The stability of possible isomers of each monomer was investigated by performing classical molecular dynamics (MD) and quantum mechanics (QM) simulations on each monomer and comparing the results. The number of stable isomers is reduced to 36 with a prevalence of MER isomerism in the central core, as previously observed by NMR experiments. The simulations on decanuclear dendrimers suggest that the stability of the dendrimer is not linked to the stability of the individual monomers composing the dendrimer but rather governed by the steric constrains originated by the multimetallic assembly. Finally, the self‐aggregation of Ru10 and the distribution of the counterions around the complexes is investigated using Molecular Dynamics both in implicit and explicit acetonitrile solution. In representative examples, with nine and four dendrimers, the calculated pair distribution function for the ruthenium centers suggests a self‐aggregation mechanism in which the dendrimers are approaching in small blocks and then aggregate all together. Scanning transmission electron microscopy complements the investigation, supporting the formation of different aggregates at various concentrations.The structure of a decanuclear photo‐ and redox‐active dendrimer based on Ru(II) polypyridine subunits, suitable as a light‐harvesting multicomponent species for artificial photosynthesis, has been investigated by means of computer modelling. The compound has the general formula [Ru{(μ‐dpp)Ru[(μ‐dpp)Ru(bpy) Molucular simulations on the structure of a decanuclear Ru‐based dendrimer capable to play the role of antenna for artificial photosynthesis is performed. Aggregation of dendrimers is also modelled and compared to experimental results obtained by SAXS, scanning tunneling electron microscopy and energy‐dispersive X‐ray analysis. Although multi‐ruthenium(II) polypyridine complexes have been extensively investigated in the last decades, this is the first time (to the best of our knowledge) that a computational analysis is applied to characterize species containing more than four Ru(II) polypyridine centers. A scanning transmission electron microscopy (STEM) study complements the investigation, supporting the formation of different aggregates at various concentrations, as indicated by the photophysical experiments formerly reported.Here we report a computational study aimed to rationalize the isomer composition and the spontaneous aggregation of Ru10 species here investigated are highly branched tree‐like species formed by one metal atom  connected by bridging ligands to other metal nuclei . Such metal dendrimers can be prepared by iterative protection/deprotection synthetic protocols, and the iterative grow process ends when terminal ligands are inserted.2+, which corresponds to the core of our dendrimers, have shown that the purified material is a mixture of the MER and FAC isomers in which the MER isomer predominates (92 %).It can be noted that the structures with a MER central core (in particular those with SDD ligands) are energetically favored (therefore more stable), according to experimental observations previously reported: in particular, NMR experiments on the complex [Ru(dpp)3 dendrimer, which has the most stable core, presents a small minimization energy, but not the lowest. Similarly, the DDD MER SYM (DD 06)3 dendrimer, consisting of the isomers with highest ΔE, has not the highest overall energy. Overall, in order to obtain the dendrimers with lowest energy, one SDD MER central core plus three different intermediate monomers with alternated SD and DD ligands seems to be the better choice.Apparently, the stability of the dendrimers is not strictly linked to the energy of the individual monomers composing the dendrimer: for instance, the DDD FAC 02 (DD 05)6− counterions in acetonitrile solution.To further investigate the structure and the stability of the lowest energy dendrimers optimized in implicit solvent, we have carried out explicit solvent simulations of SDDMER01 DD02 SD06 DD05 dendrimer complex with PFIn order to investigate the dendrimer structure, we calculated the distribution function of Ru−Ru pair distances, shown in Figure OsRu9, a decanuclear dendrimer with an osmium atom in the central unit and nine ruthenium centers in the intermediate and peripheral units; the cyan line refers to experimental SAXS data for Ru10 (the compound investigated in our simulations), and the red and blue lines refer to the theoretical spectra obtained from CRYSOL for OsRu9 and Ru10 dendrimers, respectively.In Figure We have also to keep in mind that the experimental PDDF obtained by SAXS refers to a mixture of isomers that self‐aggregate forming intermediate values of the Ru−Ru distances, whereas our data are referring to a single monomer model. Nevertheless, the first peak, obtained by simulation,−5 M).[Despite their positive charge (each Ru(II) center has positive charge +2), the aggregation (self‐assembly) is a peculiar behaviour of dendrimers based on Ru and Os polypyridine complexes in acetonitrile solution, even at relatively low concentration (about 5×10−5 M).16, 31To investigate the process of self‐assembling of different dendrimers we have selected two different modelling scenarios:a system with 9 dendrimers, all different from each other, initially located faraway from each othera system with 4 dendrimers, all of the same type (SDD MER 05+SD02+DD03+SD06) in a box with explicit acetonitrile solvent.Both systems upon dynamics arrive to a more compact geometry, leading to the aggregation of the dendrimer polymers, as shown in the snapshots of Figure By analizing the distribution function of Ru−Ru distances related to the first case after 200 ns, shown in Figure Table Ru10 have been made using different concentration in acetonitrile solutions. A drop of Ru10 acetonitrile solutions with concentration lower than 5 x10−6 m, deposited on gold surfaces and upon solvent evaporation, yielded images of domains of fractal‐type aggregates  of dendrimers, and it is followed by aggregation of pre‐aggregated smaller systems into larger entities. The above proposed mechanism of aggregation is confirmed by scanning transmission electron microscopy (STEM) experiments. Actually, STEM experiments on s Figure a. The com Figure b.In this case, as evidenced by the EDX analysis . the percentage of F is slightly higher (about 4 %) with respect to the less concentrated sample, the percentage of P remains substantially unchanged (about 1 %) and that of the Ru is, on average, of about 0.7 %.6− counteranions, the distribution function of distances between ruthenium centers and PF6− anions shows, as in the case of a single dendrimer, that the anions arrange near the dendrimer molecules. This can be observed in PDDF graph  showing the distribution function of distances between Ru(II) centers and PF6− anions after 200 ns. Peaks at 0.61 nm, 1.08 nm, 1.51 nm and 3.6 nm are evident. These peaks show that most of PF6− anions are arranged as close as possible to Ru atoms (i. e. 0.61 nm considering the presence of ligands).With regard to the position of PFThe case of four dendrimers (SDD MER 05+SD02+DD03+SD06), in acetonitrile solution is briefly reported, because it is very similar to the case of the aggregation of the nine dendrimers previously discussed. We analyzed the distribution function of Ru−Ru distances after 50 ns, shown in Figure 2+−PF6− is also absolutely similar to that obtained in the case of the nine dendrimers aggregation in implicit solvent. In the case of the four dendrimers, in explicit solvent we obtained three peaks at 0.57 nm, 0.94 nm and 1.56 nm. These peaks roughly match the corresponding peaks in the case of nine different dendrimers in implicit solvent: 0.61 nm, 1.08 nm, 1.51 nm. Overall, since the behavior of dendrimers in simulations containing acetonitrile molecules is absolutely similar to that in implicit solvent simulations, we can infer that the implicit solvent model provides a good approximation of the molecules behavior during the aggregation process.The distribution of the distance RuRu10. To allow to face a very complex problem, most of the work has been focussed on geometrical isomerisms , leaving aside the Δ and Λ stereoisomerism of each metal building block.In this work we presented the first attempt to computationally tackle the structure of light‐harvesting metal polypyridine dendrimers, already known to be involved in photoinduced water oxidation processes relevant for artificial photosynthesis. Focus is here on a representative compound, 6); therefore, particular dendrimers have been composed and investigated in search of some more general rule.Thanks to good agreement between QM and MD simulations (with force field properly reparameterized) the number of stable building block isomers is reduced to 36, but the number of possible combinations in a decanuclear dendrimer still remains high  are energetically favored, according to the experimental NMR observations;if the central core has a FAC structure, more stable dendrimers are obtained when the intermediate monomers have dpp ligands of the same type as those of the central monomer ;to obtain the dendrimers with lowest energy, one SDD MER central core plus three different intermediate monomers with alternated SD and DD ligands has proven to be a good choice;the stability of the dendrimer is not strictly linked to the energy of the individual monomers composing the dendrimer.Ru10. On this regard, MD simulations, in acetonitrile solution and with PF6− counterions shows that the distances between the Ru atoms in the calculated structure of dendrimers, calculated through the pair distance distribution function, reflect those experimentally obtained by the SAXS.Wa have also faced the self aggregation of Finally, some groups of dendrimers were investigated both in gas phase and in acetonitrile solution. In all cases, both the movies of trajectories and the calculated pair distance distribution function (for the Ruthenium pairs) confirm the aggregation between the dendrimers and suggest its mechanism: the dendrimers are approaching in small blocks and then aggregate all together.Summarizing, our results show the succesfully application of computational methods to investigate the structure, as well as the self‐aggregation behavior, of quite large photo‐ and redox‐active artificial metal‐based dendrimers, although limiting the study, in this first attempt, to geometrical isomers and neglecting stereoisomers. Since smaller dendrimers aggregate with molecular water oxidation catalysts to give arise to functional systems for artificial photosynthesis,15 our rContains details on the computational aspects of the study and STEM/EDX experiments: 11 figures and 2 tables).The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Hesperin (AtHESPERIN) is the first circadian deadenylase discovered in plants. Herein, we determine its active site and provide information on the catalytic mechanism. We analyzed the kinetics of deadenylation reactions and modified Hanes–Woolf formalism together with application of molecular docking to study the effect of modulators on deadenylation reactions and oligomeric conformation of Hesperin. The results classify AtHESPERIN in the exonuclease–endonuclease–phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover. in silico results classify AtHESPERIN in the exonuclease–endonuclease–phosphatase deadenylase superfamily and contribute to the understanding of the intricate mechanisms of circadian mRNA turnover.The 24‐h molecular clock is based on the stability of rhythmically expressed transcripts. The shortening of the poly(A) tail of mRNAs is often the first and rate‐limiting step that determines the lifespan of a mRNA and is catalyzed by deadenylases. Herein, we determine the catalytic site of Hesperin, a recently described circadian deadenylase in plants, using a modified site‐directed mutagenesis protocol and a custom vector, pATHRA. To explore the catalytic efficiency of AtHESPERIN, we investigated the effect of AMP and neomycin, and used molecular modeling simulations to propose a catalytic mechanism. Collectively, the biochemical and  DEPCdiethyl pyrocarbonateEEPexonuclease–endonuclease–phosphataseGSTglutathione S‐transferaseMTGmonothioglycerolSECsize exclusion chromatographySPCsingle point charge2+ coordination and contribute to substrate positioning within the catalytic pocket  at 85 °C for 3 min. Reaction products were analyzed in denaturing 20% acrylamide : bisacrylamide/8 m urea–PAGE in a Mini‐PROTEAN Tetra cell system . All assays with Cy3‐labeled RNA were visualized using the Alliance 4 imager  unless otherwise indicated.The measurement of enzymatic activity of AtHESPERIN using fluorescence was based on Maryati et al.  with somInhibition by either AMP  or neomycin B sulfate  was tested with both assays at various concentrations. The Cy3‐labeled RNA assay in the presence of neomycin was visualized using ImageQuant Fluor imaging system [Cytiva ]. All reactions were performed with the addition of 0.1 U RNasin  and DEPC‐treated water.grafit6 /10 (25 μg). The reactions were set up and incubated at 25 °C for 60 min; after incubation, they were immediately analyzed in a 7% PAGE under nondenaturing conditions for acidic proteins with appropriate molecular weight standards . For the native PAGE analysis of AtHESPERIN in the presence of urea, 16 μg AtHESPERIN was incubated with increasing concentrations of urea, and then samples were analyzed in a 7% PAGE under nondenaturing conditions for acidic proteins with appropriate molecular weight standards. More specifically, BSA was used as an indicator of AtHESPERIN’s dimeric and trimeric conformations (BSA runs as two bands of 66 and 132 kDa), and urease was used to estimate the molar mass of higher‐order oligomers of AtHESPERIN [urease runs as a trimer (272 kDa) and a hexamer (545 kDa)].The effect of inhibitors or poly(A) on AtHESPERIN’s conformation was assessed by native PAGE as described previously . In brieProtein samples before and after the SEC run were analyzed by SDS/PAGE and then transferred onto a Porablot poly(vinylidene difluoride) 0.22‐μm membrane (Macherey‐Nagel) in Towbin transfer buffer  supplemeprocheck.The homology modeling of the AtHESPERIN deadenylase was performed using the Molecular Operating Environment Suite . The 3NGamber99 force field  plot. Hesperin is an allosteric enzyme; therefore, plotting of kinetic data requires adaptations, as described previously  [v versus [S]) again yields curved lines and for this reason, we raised [S] to the same power as in a Lineweaver–Burk plot to produce a straight line. Plotted data produced straight lines when [S] values were raised to the power of 3, in line with previous works  , 25. Plous works . ApplicaKM concentration of both AMP and neomycin B, and the reactions were analyzed in native PAGE  shares significant sequence identity with the catalytic region of human PARN [A. thaliana PARN is still pending.Taken together, the previous observations suggest that AtHESPERIN has a stable oligomeric conformation, mainly trimeric, not owing to weak hydrophobic interactions. AtHESPERIN together with PARN are the only described allosterically regulated deadenylases in plants and higher eukaryotes, respectively, thus far. Notably, man PARN , yet a sA8MS41) has conserved the 3D arrangement of the template structure by reproducing the beta‐sheet network and the main two alpha‐helices, flanking the catalytic site. The human CNOT6L (PDB: 3NGQ) [The homology model of Hesperin (accession number UniProtKB: B: 3NGQ)  was usedAMP was docked in the catalytic sites of both the template structure and the model of AtHESPERIN. The adopted AMP conformation, as well as the established interaction patterns, was conserved in the AtHESPERIN model, thus supporting the viability of the model Fig. .ligplot analysis [Next, to further analyze the interaction between AMP and AtHESPERIN, as well the impact of the selected mutations, we had to identify the nature of the interaction. In particular, we wished to discriminate between direct hydrogen bonding and long nonbonded interactions. The analysis  revealedin silico mutation of any of the earlier‐mentioned residues to alanine resulted in the loss of interactions and significant changes in the physicochemical nature of the catalytic site and docking pattern. The loss of interactions between AtHESPERIN and AMP upon any combination of the E114A, D287A, D346A or H385A mutations is shown in Fig. The 3, Fig. 3 was capable of adopting only a single conformation in the docking experiment. Because only one conformation was acquired, we investigated the electrostatic surface  are in close proximity in the 3D conformational space of the catalytic site and initially coordinate the two Mg ions. Then, the imidazole ring of His385 acts as a base and attracts a proton from a water molecule. Consequently, the protonated histidine becomes activated for nucleophilic attack, while the neighboring Glu114 deprotonates the pentacoordinate intermediate, thus achieving hydrolytic cleavage of the phosphodiesteric bond. It is a well‐defined and coordinated mechanism, making the deadenylation reaction very dependent on the 3D conformation of the full enzyme that is absolutely essential to be conserved to coordinate the four catalytic residues in the exact optimal spatial positions in the proximity of the catalytic site of AtHESPERIN to achieve maximal efficacy.Based on the previous data on the identification of catalytically important amino acids Fig.  and consace Fig.  of the eace Fig. . The anaTo characterize AtHESPERIN and determine its active site, we modified established site‐directed mutagenesis protocols and developed pATHRA, a custom expression vector. The modified protocol is efficient for both single and successive amino acid substitutions.2+ from the active site could account for loss of activity, as has been shown for CNOT6L [AtHESPERIN is an oligomeric EEP deadenylase and shares important features with other EEP members. Kinetic analysis showed inhibition by the reaction product (AMP) and neomycin B. Further, native PAGE does not indicate loss of AtHESPERIN’s quaternary structure, because the presence of poly(A), AMP and neomycin separately yield an identical band migration pattern Fig.  and may r CNOT6L .2+ or stabilizing substrate binding within the active site. Molecular modeling analyses confirmed that any of the earlier‐mentioned mutations result in the loss of interactions and consequently a thermodynamically less stable molecular system. As a result, these residues are essential to the function of AtHESPERIN. Collectively, our biochemical and in silico data suggest a cleavage mechanism for AtHESPERIN  and overexpression mutant lines, which exhibit both developmental structural phenotypes and an altered response to stress conditions . The datThe authors declare no conflict of interest.NAAB conceived and designed the project; RAAB, AK, VD, EP and MR performed the experimental work and acquired the data; DV performed the molecular modeling analysis; RAAB, DDL, VD and NAAB analyzed the data and wrote the paper.Fig. S1. (A) Schematic representation of AtHESPERIN showing the conserved EEP nuclease domain in blue and the positions of the mutated amino acids. (B) Sequence map of AtHESPERIN. Blue characters represent the catalytic domain, and red characters in bold the mutated amino acids.Click here for additional data file.


This paper considers the key issues surrounding the virtual assessment of clients for medicolegal purposes. Our main hypothesis was that, within certain defined parameters, the vIME technique can deliver reliable and accurate assessments. To explore this, a systematic literature search focusing on advanced device‐based range of motion measurement was conducted, along with an historical snapshot of observation‐based range of motion measurement considering application to remotely performed IME. While some specialists are of the view that observational measurement may be applied reliably to some joints when conducted by experienced orthopaedic surgeons, evidence for this is scant. The results, instead, support the notion of using task substitution, that is specialists appropriately assisted in conducting vIMEs by musculoskeletal trained allied health practitioners, regardless of the measurement tool, for permanent impairment assessments. Moreover, self‐performed examinations by injured individuals using advanced technology are not reliable in this setting. Our final contention is that remote examinations with limited clinical assessment have utility for legal matters, such as the assessment of causation of injury, treatment advice or approvals and fitness for pre‐employment tasks or safe variations, with objective clinical adjunct support such as Picture Archiving and Communication System‐based modern radiology systems.Remote telehealth practices were forced to advance 10 years in a few short weeks in March 2020 due to the onset of a global pandemic. In the sphere of non‐clinical medicine, a dramatic element of uncertainty entered the psyche of doctors and lawyers in relation to the validity of remote or  This paper addresses a previously unexplored gap in considering the potential for remote or virtual independent medical examinations (vIME), and is a step towards demonstrating that best practice vIME techniques, within certain defined parameters, can deliver reliable and accurate assessments. The key elements of this manuscript include: a systematic literature search focusing on advanced device‐based range of motion measurement, and an historical snapshot of observation‐based measurement considering application to remotely performed IME. In clinical musculoskeletal medicine, range of motion (ROM) tells us many important pieces of information about a patient's condition. By observing the movements of different body parts, we are able to determine the primary physical challenges following an injury and measure change objectively over time.There is a barrier of distance affecting regional Australia when it comes to medical care. Telehealth represents a contemporary option that complies with travel restrictions and need for safe social distancing stemming from the COVID‐19 pandemic. Telehealth practices were forced to advance 10 years in a few short weeks in March 2020,The primary objective of this paper is to examine this topical, contentious issue and come to some conclusions to assist clinicians using telehealth for medicolegal purposes and determine the most reliable method(s) for performing clinical examinations that will also satisfy legal requirements. We use terminology recently adopted to reflect this change in examination technique, i.e. virtual IME (vIME).over the past 15 years, presented in the body of this paper, allows an evaluation of whether new technologies are able to advance the reliability of vIME assessments.Measurement of ROM has been in ongoing development for many decades Fig. ; its valDue to the lack of evidence relevant to the vIME setting, hypotheses were formed by integrating the evidence from the general orthopaedic setting with the clinical experience of two orthopaedic surgeons (PS and BH). To assist clinicians using telehealth for medicolegal purposes, we extrapolated the findings to the vIME setting, formulating scenarios upon which the legal system can rely.unassisted examinations within the vIME setting for permanent impairment assessment, where rigorous physical examination and precise measurements are required  Guides' permanent impairment textbook tables and algorithms). We predict that the evidence will suggest that unassisted vIME, where the finer aspects of the examination are less critical, should be limited to certain types of assessment, such as causation of injury, treatment advice and return to work assessment.Our key hypothesis is that the current evidence is unlikely to support performing with or without the assistance of a musculoskeletal‐trained allied health practitioner (AHP) on site with the injured individual. We examined whether certain modern technologies are suitable to be used during assessments of the injured individual alone in a remote location (i.e. home‐based vIME assessment), or whether such tools and technologies require the physical presence of an AHP with musculoskeletal knowledge and positional training to allow the IME practitioner to achieve the required measurement standards of the permanent impairment guides.We examined the conditions under which observational goniometry (i.e. VE), traditional goniometry and modern manual or electronic goniometrical devices and smartphone Apps are consistently accurate . A key consideration is whether there is adequate evidence that the method of measurement can be performed reliably in the vIME setting with clinician guidance, Standard medical examination for orthopaedic conditions requires musculoskeletal measurement to determine patient recovery. Similarly, the extent of permanent impairment is determined by converting a patient's musculoskeletal injury into a ‘percentage of bodily function loss’ to help lawyers, courts and tribunals determine financial compensation. It is the role of doctors to determine the ROMs and plot these into the predetermined ROM algorithms using the AMA guides.A systematic search of papers published from January 2005 to end of May 2020 and limited to English was undertaken within the MEDLINE‐Ovid database.The search terms and their combination were:range of motion/ or range of movement/ or goniomet*/ or visual estimation/ or visual inspection/ or visual assessment/.mpANDremote/ or virtual/ or video/ or mobile application/ or smartphone/ or telehealth/ or telemedicine/ or teleorthopedic/ or telerehabilitation/.mpANDreliability/ or valid*/ or accuracy/ or quality/.titlother types of quality or functional movement‐based assessments (i.e. a lack of focus on joint ROM assessment). Commonly excluded were papers examining the reliability, validity or accuracy of other types of technologies, including 3D motion analysis systems, and any commentary or opinion‐pieces. Full text was sought for those articles still remaining .n = 72), the abstract review of 70 studies resulted in 67 full‐text articles being sourced.The primary MEDLINE database search identified a total of 142 articles. After excluding those not meeting inclusion criteria Observational only or other ROM measurement  that could be conducted Assisted measurement with a musculoskeletal‐trained AHP  on site with the patient, using a measurement tool (goniometer or App).Information extracted and presented includes conditions or moderating factors shown by the results to influence the accuracy/reliability as well as limitations and information pertaining to the suitability of using the tool(s) in the vIME / telehealth setting.Scenario 1:Observational only methods of ROM assessment relevant to the vIME setting performed without the assistance of a musculoskeletal (MSK)‐trained AHP.The data presented in Table Six studies were identified that presented preliminary evidence of validity for tools  suitable for measuring ROM in the vIME  setting. Reliability was not consistently reported to be high. Although suitable for the telehealth environment, it should be noted that all but one of the techniques  smartphone Apps  may not have in‐built technology (i.e. inclination sensors or accelerometer technology) required by the non‐photographic ROM Apps). The majority of studies reported also recommend certain conditions required to maximize accuracy, reliability and/or validity of ROM measurement when using these instruments. Anatomical expertise or experience to facilitate standardized instrument placement , and/or knowledge of anatomy  specialized equipment to aid placement of the device or to hold part of a body in the correct position during the measurement. This often requires substantial initial set‐up time and effort , despite recent technological advances. There is no doubt, however, that such Apps will continue to evolve with the development of further smart phone technology. Moreover, in the instance of permanent impairment assessments, there is the additional risk of unreliability if the claimant is required to facilitate their own assessment in which they have a potential conflict of interest.We have identified the specific circumstances that are likely to maximize the accuracy and reliability of ROM measurement in the vIME setting. There is convincing evidence when assisted by a musculoskeletal‐trained AHP, even for permanent impairment assessment where rigorous physical examination and precise measurement are required. The accumulated evidence suggests that a MSK‐trained AHP (e.g. physiotherapist or accredited exercise physiologist) using any valid goniometric instrument or App would be likely to reach the same conclusions as a highly skilled surgeon, assuming familiarity with the device underpinning the process of successful measurement. In this circumstance, the MSK‐trained AHP would conduct the ROM assessment in the presence of the claimant under direct instruction of the clinician observing the assessment remotely.Our results have provided support for using modern manual or electronic devices, or ROM Apps in the vIME setting The vIME environment does have some limitations that are impossible to overcome. One critical aspect unique to the IME setting is the somewhat subjective assessment of ‘participation’ by the injured individual, which is challenging to observe even in the gold standard face‐to‐face circumstances. Although a MSK‐trained AHP can assist with the measurement aspect, they may not have the training to appreciate these subjective nuances that experienced medicolegal specialists can observe and evaluate. For this reason, MSK‐trained AHP assisted vIME may not be appropriate for permanent impairment judgements where, for example, a neurological examination is required that could lead to an injury category change by some percentage points. Moreover, spine conditions with radiculopathy present a limitation to virtual examination that either requires the examiner to use a different diagnostic technique or a different model for permanent impairment assessment.Our results have provided sufficient evidence that assisted examination represents the best available option when telehealth is required. In this case performed by a suitably trained  MSK practitioner supervised remotely by the IME examiner in a COVID‐19‐safe protocol setting. The importance of such assistance is emphasized in most of the examined literature demonstrating that the patient's position during the examination is critical to maximize the accuracy of angular measurements.assisted remote examination will represent the ‘gold standard’ for Virtual IMEs. When directed assistance is provided by a well‐trained MSK health practitioner using a valid measurement tool, this review demonstrates that those measurements have satisfactory reliability for forensic purposes.The accumulation of evidence supports that ‘Normal’ clinical practices seem a long way off in the midst of this COVID‐19 global pandemic. Potentially placing a qualified assistant at risk of exposure at the patient‐end raises professional ethical considerations surrounding ‘task substitution’; however, we are fast becoming accustomed to employing a COVID‐19 safe environment using detailed safety protocols to avoid exposing health practitioners to unnecessary risk.It is inappropriate to allow injured individuals to participate in their own assessment of loss of physical function using a smartphone measurement device in a system within which there are positive compensation benefits associated with poor participation. With this in mind, the evidence presented suggests that the vIME is likely to be a valid and viable setting for most non‐impairment categories to achieve reliable, accurate and timely assessments of legal importance such as causation, ongoing treatment, or fitness to return to pre‐injury duties. Investigative diagnostic adjuvants such as PACS radiology  also reduce the reliance on face‐to‐face physical examination.Inevitably, the reliability of the vIME will be tested by cross‐examination. Adherence to robust protocols and retention of carefully documented assessment records will be vital for confidence in the reliability of results that are presented.In conclusion, there is sufficient evidence to suggest that assisted medical examination in a vIME setting represents a satisfactory modality for permanent impairment assessments with certain limitations as discussed. The next step is the development of detailed guidelines and protocols to maximize both efficiency and consistency of methodology.Peter Steadman: Conceptualization; supervision; writing‐original draft; writing‐review & editing. Dianne Sheppard: Data curation; formal analysis; investigation; methodology; project administration; writing‐original draft; writing‐review & editing. Janette Henderson: Data curation; formal analysis; project administration; resources; validation; visualization. Brett Halliday: Writing‐review & editing. Ian Freckelton: Writing‐review & editing.PS, DS, JH and BH are all employees of MedHealth Pty Ltd. in Australia. MedHealth is a national provider of IME.Data S1. Eligibility criteria.Click here for additional data file.Figure S1. A detailed timeline that provides an historical ‘snapshot’ of the variety of methods used to assess ROM over the last 120 years.Click here for additional data file.Figure S2. PRISMA flow chart of primary and secondary literature search, screening, selection and inclusion.Click here for additional data file.Table S1. Studies examining the reliability and validity of ROM1 assessments conducted with measurement tools or App that do NOT require musculoskeletal‐trained allied health practitioner (AHP) assistance, including visual estimation.Click here for additional data file.Table S2. ROM techniques and technologies (goniometers or Apps)1 with potential to be administered in the vIME setting with assistance of a musculoskeletal‐trained allied health practitioner (AHP) on site.Click here for additional data file.


Initial use of drugs such as tobacco and alcohol may lead to subsequent more problematic drug use—the ‘gateway’ hypothesis. However, observed associations may be due to a shared underlying risk factor, such as trait impulsivity. We used bidirectional Mendelian randomization (MR) to test the gateway hypothesis.Our main method was inverse‐variance weighted (IVW) MR, with other methods included as sensitivity analyses (where consistent results across methods would raise confidence in our primary results). MR is a genetic instrumental variable approach used to support stronger causal inference in observational studies.n = 1749–1 232 091).Genome‐wide association summary data among European ancestry individuals for smoking initiation, alcoholic drinks per week, cannabis use and dependence, cocaine and opioid dependence  = 1.34; 95% CI = 1.24–1.44; P = 1.95 × 10−14] and cannabis dependence . We also found evidence of an effect of cannabis use on the increased likelihood of smoking initiation . We did not find evidence of an effect of drinks per week on other substance use outcomes, except weak evidence of an effect on cannabis use . We found weak evidence of an effect of opioid dependence on increased drinks per week .We found evidence of causal effects from smoking initiation to increased drinks per week [(IVW): β = 0.06; 95% confidence interval (CI) = 0.03–0.09; Bidirectional Mendelian randomization testing of the gateway hypothesis reveals that smoking initiation may lead to increased alcohol consumption, cannabis use and cannabis dependence. Cannabis use may also lead to smoking initiation and opioid dependence to alcohol consumption. However, given that tobacco and alcohol use typically begin before other drug use, these results may reflect a shared risk factor or a bidirectional effect for cannabis use and opioid dependence. Illicit substance use and substance use disorders result in a substantial global burden on a range of health conditions , 2. IdenrG = 0.35–0.66) [The gateway hypothesis, in its simplest form, is the theory that initial use of legal ‘gateway’ drugs, including tobacco and alcohol, may lead to illicit drug use such as cannabis, cocaine and opioids , 4, 5. P35–0.66) , 25, 26.While these studies may support the gateway hypothesis it is equally plausible that there are underlying shared risk factors; for example, risk‐taking or impulsive behaviours. Previous studies have reported an association of attention deficit hyperactive disorder (ADHD) with substance use outcomes , 28 and Mendelian randomization (MR) is a well‐established method for causal inference based on instrumental variable (IV) analysis, which attempts to overcome issues of residual confounding and reverse causation , 34, 35.We applied this two‐sample MR approach to investigate the possible causal effect between both smoking initiation and alcohol consumption (defined as drinks per week) and substance use outcomes of cannabis use and dependence, cocaine dependence and opioid dependence. We refer to these outcomes as ‘illicit substance use’, although we acknowledge that cannabis is not illegal in all jurisdictions. We also examined the association between smoking initiation and alcohol consumption. We used a bidirectional approach Fig.  to assesWe used GWAS summary statistics obtained from several consortia and other samples, the details of which are shown in Table The smoking initiation GWAS  identifiThe drinks per week GWAS  identifiThe cannabis use GWAS  identifiThe cannabis dependence GWAS  did not The cocaine dependence GWAS  identifiThe opioid dependence GWAS  did not Units for all binary measures were in log odds ratios (ORs) and for the continuous drinks per week measure were per standard deviation (SD) increase in the number of drinks per week.−05.MR was used to assess whether relationships may be causal by using genetic variants as IV proxies for the exposures. Further details can be found in the Supporting information. Two‐sample MR was conducted in R (version 4.0.0)  using thP‐value as a continuous measure of statistical evidence [exposure–SNPoutcome associations for each SNP included in the instrument. If there is evidence of heterogeneity this may indicate possible horizontal pleiotropy.Multiple MR methods were used to assess the causal effects of: (i) the exposure of smoking initiation/alcohol consumption on illicit substance use outcomes and (ii) illicit substance use exposures on smoking initiation/alcohol consumption. These were inverse‐variance weighted (IVW) , MR‐Eggeevidence  and consThe MR‐Egger method tests whether there is overall directional pleiotropy by not constraining the intercept, where a non‐zero intercept indicates directional horizontal pleiotropy. We also used the Rucker's Q‐test to assess heterogeneity in the MR‐Egger estimates for individual SNPs, similar to the Cochran's test. The weighted median method provides an estimate under the assumption that at least 50% of the SNPs are valid instruments (i.e. satisfy the IV assumptions). Finally, the mode‐based approaches provide an estimate for the largest cluster of similar SNPs, where the SNPs not in that cluster could be invalid, with the weighted method taking into account the largest weights of SNPs. Additionally, we estimated effects for single SNP and leave‐one‐out analyses and plotted these results where there was evidence for a causal effect.F‐statistic, unweighted and weighted I‐squared values for each of the analyses [F‐statistic represents instrument strength, where a value under 10 may indicate a weak instrument [I‐squared value falls between 0 and 1 and indicates the amount of bias in the ‘no measurement error’ (NOME) assumption in the MR‐Egger estimate. If bias was apparent, we ran simulation extrapolation (SIMEX) corrections and present these in place of the MR‐Egger results; if the bias was too large, neither were presented (see Supporting information for further details).We also estimated the mean analyses . The F‐sstrument . The I‐sFinally, we conducted multivariable MR (MVMR) to investigate whether the causal effect of smoking initiation was independent of that for the drinks per week exposure for any illicit substance use outcomes where both exposures were associated with the outcome. MVMR is an extension of MR that estimates the causal effect of multiple exposures on an outcome and assesses whether each exposure is independent of the others . Please P‐value = 9.44 × 10−06). The I‐squared values . Results were in a consistent direction among MR analyses . Results were in a consistent direction for the SIMEX‐adjusted MR‐Egger and weighted median methods  or opioid dependence  with any of the MR analyses, except for weak evidence for the SIMEX‐adjusted  for all MR analyses except MR‐Egger. Results were in a consistent direction across MR analyses , cannabis dependence , cocaine dependence  or opioid dependence  on smoking initiation for any of the MR analyses.We did not find any evidence of a causal effect of drinks per week, , although there was some evidence of a causal effect with the other MR analyses. We did not find evidence of a causal effect on cannabis dependence , cocaine dependence  or opioid dependence .When examining whether or not there was evidence for causal effects of alcohol consumption (drinks per week) on the illicit substance use phenotypes , cannabis dependence  or cocaine dependence  on drinks per week.For the reverse direction , although the effect size was very small and this was not found for any other MR analyses . This result was similar to that from the two‐sample MR model. However, there was no evidence of a direct effect of drinks per week on cannabis use .We conducted MVMR analysis for cannabis use only due to evidence of a causal effect of smoking initiation on cannabis use and weak evidence of a causal effect of drinks per week on cannabis use. We found evidence of a direct effect of smoking initiation, independent of drinks per week on cannabis use . As these genetic instruments may overlap, this does not help us to disentangle the reason behind this relationship.We also found evidence for a potential causal pathway from cannabis use to smoking initiation. It has been previously suggested that cannabis use may act as gateway to tobacco use, possibly due to the form in which cannabis is used, i.e. if smoked with tobacco . HoweverThere are several potential reasons for our results: (1) a causal effect of smoking initiation on cannabis use, (2) a causal effect of cannabis use on smoking initiation, (3) a bidirectional effect, (4) an underlying shared risk factor, (5) horizontal pleiotropy  and (6) confounding due to LD. Without further understanding of the biological function of these genetic variants it is difficult to conclude which of these explanations  could be true, and this has been discussed previously in relation to mental health behavioural risk factors , 58. PreWe found a potential causal effect of smoking initiation on increased drinks per week, but did not find an association in the reverse direction. It is plausible that an underlying risk‐taking behaviour may affect alcohol consumption via smoking. However, a biological mechanism behind this association should also be considered and studied further. Finally, we saw weak evidence of a causal effect of opioid dependence on increased drinks per week; however, due to the low power for the opioid dependence GWAS and the small effect size we would interpret this with caution. Opioid dependence (compared with ever use) is less probably explained by underlying risk‐taking behaviour. Therefore, research into alternative shared risk factors is warranted. It may be the case that opioid dependence has a causal effect on increased alcohol use, and this also warrants further investigation.P‐value threshold of 1 × 10−05 due to a low number of genome‐wide significant SNPs. Therefore, these instruments may be less robustly associated with the exposure, and pleiotropy could be introduced. For our results with the dependence variables as exposures the CIs were very narrow, which could be a result of the relaxed P‐value thresholds used. However, the absence of evidence here does not mean we can exclude the possibility of an effect for this relationship. Furthermore, the lower number of SNPs used for the dependence exposures may mean that the instruments are weak, which may be particularly problematic for MR‐Egger. Additional caution should be taken when interpreting this result for our finding of an effect of opioid dependence on drinks per week, as the opioid dependence exposure is a dichotomized variable for an underlying latent risk factor. Thus, the estimate here is less interpretable than for our other results and instead focus should be upon the direction and evidence of an effect as opposed to the effect size. Therefore, our dependence results should be interpreted with caution revisited once larger GWAS become available.Our study is the first, to our knowledge, to examine whether causal pathways may exist between smoking initiation/alcohol consumption and various illicit substance use phenotypes using an MR approach. However, there are several limitations to note; for example, some of our analyses may be limited in their power to detect a causal effect. This is particularly the case where the dependence measures were the exposures, as the GWAS discovery samples were much smaller than those for drinks per week, cannabis use and smoking initiation. Additionally, we used a less stringent I‐squared estimate was low. In cases where the IVW shows evidence for a causal effect but results are inconsistent across the sensitivity analyses, this may be indicative of pleiotropy. However, inconsistent effects across sensitivity analyses and no evidence from the IVW is more likely to reflect a lack of evidence for an effect.We also found some evidence of heterogeneity and horizontal pleiotropy for different analyses, meaning that these results should be interpreted in light of this, as some of the SNPs used may be associated with the outcome other than via the exposure. However, the additional MR analyses, which account for this, were generally in the same direction as our main results, although we were unable to formally test for directional pleiotropy in some cases where the Another consideration is that the MR instruments used may not be valid for smoking, as they may be picking up risk‐taking behaviours more than smoking itself . TherefoP‐values and biasing the MR estimate towards the null. Thus, interpreting the direction of effect as opposed to the effect size itself is more valid here. The effect estimate may also be biased by trait heterogeneity; for example, different aspects of substance use behaviours may be associated with the same genetic variants and therefore it is difficult to gain a precise estimate for a single aspect of any substance use behaviour.The MR analysis itself is subject to several limitations . For exaFinally, our results should be considered in the context of the multiple potential causal pathways that we have investigated.While, to some extent, our findings support the gateway hypothesis they also point to a potential underlying common risk factor, and with better‐powered GWAS or those with more precise instruments and additional research we may be able to interrogate this further. Triangulating our results with other approaches would help to answer this question , 64. By None.Zoe Reed: Data curation, formal analysis, investigation, methodology, resources, software, visualization. Robyn Wootton: Methodology, resources, visualization. Marcus Munafo: Conceptualization, funding acquisition, methodology, project administration; supervision.Table S1 Mean F‐statistic, weighted and unweighted I‐squared results for all of the MR analyses.Table S2 Two‐sample Mendelian randomisation results with smoking initiation as the exposureTable S3 Two‐sample Mendelian randomisation results with smoking initiation as the outcomeTable S4 Two‐sample Mendelian randomisation results with drinks per week as the exposureTable S5 Two‐sample Mendelian randomisation results with drinks per week as the outcomeFig S1 Mendelian randomisation results with smoking initiation as the exposure and drinks per week as the outcome.Fig S2 Forest plot of single SNP analysis using the Wald ratio with smoking initiation as the exposure and drinks per week as the outcome.Fig S3 Funnel plot of individual Wald ratios for each SNP against their precision with smoking initiation as the exposure and drinks per week as the outcome.Fig S4 Leave‐one‐out analysis plot with smoking initiation as the exposure and drinks per week as the outcome.Fig S5 Mendelian randomisation results with smoking initiation as the exposure and cannabis use as the outcome.Fig S6 Forest plot of single SNP analysis using the Wald ratio with smoking initiation as the exposure and cannabis use as the outcome.Fig S7 Funnel plot of individual Wald ratios for each SNP against their precision with smoking initiation as the exposure and cannabis use as the outcome.Fig S8 Leave‐one‐out analysis plot with smoking initiation as the exposure and cannabis use as the outcome.Fig S9 Mendelian randomisation results with smoking initiation as the exposure and cannabis dependence as the outcome.Fig S10 Forest plot of single SNP analysis using the Wald ratio with smoking initiation as the exposure and cannabis dependence as the outcome.Fig S11 Funnel plot of individual Wald ratios for each SNP against their precision with smoking initiation as the exposure and cannabis dependence as the outcome.Fig S12 Leave‐one‐out analysis plot with smoking initiation as the exposure and cannabis dependence as the outcome.Fig S13 Mendelian randomisation results with cannabis use as the exposure and smoking initiation as the outcome.Fig S14 Funnel plot of individual Wald ratios for each SNP against their precision with cannabis use as the exposure and smoking initiation as the outcome.Fig S15 Forest plot of single SNP analysis using the Wald ratio with cannabis use as the exposure and smoking initiation as the outcome.Fig S16 Leave‐one‐out analysis plot with cannabis use as the exposure and smoking initiation as the outcome.Fig S17 Mendelian randomisation results with opioid dependence as the exposure and drinks per week as the outcome.Fig S18 Forest plot of single SNP analysis using the Wald ratio with opioid dependence as the exposure and drinks per week as the outcome.Fig S19 Funnel plot of individual Wald ratios for each SNP against their precision with opioid dependence as the exposure and drinks per week as the outcome.Fig S20 Leave‐one‐out analysis plot with opioid dependence as the exposure and drinks per week as the outcome.Click here for additional data file.


The COVID‐19 pandemic has resulted in unprecedent changes to clinical practice, and as the impact upon delivery of exercise services for people with cystic fibrosis (CF) in the United Kingdom was unknown, this was characterised via a national survey.An electronic survey was distributed to healthcare professionals involved in the exercise management of CF via established professional networks.In total, 31 CF centres participated. Findings included significant reductions in exercise testing and widespread adaptation to deliver exercise training using telehealth methods. Promisingly, 71% stated that they would continue using virtual methods of engaging patients in future practice.These findings highlight adaptation to the COVID‐19 pandemic and the need to develop sustainable and standardised telehealth services to manage patients moving forwards. CFcystic fibrosisMDTmultidisciplinary teamNHSNational Health ServicePAphysical activityUKUnited Kingdom1Participation in regular physical activity (PA) and exercise is beneficial for people with cystic fibrosis (pwCF) and is an integral part of their clinical management. Regular exercise testing and reviews of exercise training programmes are therefore recommended to occur at least annually.Upon the emergence of the SARS‐CoV‐2 coronavirus‐2019 (COVID‐19) pandemic, restrictions were imposed by the U.K. Government to limit transmission and included additional constraints for clinically vulnerable individuals, such as pwCF.As with many services within the National Health Service (NHS), CF multidisciplinary care teams (MDTs) have been forced to adapt and deliver services virtually where possible .Understanding service change has applicability beyond the current pandemic. Given that pwCF are regularly advised to segregate from one another for infection control reasons, and ‘shield’ at home when unwell, any positive or sustainable changes to clinical practice may be appropriate for continued implementation. This online survey therefore sought to identify the impact of the COVID‐19 pandemic upon the delivery of exercise services within CF clinics in the United Kingdom.2Questions asked within this survey formed part of a wider survey related to exercise services in CF MDTs across the United Kingdom , a replication of a previous survey.The survey was distributed via email, by the Association of Chartered Physiotherapists in CF, the U.K. CF and Exercise Technicians Network, and U.K. CF Medical Association to their respective memberships. It was asked that a single member of each MDT  completed the survey on behalf of their site, to ensure a single response per centre. The survey was distributed in January 2021 and remained open for 6 weeks, to maximise the response rate. This survey was hosted using an online platform , chosen because of its compatibility with both computers and smartphones, whilst also ‘whitelisting’ IP addresses for compliance with data protection regulations.This study was approved by the University of Exeter Sport and Health Sciences Ethics Committee (200 708‐A‐01). All respondents provided consent to participate via a series of check‐boxes, confirming they understood the study and were providing information on behalf of their centre.Data are presented as frequency statistics, and free‐text responses are provided to emphasise predominant themes within responses.3n = 31 respondents from across the United Kingdom, representing specialist  and network (n = 7) centres, covering adult (n = 11), paediatric (n = 16) and mixed (n = 4) care centres. In total, n = 27 respondents were physiotherapists  within their respective CF MDTs. The remaining responses were completed by Therapy Assistants/Technicians (n = 2), Exercise Therapist (n = 1) and Exercise Practitioner (n = 1).The survey was completed by The majority of respondents stated the pandemic restricted their ability to undertake exercise testing (97%) and exercise training 71%), with the relative frequency of both being negatively affected  stated that they would maintain some form of telehealth  within their clinical practice  felt they were able to confidently answer these questions. Respondents themselves highlighted a number of questions and comments, across numerous themes, including short‐term and long‐term care for pwCF, guidelines for ongoing practice and the changing nature and direction of engagement with exercise . All respondents provided consent to participate via a series of check‐boxes, confirming they understood the study and were providing information on behalf of their centre.Data S1. Supporting informationClick here for additional data file.


Stachyris strialata) and Nonggang Babblers (S. nonggangensis) in a non‐captive environment in southern China. To our knowledge, these observations represent the first record of interspecific allopreening in the family Timaliidae. We suggest that this understudied behavior is most likely related to the dominant–subordinate relationship between these two species: either the dominant species preening the subordinate species to assert dominance or the subordinate species preening the dominant species to reduce tensions by appeasement. We also suggest interspecific allopreening may not be as rare as we thought if we study this behavior under circumstances where different species are close to each other. This study contributes to our understanding of not only the potential mechanism(s) behind interspecific allopreening but also the behavioral ecology of the vulnerable Nonggang Babbler.Allopreening occurs in many species of birds and is known for providing hygienic and social benefits. While this behavior has been extensively studied among conspecifics, its occurrence among different species remains little known. Outside of captive environments, only a few records of interspecific allopreening exist. In this study, we describe our observations of the interspecific allopreening behavior between Spot‐necked  and Nonggang Babblers (S. nonggangensis) in a non‐captive environment in Guangxi, China. We suggest that this understudied behavior is most likely related to the dominant‐subordinate relationship between these two species. We also suggest interspecific allopreening may not be as rare as we thought if we study this behavior under circumstances where different species are close to each other.Interspecific allopreening is a rare and understudied behavior in birds. In this Nature Note, we describe our novel observations of the interspecific allopreening behavior between Spot‐necked ( This be Cortés, .Stachyris strialata, hereafter “SNB”) and Nonggang Babblers . While SNB occurs from southern China to southern Sumatra , which are bird‐feeding stations built by local people as a form of bird photography tourism.Despite these records, we still lack an understanding of the mechanism(s) underlying interspecific allopreening and the associated allopreening invitation display. Here, we report the first documented record of interspecific allopreening between two gregarious babblers in the family Timaliidae in a non‐captive environment. These species are Spot‐necked  near Nonggang National Nature Reserve in Guangxi, China    , with feather erection and eye pinning. We also observed the SNB nibble many times right after preening the NB, as if it were eating the ectoparasites from the NB.3During our observations, the allopreening from SNB to NB included many self‐accessible body parts such as the flank and breast regions. While allopreening on self‐inaccessible regions such as the head and nape tends to serve a hygienic function, allopreening on self‐accessible regions tends to serve a social function ; investigation (lead); methodology ; writing – original draft ; writing – review and editing . Zhuyang Zhang: Conceptualization ; investigation (supporting); methodology ; writing – original draft ; writing – review and editing .The authors declare no conflicts of interest.


Opuntia ficus‐indica and Euphorbia tirucalli. Using two different algorithms and five predictor sets, we created distribution models for these exemplar species and produced an updated map of global inter‐annual rainfall predictability. No single predictor set produced markedly more accurate models, with the basic bioclim‐only predictor set marginally out‐performing combinations with additional predictors. Minimum temperature of the coldest month was the single most important variable in determining spatial distribution, but additional predictors such as precipitation and inter‐annual precipitation variability were also important in explaining the differences in spatial predictions between SDMs. When compared against previous projections, an a posteriori approach correctly does not predict distributions in areas of ecophysiological tolerance yet known absence . An updated map of inter‐annual rainfall predictability has successfully identified regions known to be depauperate in succulent plants. High model performance metrics suggest that the majority of potentially suitable regions for these species are predicted by these models with a limited number of climate predictors, and there is no benefit in expanding model complexity and increasing the potential for overfitting.Identifying the environmental drivers of the global distribution of succulent plants using the Crassulacean acid metabolism pathway of photosynthesis has previously been investigated through ensemble‐modeling of species delimiting the realized niche of the natural succulent biome. An alternative approach, which may provide further insight into the fundamental niche of succulent plants in the absence of dispersal limitation, is to model the distribution of selected species that are globally widespread and have become naturalized far beyond their native habitats. This could be of interest, for example, in defining areas that may be suitable for cultivation of alternative crops resilient to future climate change. We therefore explored the performance of climate‐only species distribution models (SDMs) in predicting the drivers and distribution of two widespread CAM plants,  Using two different algorithms and five predictor sets, we created distribution models for two example succulent species and produced an updated map of global inter‐annual rainfall predictability. High model performance metrics suggest that the majority of potentially suitable regions for these species are predicted by these models with a limited number of climate predictors, and there is no benefit in expanding model complexity and increasing the potential for overfitting. Many parts of the world are likely to experience warmer climates and reduced and/or more variable precipitation in the decades ahead, so there is interest in determining which organisms may be relatively well adapted to these future climate regimes. A group of plants that are particularly characteristic of warm, semi‐arid parts of the world with strong seasonal rainfall patterns are succulents using the specific mode of photosynthesis known as Crassulacean acid metabolism (CAM). By virtue of being able to fix most of their carbon dioxide from the atmosphere at night rather than during the day time, CAM plants typically show high water‐use efficiency and can survive in environments with high daily temperatures and relatively limited water availability Cushman, . The enva posteriori models  Mill. And Euphorbia tirucalli L. as example species. In doing so, this study attempts to define the best, minimal predictors of plant distribution so that models have the greatest predictive power without being over‐parameterized  have been commonly employed as predictive tools to quantify relationships between species occurrence datasets and measurements of environmental variables   CABI,  and founOpuntia ficus‐indica and Euphorbia tirucalli growth. In doing so, this study aims to first predict the current locations with suitable biotic conditions for the occurrences of O. ficus‐indica and E. tirucalli using different SDMs tested in this study. Second, the results will help identify the most important set of variables that help define the environmental niche of two CAM species of interest. While the natural distribution of both species has generally been restricted to semi‐arid regions as outcompeted by other plants, their natural ecological requirements permit them growing in wetter areas, and competition factors have largely restricted the spread of the species to regions with annual rainfall <500 mm , while E. tirucalli is native to Africa  is commonly considered to provide a measure of overall water availability, a central component to all vegetative growth. Based on global raster data from 1970 to 2000 AD, a global aridity index based upon the implementation of the Penman–Monteith reference evapotranspiration equation . Prior to final environmental parameter selection for each of the five SDM combinations, Pearson’s correlation coefficient tests were conducted to test for covariance between the variables  as opposed to the realized niche   datasets are created as a replacement for true absence records  scores calculated for each of the individual models, the TSS and AUC scores of the ensemble models were compared to determine the relative best performing model and identify whether the additional parameters used in SDMs 2–5 increased the predictive accuracy of SDM 1 (bioclim‐only predictors). As discussed in Komac et al. , the AUC2.6Individual variable importance was approximated using the Variables_importance function of the “biomod2” package  measurements for each of the scenarios. Ensemble results from SDM scenario 1 are presented in Figures O. ficus‐indica and E. tirucalli distribution projections . Among both species and predictor scenarios, all models performed well with overall TSS scores >0.91 across all ensembles , central America and countries in southern and eastern Asia . Meanwhile the areas predicted most suitable for E. tirucalli growth are more restricted to the tropics, especially sub‐Saharan Africa, Brazil and northern South America, India, northern Australia and south China. The higher latitudes and hyper‐arid Sahara were predicted unsuitable for both species.At a global scale, ensemble models across all five predictor scenarios indicated that both species have potential distributional ranges in the tropics and mid‐latitudes. The areas predicted most suitable for O. ficus‐indica growth. By comparison, SDM 4 produces the largest suitable area estimates for the E. tirucalli projections, as well as demonstrating increased estimated suitability values in the continuous dataset for SDM 4 relative to SDM 1.When the deviation in environmental suitability is compared between SDM scenarios Figures  and 6, t3.2O. ficus‐indica and E. tirucalli, the minimum temperature of the coldest month shows a significantly higher variable importance factor than any of the other environmental parameters across the SDM scenarios. Equally, both species show similarity in response to annual precipitation, which demonstrates second greatest individual variable importance, except for when modeled in scenarios 3 and 5—when the Aridity index and R‐index, respectively, show high levels of variable importance and a reduction in the relative importance of annual precipitation.Results from individual variable importance analysis were calculated based on the weighted mean ensemble models for each of the five SDM scenarios and across the two species of interest Tables  and 6. A44.1Results from the ensemble model evaluative performance and individual variable importance analysis suggest that for both species there is not any overall model improvement with the inclusion of either the aridity index, Hellmann–Eberle quotient, cloud cover conditions or R‐index  over the primary four bioclim variables (SDM1); and that the dominant variable of importance in explaining the spatial variability in ecological niche is the minimum temperature of the coldest month. With this in mind, it seems there is little benefit in the inclusion of additional predictors beyond the four bioclim parameters, regardless of which additional parameters were to be considered. With results not differing significantly between the SDM scenario analyses, it suggests that the most important bioclimate predictors (SDM 1) primarily shaped the patterns across all models produced. These results of variable importance are in agreement with von Willert et al.  who consFollowing the role of extreme cold temperatures, moisture availability measured either through annual precipitation or the aridity index or R‐index is shown to be the second most important independent variable on overall model performance. When an alternative precipitation metric is included in the model , the relative importance of annual precipitation is reduced. The compound variable, aridity index, is defined as the ratio between annual precipitation and PET—reflecting the amount of moisture potentially available for vegetation growth. Equally, the R‐index as calculated as the ratio between AET and PET, provides a measure of water supply in relation to water demand Yao, ; unsurprinteractions between the variables or combined effects of the variables—a key tenet of SDM approaches. For example, while cloud cover has in general shown low levels of individual variable importance, Figure E. tirucalli, albeit likely through interactions with other variables. Equally, despite the consistently high TSS values and lack of variability between the different SDM predictor sets studied  across Africa, the potential yields will vary within these locations/SDM projections and hence a combination of both the continuous scale likelihood and the binary cut‐off values is useful in assessing the true scale of potential niche that could be used for growing these species.This being said, while the results in the spatial deviation of individual SDM scenarios from SDM 1 projections Figures  and 6 su4.2O. ficus‐indica and E. tirucalli. Given the overall lack of variability found between the five SDM scenarios and the equally high performance of the bioclim‐only SDM 1 model, the following section opted to only compare the results from the O. ficus‐indica SDM 1 model with existing methods from previous literature focusing specifically on Africa as an example region. Figure O. ficus‐indica growth according to the parameters detailed in Louhaichi et al.  also highlighted the importance of precipitation predictability in the distribution of succulents. As noted above, Ellenberg  examinedBy comparison, the updated Hellmann–Eberle quotient and “Ellenberg index” maps produced using a much longer period of climate data (58 years) in this study have successfully identified regions that are well‐known areas depauperate in succulents, like central Australia and large parts of Kalahari/Namib deserts. Additionally, it is highlighting other areas that agree well with observation—parts of the Arabian Peninsula, Horn of Africa, Saharan desert, and in South America the Atacama. This updated visualization based on a longer time series than previously studied suggests that perhaps high variability in annual precipitation levels over the long term is key to explaining succulent absence, such as the lack of endemic terrestrial species with CAM in arid Australia.5O. ficus‐indica and E. tirucalli when considered alongside existing land uses and primary ecosystems. The high model performance metrics of SDMs made using successfully invasive distribution‐unlimited species gives us confidence that most of the fundamental niche of O. ficus‐indica and E. tirucalli can be explained by the models produced in this study, and given the negligible variability between the different scenarios, there is no benefit in expanding model complexity and increasing the potential for over‐fitting by including additional environmental predictors. While the minimum temperature of the coldest month was found to be the key variable of importance in determining the spatial variability of O. ficus‐indica and E. tirucalli, these results are based on the individual performance of each parameter as opposed to combined effects and nonlinearities between the environmental predictors. An updated global map of Hellmann–Eberle quotient based on a much longer period of climate data (ca. 60 years), supports the ideas of Ellenberg ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Writing – original draft (lead); Writing – review & editing (lead). Andrew J.A.C. Smith: Formal analysis (supporting); Funding acquisition ; Investigation (supporting); Supervision (supporting); Writing – review & editing (supporting). David S. G. Thomas: Funding acquisition ; Investigation (supporting); Supervision (supporting).The authors declare no competing interests.Supplementary MaterialClick here for additional data file.


Binary mesocrystals offer the combination of nanocrystal properties in an ordered superstructure. Here, we demonstrate the simultaneous self‐assembly of platinum and iron oxide nanocubes into micrometer‐sized 3D mesocrystals using the gas‐phase diffusion technique. By the addition of minor amounts of a secondary particle type tailored to nearly identical size, shape and surface chemistry, we were able to promote a random incorporation of foreign particles into a self‐assembling host lattice. The random distribution of the binary particle types on the surface and within its bulk has been visualized using advanced transmission and scanning electron microscopy techniques. The 20–40 μm sized binary mesocrystals have been further characterized through wide and small angle scattering techniques to reveal a long‐range ordering on the atomic scale throughout the crystal while showing clear evidence that the material consists of individual building blocks. Through careful adjustments of the crystallization parameters, we could further obtain a reverse superstructure, where incorporated particles and host lattice switch roles. The self‐assembly of anisotropic nanoparticles into large superstructures is a key component in the research of novel metamaterials. While the formation of mono‐phase mesocrystals has been achieved in recent years, the self‐assembly of binary structures from such particles remained highly challenging. A simple method enables the formation of binary mesocrystals from anisotropic iron oxide and platinum nanocubes in two and three dimensions. This shows that the nanoparticles not only arrange within a highly ordered superlattice, they also orient themselves along their crystallographic planes. The combination of both measurements proves that our superstructures are a nanostructured material which consists of individual nanoparticles with a defined long‐range order on the atomic scale in at least one direction. Based on these results, we further specified the observed binary superlattices to be binary mesocrystals rather than colloidal crystals that do not necessarily have the unique feature of a long‐range particle orientation based on their atomic lattice planes. In addition, we investigated the orientation of the incorporated PtNC with this method as illustrated in Figure 3O4 signals, allow a determination of the particle orientation within a crystallographic reference frame regardless of a provided zone axis as illustrated in Figure The previously described fragmentation of the crystals also enabled TEM imaging alongside the edges of broken crystal pieces, displaying the presence of high‐contrast platinum particles embedded within an IONC matrix. Complementary SAED measurements also show a preferred direction of the wide‐angle diffracted electrons in form of high intensity arcs Figure , which aComplementary to the formation of IONC‐based mesocrystals, which served as the primary host lattice for the binary mesocrystals in this work, we reported on the formation of pure PtNC‐based mesocrystals. It was therefore standing to reason to investigate the possibility of the formation of a “reverse” binary mesocrystal by incorporating IONCs into a PtNC host lattice. The experiments were conducted using the same particles and solvents but inverted particle concentration ratios. We found that the formation of PtNC host lattice binary particle assemblies cannot be obtained at the higher concentration levels at which the IONC host lattice binary mesocrystals were formed. The concentration of the particle mixture had to be significantly reduced by a factor of 5, to avoid mono‐phase formation of pure IONC‐ and pure PtNC‐based mesocrystals separately. We hypothesize here that the reason behind the impossibility to form reverse binary mesocrystals at high particle concentrations lies in the formation kinetic discrepancy between PtNC and IONC as reported in our preceding research.32, 44Nevertheless, the self‐assembly of a less concentrated binary particle mixture resulted in the formation of 3–5 μm sized, uniform superlattices, which are much smaller in size than the compared IONP host lattice binary mesocrystals. As previous research has shown that the obtained mesocrystal size is directly connected to the concentration of the parent particle dispersion,We were able to define the optimal particle size, shape, concentration and stabilizer for a predominant host lattice mesocrystal structure to incorporate tailored particles of another material. Ultimately, the formation of highly ordered binary mesocrystals from anisotropic nanoparticles in two or three dimensions depending on the synthesis method—solvent evaporation and slow gas‐phase diffusion, respectively—could be demonstrated.Furthermore, our observations reveal the importance of individual particle concentrations as a critical role in the development of a binary mesocrystal. We herein propose the concept of a critical particle concentration (CPC) as the driving force towards individual mono‐phase mesocrystal formation at different crystallization times. It appears thus essential that the concentration of the particle type undergoing incorporation into a host lattice must not exceed its CPC otherwise it will lead to a mono‐phase mesocrystal formation considerably earlier than by the host lattice particles hindering the formation of a binary superstructure.While our extended experiments clearly demonstrate the feasibility of the formation of an inverse binary superlattice, they also illustrate that a variety of factors may play a crucial role in their formation as indicated by the presence of particle clusters and the overall mesocrystal habit. As a result, multiple open questions remain, such as the influence of the particle mixing ratio on the binary mesocrystal structure, the transition regime between tetragonal and cubic packing and the possibility to obtain binary and even ternary mesocrystals just to name a few. These topics need to be addressed in future work, but it is already clear that 3D binary mesocrystals open up a completely new range of possible complex multicomponent superstructures with new features, packing orders as well as physical and chemical properties that are yet to be discovered.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The number of herbicide‐resistant weeds differs across the globe but the reasons for this variation are poorly understood. Taking a macroecological approach, the role of six drivers of herbicide resistance in a country was examined for barley, maize, rice and wheat crops worldwide. Drivers captured agronomic measures  as well as sources of sampling bias that result in under‐reporting of herbicide resistance .Depending on the crop, best subset regression models explained between 60% and 80% of the variation in herbicide‐resistant weeds recorded in countries worldwide. Global prevalence of herbicide‐resistant weeds is likely underestimated, especially in countries with limited capability in herbicide research. Numbers of resistant weeds worldwide will continue to increase. Agricultural intensification, captured by fertilizer and herbicide input, as well as further expansion of crop harvested area are primary drivers of future herbicide‐resistant weeds.Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Because the evolution of herbicide resistance lags behind the selection pressures imposed by fertilizer and herbicide inputs, several countries  appear to exhibit a ‘herbicide resistance debt’ in which current agronomic conditions have set the scene for higher numbers of herbicide‐resistant weeds than currently observed. Future agricultural expansion will lead to more herbicide‐resistant weeds, especially in developing countries as their economies grow and where herbicide resistance is currently under‐reported. A global strategy for increasing national capability in herbicide resistance research is needed. © 2022 The Author.  The drivers most frequently included to explain global variation in the number of herbicide‐resistant weeds were those related to biases in sampling effort (time since first record and research effort) suggesting global prevalence of herbicide resistance is underestimated. Among tAlthough agronomic variables may impact herbicide resistance directly, variation among countries could also reflect sampling effects. For a particular crop, it might be expected that the longer the time since herbicide resistance was first recorded in a country, the greater the likelihood that other weed species have also become resistant. This is in part because the first record may reflect a tipping point in crop management associated with increased selection pressure for herbicide resistance,Although there are clear expectations as to how these explanatory variables might influence the number of herbicide‐resistant weeds recorded in different countries, there has been no attempt to tease them apart and determine whether there are consistent national‐scale drivers of herbicide resistance. Such information is crucial to identify those countries that are on a future trajectory towards increased numbers of herbicide‐resistant weeds. The results might also point to the need for national guidelines for herbicide resistance management rather than the decision being left to the individual farmer. Given this background, the overall objective of this study was to use a macroecological approach to explain global variation among countries in herbicide resistance, examine how herbicide resistance varies among different cereal crops worldwide, and to identify the relative importance of agronomic variables and sampling effort in these patterns.22.1www.weedscience.org) on 19 June 2021. Selection of the target crops was based on each having a minimum of 15 countries where the crop was currently grown and in which at least one herbicide‐resistant weed had been recorded. As a result, analyses focused on the four globally important cereal crops: barley (Hordeum spp. and H. vulgare), maize (Zea mays), rice (Oryza sativa and O. glaberrima) and wheat .Data on the total number of herbicide‐resistant weed species recorded in different countries was retrieved from the International Herbicide‐Resistant Weed Databasefao.org/faostat). The FAOSTAT database contains multiple variables describing different aspects of global agriculture including production, agrochemical inputs, trade, agri‐environment indicators, investment and macrostatistics. To avoid overfitting models, an informed, hypothesis‐led approach was adopted for variable selection. A long‐list of socio‐economic and agronomic variables was further trimmed by excluding highly correlated (r > 0.7) variables . This selection process led to a set of four explanatory variables that exhibited relatively low collinearity (Table 2), nitrogen‐based fertilizer input per area of cropland (t km−2), herbicide input per area of cropland (t km−2), and the harvested area (ha) of the specific target cereal crop. Data for each of these variables were averaged over the period 1990–2018 (coinciding with the latest data in FAOSTAT).Socio‐economic and agronomic explanatory variables were either extracted directly or derived from data archived in the Food and Agriculture Organisation FAOSTAT databasety Table : human p10‐transformed to reduce any heteroscedastic biases and improve the linearity of the relationships in the regression models.For each country, the earliest record of herbicide resistance in each of the four cereal crops was extracted from the International Herbicide‐Resistant Weed Database. This date was used to calculate the number of years before 2021 that herbicide resistance first appeared in order to provide a measure of the time since first record of herbicide resistance. To capture research effort on herbicides in each country, the following search term was used: ((herbicide and weed) NOT ‘herbicide resistan*’) across the titles, abstracts and keywords of articles archived in the Clarivate Analytics Web of Science over the same period as the FAOSTAT data (between 1990 and 2018). The Boolean operator excluding articles addressing herbicide resistance from the search aimed to limit the risk of circularity where more research is undertaken on herbicide–weed interactions when there are more frequent occurrences of herbicide resistance in a country. Although searching for terms in English may limit the range of publications examined, it does capture the bulk of international peer‐reviewed agronomic literature.2.210‐transformed) that might lead to variation in the trends encountered. Subsequently, separate best subset regression analyses were undertaken for each target cereal crop using only the countries where the target crop was grown and for which there was a least one record of a herbicide‐resistant weed in that crop. An information theoretic approach to model selection was used to identify the relative importance of the different explanatory variables in the overall variation in the number of herbicide‐resistant weeds found in different countries. Information theoretic model selection using Akaike’s information criterion (AIC) offers significant advantages over standard regression analysis when applied to problems of complex causality that may include nonlinear relationships and collinearity among independent variables.An initial analysis of variance (ANOVA) was undertaken across all four crops to identify any differences in the mean values of the six explanatory variables  and each model ranked in relation to ΔAICc.on Table , the anaR2.The best subset analysis enabled the relative importance of the six explanatory variables to be gauged. The selection of the most parsimonious model among the best subset targeted the model that included the fewest explanatory variables but that also showed low collinearity (variance inflation factor <2) and retained similar predictive power as more complex models as determined by the tenfold cross‐validated 33.1The average number of herbicide‐resistant weed species found per country was low ranging from 3.11 in barley to 6.14 in wheat, but the almost twofold difference among the four cereal crops was of only borderline significance Table . The cou3.2R2 and measures of collinearity than a full model , rice (r = 0.45 p= 0.0012) and wheat . Thus, it is conceivable that population density might act as a proxy for fertilizer input.Fertilizer input was negatively related to the number of herbicide‐resistant weeds in a country for maize, but positively related in wheat Table . The inc3.4R2 values, and thus may provide some insights into future herbicide‐resistant weed risks. For each crop, the top three countries for which predictions overestimated the number of herbicide‐resistant weeds, and thus potentially indicate a higher future risk, were identified using the residuals from the fit of the most parsimonious model: barley , maize , rice  and wheat . Although these countries represent a wide range of climates and levels of economic development, they all have relatively few herbicide‐resistant weeds recorded to date  that is applied.Nevertheless, it should be borne in mind that herbicides and fertilizers are not applied to all cropland at a similar rate, nor are all herbicides used in similar amounts5In summary, despite the evolution of herbicide resistance largely reflecting specific local management contexts,The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Table S1. Pearson correlation coefficients between explanatory variables (log10 transformed) for each of four cereal crops. Values in bold are significant at the p < 0.01 level, while those in italics are significant at the p < 0.05 level.Click here for additional data file.