrate to which more material is attached; the locations of previous depositions shape the possibilities for further growth of the mound.Macrotermes michaelseni building activity at the mound surface, expanding the mound, varies seasonally and with time of day. Almost all expansion occurs during or soon after the rainy season, when soil brought up by workers from below the nest is much wetter; in the dry season, when deep soils have much lower water content, little or no mound expansion occurs  with wide-field touch or force sensing may convey a richer, more salient landscape profile for such a construction algorithm. Nevertheless, the robot used here produced the observed building patterns despite its sensory limitations and imprecise navigation. Such robustness to unreliability of individual agents and actions is necessary to collaborative construction algorithms, for engineered systems no less than for social insect colonies, whose successful operation likewise cannot depend on unerring precision by each insect.A template-based feedback process like the one considered here could potentially provide a novel mechanism to help coordinate environmentally-responsive building in collective robots. It requires no direct communication between agents, nor do agents respond directly to perceived configurations of material depositions, as they do in many insect-inspired models (Theraulaz and Bonabeau, The original contributions presented in the study are included in the article/NC, PB, JW, and RN: conceptualization. NC, PB, and JW: methodology. NC: robot hardware, software development, robot experiments, and visualization. JW: simulations and project administration. NC and JW: analysis and writing. RN and JW: supervision and funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The emergence of graphene and other two-dimensional materials overcomes the limitation in the characteristic size of silicon-based micro-resonators and paved the way in the realization of nano-mechanical resonators. In this paper, we review the progress to date of the research on the fabrication methods, resonant performance, and device applications of graphene-based nano-mechanical resonators, from theoretical simulation to experimental results, and summarize both the excitation and detection schemes of graphene resonators. In recent years, the applications of graphene resonators such as mass sensors, pressure sensors, and accelerometers gradually moved from theory to experiment, which are specially introduced in this review. To date, the resonance performance of graphene-based nano-mechanical resonators is widely studied by theoretical approaches, while the corresponding experiments are still in the preliminary stage. However, with the continuous progress of the device fabrication and detection technique, and with the improvement of the theoretical model, suspended graphene membranes will widen the potential for ultralow-loss and high-sensitivity mechanical resonators in the near future. A resonator measures the target parameters by detecting the frequency signal. It has the advantages of high resolution, good stability, and strong anti-interference ability , which iThe emergence of graphene and other two-dimensional materials brings new opportunities for the miniaturization of resonators. Graphene has a high elasticity modulus, low surface density, and high surface area-to-volume ratio, leading to a significant enhancement in resonant frequency and sensitivity to external parameters, such as force, pressure, and mass . HoweverHere, we review the state-of-the-art theory and experiments of graphene resonators. Firstly, we summarize and analyze the fabrication processes of graphene and devices. Then, the electrical, optical, and piezoelectric excitation and detection schemes of the resonator are described. From theoretical analysis and experimental verification, the resonant performance including frequency tuning, loss, quality factor, and the non-linear effect are briefly introduced. Finally, the device applications of graphene resonators in the sensor, pneumatic measurement, and sensitive detection of graphene intrinsic properties are discussed.2·V−1·s−1 at room temperature [Since graphene was discovered by mechanical exfoliation for the first time in 2004, a variety of preparation methods of graphene have been developed . Among tperature . In addiperature .In resonator devices, graphene is usually placed in two ways. The first is to etch and evaporate metal electrodes followed by covering graphene on the pre-fabricated substrate through exfoliation, transfer, or direct growth. The first graphene resonator was successfully realized by exfoliating graphene onto the substrate with preset channels and electrodes, and graphene was attached to the silicon oxide wafer by van der Waals force . This meThe second way is to place graphene on the target substrate by exfoliation, transfer, or growth firstly; then deposit metal electrodes to contact and fix the graphene; and finally suspend graphene by etching or other nano-fabrication techniques, as shown in The electrode of the graphene resonator can be divided into the gate electrode and source-drain electrode. The source and drain electrodes not only play the role of electrical contacts but also fix the suspended graphene film to retain the tensioned architecture. Au or other metals are deposited by plasma vapor deposition, thermal evaporation, or other means after ultraviolet lithography or electron-beam lithography. In addition, in order to improve the adhesion between the contact metal and graphene, Ti  or Cr 1 is usualWhen the resonator is excited by electrical input, the gate electrode is required to generate electrostatic force. A simple way is making by use of the substrate directly. In this case, the heavily doped Si substrate covered with silicon oxide on top is usually working as the gate ,15,26. HThe etching process, which can be divided into dry etching and wet etching, is the most common approach used to suspend graphene. Wet etching is a pure chemical process, in which buffered oxide etchant  is often used to etch silicon dioxide ,17,19. HCompared with wet etching, dry etching has advantages of high precision, anisotropy, and fast etching speed. Reactive ion etching (RIE) combining ion bombardment and chemical reaction is the most widely used method in the dry etching of silicon dioxide ,11,22. FSU-8 3005 (SU-8) is spun on the silicon oxide wafer and patterned by the electron beam (EB), and then the graphene is exfoliated to SU-8 followed by the SU-8 developed to suspend graphene ,24. JungThe excitation methods for graphene resonators can be summarized into electrostatic, optical, and piezoelectric excitation schemes. In the devices driven by electrostatic force, a voltage potential difference between the gate electrode and graphene is applied to generate the electrostatic force and to drive the resonance of the graphene ,29, as sThe gate voltage consists of two parts: a constant DC voltage maintains the tension of the graphene membrane and another RF AC voltage drives the graphene to a resonance frequency. Alternatively, when the gate electrode is grounded, the same effect can be achieved by applying the drive voltage to either the source or the drain electrode . Jung etIn an optically excited device, an intensity-modulated laser spot is focused on the surface of the graphene, regulating graphene temperature via light absorption of the graphene and causing periodic contraction/expansion, as well as thus vibrations of the graphene, as shown in In addition to direct excitation, parametric excitation is also widely used in electrical or optical excitation schemes ,38, whicAlthough pristine graphene is not a piezoelectric material, in recent years, piezoelectric excitation has been successfully applied to graphene resonators by indirect driving approaches ,41. The The small size of the graphene resonator makes the resonance signal weak and difficult to detect, thus the detection method needs to be carefully designed and improved. Electrical and optical detection methods are the most widely used schemes to detect the resonance signals.It is worth noting that the widely used frequency mixing technique will lead to the decrease of bandwidth and measurement speed of the graphene resonator. Alternatively, the direct RF readout technique has significant advantages in the high-speed detection for real-time applications. A common RF readout scheme for graphene resonators is to detect the RF current, combining the operation principles of graphene field-effect transistors and mechanical resonators . HoweverThe optical detection scheme, with advantages of high sensitivity and simplicity without electrical contacts, is also an important route for the effective detection of the resonance signals . InterfeCompared to conventional Micro-Electro-Mechanical System (MEMS) resonators, graphene can withstand very high strain, suggesting an extremely wide tunable range of the resonance frequency of graphene resonators. Electrostatic tuning of the frequency can be easily achieved by applying a dynamic gate voltage. Kang et al. investigated the effect of the initial strain-induced tension and deflection-induced tension generated by a gate voltage on the resonant frequency and tuning range by molecular dynamics simulation ,50,51. TDue to the excellent conductivity and thermal stability of graphene, Ye et al. achieved frequency tuning by Joule heating of graphene . The curQ value can be obtained by spectral measurements or time-domain measurement. It is worth mentioning that the time-domain measurement can effectively avoid the spectral spreading effect in spectral measurements [The loss is an important parameter in determining the performance of a resonator and is usually represented by quality factor urements . Table 1Q value increases accordingly [A number of mechanisms can lead to the reduction of the quality factor. Imboden et al. summarized the losses in graphene resonators into the extrinsic and the intrinsic losses according to their source . Extrinsordingly , which iQ factor, Eichler et al. reduced the driving force until the motion became almost undetectable and they eventually obtained a graphene resonator with an ultra-high quality factor of 100,000 at 90 mK [The source of non-linear damping is a complex problem. One explanation is that it mainly comes from two aspects: general loss path and geometric non-linearity . For exaat 90 mK .Due to the fact that graphene is an atomically thin membrane, the out-of-plane deflection of graphene is much larger than its thickness in a vibrating graphene sheet, which means that it is easy to trigger the non-linear effect in graphene resonators . Such no2rtε5/2) . As a reic range . Parmar ic range .Considering the non-linear effect, the Newtonian equation of the resonator can be expressed as follows :3)md2xmd2x(3)mdng force . If α issonators . This woGraphene has excellent mechanical properties, such as ultra-high Young’s modulus and high fracture strength, as well as ultra-low surface density. In addition, the resonance properties of graphene are very sensitive to external physical parameters and the environment, such as force, attached mass, and acceleration. These characteristics make suspended graphene very suitable for various kinds of resonant sensors.Resonant mass sensors are used to detect masses through the resonant frequency shift of graphene due to the attached mass. Based on the analysis methods of classical mechanics or nano-mechanics, a great deal of theoretical calculations and simulations of graphene resonant mass sensors are studied. For example, Dai et al. investigated the effect of non-linear vibration behavior and in-plane tension on the detection sensitivity of mass using the continuum elastic model . Their rThe pressure sensors based on graphene nano-mechanical resonators have been studied by finite element simulation . The simYet, the exploration of graphene resonant accelerometers is at the preliminary stage. Kang et al. studied the resonant accelerometers based on graphene nano-ribbons  and crosIn addition to measuring and sensing the external parameters, the graphene resonator can also be used to measure the intrinsic properties of graphene. Guan et al. fabricated a graphene-based strain sensor, in which the strain is adjusted by bending the substrate . The strGraphene resonators can also provide a feasible scheme for detecting fundamental physical properties. For example, Chen et al. controlled the electron energy in the Landau level under an external magnetic field through the graphene resonators. In this way, the energy gap between the adjacent Landau levels and the energy conversion between the energy gaps are measured . In addiinewidth .Recently, graphe0ne drum resonators have been used for pneumatic testing due to the flexibility and gas tightness of graphene membranes . As showThe high tensile strength and mechanical tunability of 2D materials such as graphene have attracted more and more attention to 2D phononic crystals in recent years. The feasibility of phononic crystals based on graphene electromechanical resonators is studied theoretically and experimentally ,90. In tThe Van der Waals heterostructure is a unique structure in two-dimensional materials. By stacking different types of two-dimensional (2D) structures, the mechanical, electrical, and thermal properties of heterojunctions can be actively regulated, which brings an endless potential of two-dimensional resonators.Inoue et al. fabricated graphene-molybdenum disulfide heterojunction resonators . By stacWith the continuous discovery of new two-dimensional materials, some of them have become new candidates for nano-electromechanical resonators, such as transition metal dichalcogenides (TMD) ,66,96, hGraphene is the first two-dimensional material to be discovered. It has atomic thickness, an extremely high Young’s modulus and fracture strength, and very low surface density, which make graphene an ideal material for the fabrication of nano-electromechanical resonators. The large surface-to-volume ratio induced by ultra-thin thickness also makes graphene resonators have great potential in the application of pressure, mass, acceleration, and other ultra-sensitive detectors. Since the first fabrication of graphene resonator in 2007, after more than ten years of development, great breakthroughs have been made in the fabrication technology and weak signal detection scheme, and the theory on the resonance characteristic also has been constantly updated. The application of graphene resonators is gradually moving from simulation to experiment and from the classical field to quantum field.However, there are still many challenging issues in graphene resonators to be solved in this stage. We summarize these challenges and the main directions of future research as follows.Fabrication of high-quality grapheneThe repeatability of graphene preparation is too weak due to the uncontrollable factors such as graphene strain, doping, defects, and so on. Additionally, the fabrication of graphene with large-scale, high-quality, uniform, and controllable thickness is still a challenge.Strain engineeringThe strain has a strong influence on the resonant performance of graphene resonators. In the process of fabrication, graphene is easy to produce residual strain, which is usually harmful. Thus, the approaches to actively control the strain of graphene is important.Device integrationAfter successful fabrication of graphene resonators, it is still a challenge to integrate graphene resonators into electronic systems and necessary to establish a set of integrated processes with a high efficiency, low cost, and high success rate.Loss source of graphene resonatorsThe loss of graphene resonators is very diverse but the source and mechanism of many losses are not clear; in particular, the relationship between loss and temperature still has a lack of accurate description. Therefore, it is necessary and urgent to study the loss source of graphene.Research of non-linear effectsSince the graphene resonator has a high level of non-linearity, resonant sensors based on the non-linear behavior are also widely concerning. In particular, the non-linear vibration has been proved to improve the sensitivity of sensors, which attracts attention to application in the non-linear regime.The expansion of the application fieldIt is very attractive to further expand the application field of graphene resonators from the classical field to quantum field. Considering the atomic thickness of graphene, the graphene-based resonator can reach an extremely high frequency, small mass, and dramatical large zero-point motion, which makes it an ideal device for quantum-electromechanical systems.In general, graphene resonators promote the development of micro-nano-electromechanical systems to miniaturization, ultra-low power consumption, and ultra-high sensitivity. With the continuous progress of the device fabrication and detection technique, and with the improvement of the theoretical model, graphene resonators will certainly open the potential for wide applications in various high-tech fields.


We also report DFT data used to obtain hydrogen Gibbs free energies for adsorption on the MoS2 monolayer and on graphene of the heterostructure. The DFT data were calculated using the periodic DFT code CRYSTAL17, which employs Gaussian basis functions, under the hybrid functionals PBE0 and HSE06. Moreover, we also report the data used for Quantum Theory of Atoms in Molecules (QTAIM) and Non-covalent Interaction (NCI) analysis calculations. These data were obtained using the optimized unit cell configurations from the periodic DFT and inputted to Gamess program, thus generating files that could be read by the Multiwfn program used for QTAIM and NCI calculations.The data presented in this paper refer to the research article “Dry and Hydrated Defective Molybdenum Disulfide/Graphene Bilayer Heterojunction Under Strain for Hydrogen Evolution from Water Splitting: A First-principle Study”. Here, we present the Density Functional Theory (DFT) data used to generate optimal geometries and electronic structure for the MoS Defect MoS2/graphene heterostructures serve as hydrogen evolution catalysts (HER).We provide (a) structural and electronic information for dry and hydrated pristine and defect MoS•Electronic information shows a bandgap opening at the Dirac point region, which is affected by hydration and vacancies. Thus, this bandgap could be engineered for producing efficient HER electrocatalysts.•2-graphene interaction is var der Waals.The presence of QTAIM S-C bond critical points and NCI calculations show that MoS•2/graphene use as HER, when S and C defects are included in the lattice. These data can be further used by experimentalists to examine the needed concentration of S that produce an HER catalyst with hydrogen Gibbs energy of approximately zero.We also provide data that support the MoS12/graphene heterostructures with and without interacting waters on the S surface of the MoS2, as calculated by the PBE0 functional. We found no significant differences in the above optimal geometries for calculations using the HSE06 functional. D), minigaps (ΔE), and MoS2 bandgaps (Eg) for pristine and defect configurations of this work under the HSE06 calculations. Corresponding ED, ΔE, and Eg using the PBE0 functional are shown on 2 bandgaps Eg relative to the HSE06 calculations, as expected. H for H adsorption on MoS2 and graphene for each MoS2/graphene configuration under the PBE0 calculations. For H/MoS2, ΔGH is positive for adsorption on pristine MoS2 and negative, when S vacancies are present. 2/graphene using Multiwfn and plotted via VMD . Thus, for these calculations a molecular cluster has been used.The submitted data are grouped in five directories. Two directories contain CRYSTAL17 input and output files from electronic band structure and densities of states (DOS) calculations and from optimized geometries. Each of these directories contain information from HSE06 and PBE0 calculations, which are found in separate subdirectories. The other three directories contain HER calculations for the PBE0 functional (CRYSTAL 17 input and output files), Gamess input and output files, and QTAIM-NCI Multiwfn output files.22/graphene heterostructure under comprehensive strain by using a 2H-MoS2 three-layer 4 2 surfaces contain three water molecules per unit cell. These waters are placed on the S layer away from graphene. We built defective MoS2/graphene heterostructures by considering all combinations of single (VS) and non-adjacent double S vacancies (VS2) in the MoS2 with a single C vacancy (VC) in the graphene layer.We constructed the pristine dry MoS2/graphene configurations were obtained using the periodic DFT code CRYSTAL17 D appears at the Γ point.Optimized geometries and electronic structure of all MoS2. The Gibbs free energy Hydrogen adsorption energies QTAIM ,10 and NNone.Nicholas Dimakis: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing. Sanju Gupta: Conceptualization, Methodology, Writing – review & editing. Razeen Wadud: Visualization, Investigation, Validation. Muhammad I. Bhatti: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


Mycobacterium tuberculosis infection and tuberculosis (TB) disease across these communities. We sought to clarify whether specific communities could have been more/less vulnerable to M. tuberculosis transmission as a result of sociological variety relevant to transmission efficiency.Selected Zambian communities formed part of a cluster randomised trial: the Zambia and South Africa TB and AIDS Reduction study (ZAMSTAR). There was wide variability in the prevalence of We conducted a mixed methods secondary analysis using existing data sets. First, we analysed qualitative data to categorise and synthesise patterns of socio-spatial engagement across communities. Second, we compared emergent sociological variables with a measure of transmission efficiency: the ratio of the annual risk of infection to TB prevalence.ZAMSTAR communities in urban and peri-urban Zambia, spanning five provinces.Fifteen communities, each served by a health facility offering TB treatment to a population of at least 25 000. TB notification rates were at least 400 per 100 000 per annum and HIV seroprevalence was estimated to be high.M. tuberculosis transmission. For example, watching video shows in poorly ventilated structures posed a presumptive risk in more socially open communities, while outdoor farming and/or fishing were particularly widespread in communities with lower transmission measures.Crowding, movement, livelihoods and participation in recreational activity differed across communities. Based on 12 socio-spatial indicators, communities were qualitatively classified as more/less spatially crowded and as more/less socially ‘open’ to contact with others, with implications for the presumptive risk of M. tuberculosis transmission efficiency. To reduce transmission, certain socio-spatial aspects could be adjusted , while more structural aspects are less malleable . We recommend integrating community level typologies with genome sequencing techniques to further explore the significance of ‘social permeability’.A dual dynamic of ‘social permeability’ and crowding appeared relevant to disparities in ISRCTN36729271. We employed a novel qualitative approach to investigate disparities in transmission efficiency.We combined population (community) level socio-spatial data with actual measurement of tuberculosis (TB) infection and disease.Data were effectively cross-sectional, focusing on interactions within localised geographical boundaries, and did not capture details of extra-community travel, nor of infrequent and/or seasonal events, potentially relevant to TB transmission.Results are context specific; therefore, further investigations in other settings would be valuable to assess the generalisability of key concepts.Measures of association should be interpreted with appropriate caution and the study regarded as hypothesis generating.Mycobacterium tuberculosis, within and outside households, is critical to developing alternative approaches that could reduce ongoing M. tuberculosis transmission at the community level.M. tuberculosis transmission dynamics. We used a novel mixed methods interdisciplinary approach to conduct a secondary analysis of Zambian data sets, collected during a cluster randomised trial (CRT)—the Zambia and South Africa TB and AIDS Reduction study (ZAMSTAR)—and as part of an ancillary study, named the Contact Observations of Daily Activities (CODA).M. tuberculosis transmission efficiency .Interactions between the tuberculosis (TB) and HIV epidemics have complicated and compromised TB containment efforts in Zambia and other high burden countries. There is evidence that as the HIV epidemic ages, an increasing burden of undiagnosed TB is emerging among older adults in Zambia, underscoring the resilience of epidemic TB in the region.M. tuberculosis transmission and TB prevalence across Zambia and South Africa at a population level. The two interventions—enhanced community TB case finding and household level combined TB–HIV care—were implemented from 2006 to 2009. In Zambia, 16 communities were selected by TB notification rates in excess of 400 per 100 000 per annum and a high HIV prevalence.M. tuberculosis infection, as measured by tuberculin skin test (TST) surveys in primary school children, was highly variable, ranging from 2% to 13% across the Zambian communities and allowed for the estimation of ARI.M. tuberculosis transmission dynamics. Towards the end of ZAMSTAR, CODA sought to measure patterns of social contacts relevant to M. tuberculosis transmission and model the implied ARIs in adult age groups.M. tuberculosis transmission.ZAMSTAR (2004–2011) compared two interventions to reduce This secondary analysis used whole communities, rather than the individual, as a unit for analysis and drew on four sources of data: qualitative data collected for the purpose of the CODA study; estimates of population density from the ZAMSTAR household enumeration; estimates of ARI from ZAMSTAR TST surveys; and TB prevalence data from the final ZAMSTAR TB prevalence survey. Details of ZAMSTAR household enumeration, TST data and implied ARI estimates, TB prevalence data and linked methods have been published.M. tuberculosis ‘transmission efficiency’.We focus on the CODA qualitative data collection and analysis used for the identification of socio-spatial variables; our use of quantitative ARI and prevalence data from ZAMSTAR; and the mixed method analysis approach that investigated relationships between socio-spatial variables and calculated Following the intervention period of ZAMSTAR, Broad Brush Surveys (BBS) were conducted in 2011 across Zambian ZAMSTAR communities as part of CODA.The qualitative BBS data indicated how public places and transport were used by different age groups and genders within each community. Our focus on the broader community is justified as we understand transmission is less likely to take place within households in this particular setting.We delineated the strongest variables as broadly linked to one or more of the following aspects: (a) levels of spatial crowding, pertaining to the amount of people within shared/confined spaces and the density of physical structures; (b) the range and degree of participation in predominantly indoor recreational activity; (c) the dominance of outdoor livelihoods, for example, widespread commitment to farming or fishing dramatically shaped socio-spatial engagement in certain communities; and (d) what we have termed as ‘social permeability’, that is, the level of exchange between, or movement of, local residents and non-residents across and within geographical boundaries, with some communities clearly more socially ‘open’ to extra-community contact. Eleven qualitative indicators were identified to represent assessments of socio-spatial variability, selected on the basis of being observable (tangible) and categorical, with perceived relevance for TB transmission. The indicators were general crowding in public spaces/places; the use of overloaded and poorly ventilated transport; local residents remaining in their residential community during the day; housing density; crowding at health centres; time spent at home during the day; watching video shows; going to bars/taverns; spending time farming/ fishing; the presence of non-residents; and evidence of residents travelling out of their residential community.M. tuberculosis transmission. Karel Styblo used this ratio to help describe historical patterns in European TB epidemiology. While the values that Karel Styblo found no longer apply, the ratio remains a useful metric.As part of ZAMSTAR, TST surveys were conducted in a cohort of primary school children to calculate ARI across communities, estimating community ARIs of between 0.25%/year and 1.49%/year (using a 15-millimetre cut-off for positivity).After completing the qualitative analysis and quantitative calculations described above, we first correlated the measure of transmission efficiency with the 11 qualitative variables. Each variable was titled according to one of the four social aspects to which it most strongly indicated. For example, the variable ‘presence of non-residents’ was regarded as a strong indicator of social permeability, given the manner in which it presented in the BBS. Qualitative variables (indicators) were also ranked by a priori expectations of increasing transmission risk. For example, ‘high’>‘low’ for housing density, or ‘WMGB’>‘M’ for women, men, girls and boys spending time in bars versus only men. Previous work on these communities combining the TB prevalence results, ARI estimates and quantitative social contact data suggested men are responsible for more infections than women.10.1136/bmjopen-2020-047136.supp1Supplementary data10.1136/bmjopen-2020-047136.supp2Supplementary dataCommunity Advisory Boards (CABs) were consulted throughout the ZAMSTAR trial and formed before the implementation of interventions. In the 16 Zambian communities, CABs were formed from pre-existing Neighbourhood Health Committees (NHCs) in each community. NHCs were already representative of different geographical ‘zones’ in the trial communities, endorsed by an Act of Parliament and democratically elected. NHCs were, therefore, asked if they would like to represent public (including patient) TB and HIV interests, and sought representation across interest groups, age and gender. Although not involved in the design of the ZAMSTAR trial, CABs were closely involved from ZAMSTAR baseline research onward. Zambian research work was conducted by Zambart, a research organisation housed by the University of Zambia’s School of Medicine and with links to the London School of Hygiene and Tropical Medicine. CAB members received training in good clinical practice, research protocols and research dissemination. They were involved in the public randomisation of the trial communities and all research dissemination. Other than the CABs, representative participants involved in different components of the trial were invited to community and district dissemination events. This included BBS disseminations, carried out in the communities, using informative flyers for community members. Being a substudy of ZAMSTAR, CODA used the same CAB structures and communication channels. The community engagement lead, Musonda Simwinga, used his experience with the ZAMSTAR CABs as a platform for his own PhD on community engagement using CABs in trials, including reflections on equitable partnerships and accountability, and to develop other CAB structures for subsequent trials.23Certain communities were classified as more ‘socially permeable’ than others: socially ‘open’ to exchange across geographical boundaries and orientated toward mixing across age and gender in congregate settings. In nine communities, adults from other regions—‘outsiders’—engaged more with local community life. The presence of ‘outsiders’ appeared particularly diverse in three of these nine communities, where men, women, girls and boys visited. In contrast, there was little to no evidence of non-residents operating in the remaining six communities. In places with a strong presence of ‘outsiders’, social mixing across age groups and genders appeared widespread, although where and how this contact took place varied. For example, in community 14, bars played a greater role in bringing people together than video shows. We also distinguished between communities where both adults and children travelled out and those where mostly adults travelled out. In four communities, only men travelled out regularly. This travel could be linked to livelihood pursuits, often of an agricultural nature. Involvement in seasonal farming or fishing was particularly widespread across age and gender in four communities; in an additional seven communities, there was evidence of engagement with outdoor farming or fishing, but this was notably less intense. Residents left communities for short periods to fish and/or buy fish or tend to fields in the rainy season. There was no evidence of farming or fishing in the remaining 4 (of the 15) communities.Levels of crowding varied at health centres and in other gathering places, although the majority of communities were classified as generally ‘crowded’ by fieldworkers. The same was true for housing density: only five communities were observed to have relatively low-density housing. Importantly, public transport was often overloaded and gauged as crowded, with extended durations of travel. Ventilation during travel was ordinarily poor given that it was common practice to keep windows shut to prevent dust. In one community, panel vans with no windows were being used. Although most communities were reliant on buses, minibuses and/or vans for travel, some were substantially less dependent on public transport; residents of one community mostly travelled by foot. When assessing who remained within the geographical boundaries of their residential community during the day, communities again differed. In six communities, we found little evidence of residents remaining. Children and/or women generally remained in six of the other nine communities, but men rarely did. In three communities, local men were significantly more present.Proportionally, women generally spent more time at home . In some communities, men appeared more engaged with recreational activity. Indoor recreational activities across communities were often similar, but contact patterns differed. Children came into contact with adults at drinking places and/or at pool table clubs in seven of the communities, while in the others this was far less evident. In three communities, only men were ever seen at bars. In certain communities, women were observed with babies strapped to their backs and boys were frequently trading in eggs and/or bottles, highlighting the multifunctionality of drinking places in these areas. Bars and taverns were generally crowded and poorly ventilated, with limited small windows designed to deter theft. Watching films in small makeshift tented ‘theatres’ was a popular leisure activity in 14 communities. Shows were held in small outdoor structures, erected using poles and tarpaulin, measuring approximately 4×2.5 m. These structures were darkened as much as possible to enhance viewing, by closing gaps in the tarpaulin, which in turn limited ventilation. Audiences were ordinarily seated on a few tightly packed benches. Children watched video shows during the day, and in the evening, adults and children often watched together. All communities showed widespread commitment to their local churches. Community members of all ages spent a substantial proportion of their time at services ranging between 2 and 6 hours, which often exceeded the time spent on many of the other contact activities identified. It should be noted that most community residents are Christian and the fieldwork was conducted in the period leading up to Easter.Population density and qualitative assessments of crowding were broadly concordant, given the small sample size (number of communities). Communities sharing higher transmission efficiency could generally be classified as more crowded, whereas communities with lower transmission efficiency tended to present in more of a mixed way (across lower and higher degrees of crowding). By tracing an individual community (number) through M. tuberculosis transmission in unique and sociologically complex ways. Communities with lower measures of transmission efficiency included, but were not limited to those in which fishing and/or farming were particularly dominant across gender and age; where public transport was more ventilated and less overloaded; where less crowding took place in public space; where fewer ‘outsiders’ were present; where only men mostly travelled out of their community; where there was negligible evidence of indoor recreational activity; and where population and housing density was lower. Links between some of these socio-spatial aspects and M. tuberculosis transmission risk have been shown, using different approaches, in other sub-Saharan settings. In Malawi, Odone et al identified that TB was more common among those working in the cash economy than those in subsistence economy,25–27This interdisciplinary analysis suggests that certain communities may have been protected from M. tuberculosis and local aerosol transmission in poorly ventilated spaces. ‘Social permeability’ not only reflects the presence of non-residents within a community, but it also captures the reach of ‘mixing’ across age and gender, and patterns of locals travelling out. The latter provides additional opportunity for extra-community M. tuberculosis exposure and infection (or re-infection), outside of the residential community. It seems that this dual dynamic of spatial ‘closeness’ and social ‘openness’ is relevant to increased M. tuberculosis transmission across the communities.From a distinctly qualitative angle, our work begins to unveil and define how the sociological topography of a particular community could play an important role in shaping transmission efficiency. Two meta-observations are significant in this regard. The first observation relates to the relative vulnerability of the communities with more outwardly orientated and socioeconomically diverse features in comparison to their more socially secluded and/or agriculturally focused counterparts. Certain communities exhibited a dual dynamic of crowding and ‘social permeability’, which could be providing increased opportunity for the importation of M. tuberculosis exposure. Routine variables refer to habits or patterns of behaviour, which, although related to socioeconomic factors, are not obviously reducible to them. Watching video shows is a good example; it is a form of routine leisure activity which carries a particularly high presumptive risk for M. tuberculosis transmission because of the darkened, poorly ventilated and contained space in which it is conducted. For health policy, our distinction between ‘structural’ and ‘routine’ variables is important because the latter would be more malleable to intervention. While structural dependency on the outside world cannot be altered easily, people can be encouraged to change the way in which they conduct routine leisure activities; for instance, a concerted effort could be made to increase ventilation in these places.28The second observation relates to a distinction that needs to be drawn between what we could call ‘structural’ and ‘routine’ variables. By structural variables, we refer to socioeconomic factors that are to a certain extent beyond people’s agency. For instance, communities that are more structurally dependent on exchange and interaction with outsiders could be more vulnerable to M. tuberculosis transmission risk in communitiesOur secondary analysis has limitations. Findings are context specific and data were effectively cross-sectional. We focused on interactions within localised geographical boundaries, and did not capture interactions on the far side of extra-community travel. Furthermore, fieldwork was conducted at a particular time of year and observations took place during the daytime, which limits the generalisability of our observations accordingly—not only across but also within the study communities. We anticipate relevant socio-spatial features and their role in mediating TB transmission will vary between contexts. However, the dual dynamic of spatial closeness (crowding) and social openness (permeability) may be relevant in other settings, with further investigations valuable. The addition of pathogen genomic data might help generate or eliminate hypotheses on transmission location or context, and recent research illustrates that quantitative measures of geographic mobility could be applied to studies of social permeability.M. tuberculosis transmission. Interactions between social permeability and crowding appear relevant to increased transmission efficiency, at least in these Zambian communities. A conceptual framework differentiating structural and routine elements of high-risk socio-spatial features could provide a valuable foundation for the development of contextualised public health interventions. For example, some socio-spatial aspects carrying transmission risk are easily adjusted , while more structural aspects are less malleable . The significance of social permeability could be further explored by integrating community level typologies with genome sequencing techniques able to map strain introduction events and community transmission chains.Localised sociological distinctions offered an explanation of why certain communities might have been more (or less) vulnerable to