Small‐scale fisheries are an important livelihood and primary protein source for coastal communities in many of the poorest regions in the world, yet many are overfished and thus require effective and scalable management solutions. Positive ecological and socioeconomic responses to management typically lag behind immediate costs borne by fishers from fishing pressure reductions necessary for fisheries recovery. These short‐term costs challenge the long‐term success of these interventions. However, social marketing may increase perceptions of management benefits before ecological and socioeconomic benefits are fully realized, driving new social norms and ultimately long‐term sustainable behavior change. By conducting underwater visual surveys to quantify ecological conditions and by conducting household surveys with community members to quantify their perceptions of management support and socioeconomic conditions, we assessed the impact of a standardized small‐scale fisheries management intervention that was implemented across 41 sites in Brazil, Indonesia, and the Philippines. The intervention combines TURF reserves  with locally tailored social‐marketing behavior change campaigns. Leveraging data across 22 indicators and 4 survey types, along with data from 3 control sites, we found that ecological and socioeconomic impacts varied and that communities supported the intervention and were already changing their fishing practices. These results suggest that communities were developing new social norms and fishing more sustainably before long‐term ecological and socioeconomic benefits of fisheries management materialized. Article impact statement: Moving from overfishing to sustainability, behavior change campaigns can be used to transition fisheries from near‐term losses to long‐term gains. Small‐scale fisheries provide livelihoods for more than 90% of the planet's fishers  from the Department for International Development DFID . The SLFHousehold survey questions were kept as consistent as possible across sites and countries while still allowing for differences in local social context, language, and institutions. Question framing was highly related to the objectives and specific interventions implemented at each site and were defined in community‐based discussions during the pre‐implementation phase of each intervention. A global monitoring and evaluation team trained campaign managers in sampling protocols and worked with them to tailor survey questions to local contexts while maintaining global consistency. Households were selected randomly from households actively engaged in the intervention target fisheries and were geographically stratified across the community. Surveys were performed with heads of these households; the vast majority of these individuals were males. However, given the broader dependence of these communities on fisheries, socioeconomic changes perceived by these households were also likely noticed to some extent by other community members.For household surveys, we calculated a normalized response score for each indicator scaled from 0 to 100 . For binary questions, a positive response was a 100 and a negative response was a 0. For questions with multiple Likert‐type response options along a gradient , the most positive response was a 100, the most negative response was a 0, and responses in between were given a score scaled from 0 to 100 and proportional to the number of response options. At some sites, multiple questions relating to the same indicator were asked, in which case we averaged values across the normalized indicator scores (Supporting Information). Combining or comparing normalized scores from binary and Likert‐type questions may introduce bias because respondents to Likert‐type responses may chose extreme options less frequently than more central options Guilford . In futuAlthough direct questioning may result in biased responses, deliberate survey design and interview training can reduce some biases Catania , and dirThe sustainable ecosystem survey in Indonesia and the Philippines is a standard coral reef monitoring underwater visual survey that measures biomass of target fished families within the TURF, both inside and outside the NTZ . Structural equation modeling integrates several multivariate analyses to test hypothesized relationships among both manifest indicators  and latent indicators  Grace . Becauseχ2 values of the model with a null model in which all indicators are uncorrelated.We ran the model in the lavaan package in R Rosseel  with a pAlthough there was heterogeneity across sites, some patterns emerged when looking at the precision‐weighted estimates at the country level Fig. . All comThere was again variability across individual sites, but certain patterns emerge when looking at country‐level precision‐weighted estimates Fig. . BiomassOverall the SEM performance (CFI = 0.908) met the desired threshold of 0.9. The model suggested that community support is a strong predictor of management participation, enforcement, and catch reporting, but not of NTZ compliance or TURF compliance Fig. . EnforceFor all 3 countries, positive impacts on all community‐support indicators suggested that the Fish Forever intervention created the social conditions needed for shifting behavior toward more sustainable fishing practices. We saw this result despite neutral or negative trends in several sustainable‐ecosystem and sustainable‐livelihood indicators, suggesting that behavior change interventions can build community support and social norms even while communities incur short‐term costs and before tangible results materialize. Before and after impact trends of the intervention were generally positive or neutral for ecosystem and sustainable‐livelihoods indicators, with the exception of social trust in the Philippines and perceived 5‐year catch trends in Indonesia, which both decreased. Limited counterfactual analyses in the Philippines showed that biomass improved inside and outside the NTZ relative to the control sites, whereas political trust was the only sustainable livelihoods indicator that improved relative to control sites. The intervention sites in the Philippines did not perform as well as the control sites in terms of food security, collective efficacy, and social equity. This provides complementary information to the results of the before–after analysis, which showed positive or neutral responses for these indicators. These negative results may be partially driven by near‐term socioeconomic costs and disruptions from marine reserves and more restrictive fisheries management at intervention sites Christie . ContinuPositive changes were seen across knowledge, attitudes, and interpersonal communication indicators. Changes across this spectrum are illuminating and point toward changing social norms because knowledge alone is not enough to drive behavior change  (Appendix S1), a summary of all questions used across surveys (Appendix S2), a summary of which questions were asked multiple ways (Appendix S3), the actual survey instruments (Appendix S4‐S20), and a complete list of control‐site matching attributes and scores for each attribute at matched control and intervention sites (Appendix S21) are available online. The authors are solely responsible for the content and functionality of these materials. Queries  should be directed to the corresponding author. All data and the R code used in these analyses are available from https://github.com/emlab‐ucsb/fisheries‐behavior‐change.Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


A 66‐year‐old man with a 2‐year history of suspected scalp eczema with excessive dandruff developed painful swollen joints in the extremities. Four months after developing polyarthritis and polydactylitis, eczema gradually spread to the face. He was referred to our hospital for intractable scalp and facial eczema and polyarthritis. Based on the appearance of the head and facial skin lesions, psoriasis was suspected. Treatment with apremilast (a phosphodiesterase‐4‐inhibitor) was initiated, which swiftly alleviated the skin lesions. The joint deformities persisted, but the pain in the joints disappeared. This case implies that psoriatic arthritis should be suspected even if psoriatic skin lesions are localized to the scalp. Psoriatic arthritis was observed in a middle‐aged man with the skin lesions localized to scalp area. Because a delayed diagnosis may result in irreversible joint deformity, close collaboration with dermatologists is essential for early diagnosis of psoriatic arthritis in patients with localized skin lesions remote from the site of arthritis or dactylitis. Some patients with psoriasis show localized skin lesions on the scalp, known as scalp psoriasis. In this report, we present the case of a middle‐aged man with scalp psoriasis, who later developed polyarthritis and dactylitis without skin lesions other than scalp area.2A 66‐year‐old Japanese man with a large amount of dandruff who was suspected to have scalp eczema and had been treated by a general practitioner with an anti‐inflammatory ointment for 2 years gradually developed painful swollen joints in his right hand, right knee, and bilateral ankle joints. He experienced difficulties in walking because of the severe joint pain in his legs. Because he tested negative for serum rheumatoid factor (RF), he was given a presumptive diagnosis of gout and was treated with nonsteroidal anti‐inflammatory drugs, which partially alleviated his joint pain. About 4 months after the development of polyarthritis and dactylitis, the scalp skin lesions gradually spread to his face. He remained free from any skin lesions besides those on the face and scalp. At this stage, the patient was referred to our hospital for intractable arthritis and dactylitis with joint deformities. The manifested skin lesions and arthritis together with the clinical course of this patient is shown in Figure 3Psoriasis is a chronic inflammatory skin disease with genetic and environmental predisposing factors. Most patients with the disease develop well‐demarcated erythematous plaques with overlying silvery‐white scales, typically involving the scalp, trunk, buttocks, and extensor surfaces of the extremities.PsA is one of the most common complications associated with psoriasis and occurs in 5%‐30% of patients.Topical treatments for psoriasis include steroid ointment and vitamin D analogs. Systemic treatments for psoriasis include ultraviolet B radiation; oral administration of cyclosporin, retinoids, or apremilast; and injection of monoclonal antibodies. Patients with PsA are often treated with nonsteroidal anti‐inflammatory drugs (NSAIDs), oral disease‐modifying antirheumatic drugs , or the injection of monoclonal antibodies . Oral administration of apremilast is also a useful therapeutic strategy in these patients.This case highlights the importance of careful examination including scalp to diagnose psoriatic arthritis and dactylitis. Close collaboration with dermatologists is essential for early diagnosis and prompt management of PsA in patients with localized skin lesions.4None.5The authors have stated explicitly that there are no conflicts of interest in connection with this article.6Written informed consent was obtained to publish this case report.


We demonstrate that a molten globule‐like (MG) state of a protein, usually described as a compact yet non‐folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, BSA hydrogels made from MG‐BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below the MG‐state value dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR‐active stearic acid derivatives , the MG‐state based hydrogel is the least long‐lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules. These bio‐ and polymer‐physically unexpected findings may lead to new bio‐compatible MG‐based hydrogels that display novel properties in comparison to conventional gels. Hydrogels based on proteins or synthetic molecules are good candidates for this task since one of their main common features is biocompatibility. Their high water content and soft nature make them similar to natural extracellular matrices and minimize tissue irritation and cell adherence.There is substantial debate on its nature, ranging from MG states either reflecting a general intermediate in protein folding to being unique for every protein or simply a low‐energy misfolded species. In this work, further complexity is added to the current discussion around MG states by demonstrating that molten globule‐like state of protein, BSA, can be preserved in hydrogels, formed by the protein itself. In the context of our paper, one may define that molten globules are partially folded conformations of proteins that have near native compactness, substantial secondary structure, slowly fluctuating (little detectable) tertiary structure and an increased solvent‐exposed hydrophobic surface area relative to the native state.2In the following, the presence of the molten globule‐like states both in acidic and basic pH ranges of bovine serum albumin (BSA) are investigated in high concentration environments with the help of continuous‐wave electron paramagnetic resonance (CW EPR) spectroscopy. We first develop the spectroscopic analysis for a 5 wt% albumin solution (below the gel point) and then, for the first time, describe the same experiments performed with BSA hydrogels at high concentrations (20 wt%). We present evidence that the MG state observed in individual proteins persists in the gel state of the protein and in fact dominates its properties.In case of human serum albumin (HSA), there is a molten globule state, which can be obtained under acidic conditions at pH 2.0.aiso) peaks of freely tumbling 16‐DSA appear from pH 11.29 onwards and the intensity of these peaks increases with increasing pH, while the broad spectrum of albumin bound 16‐DSA (indicated by the apparent hyperfine coupling constant Azz′ in the spectra) decreases in intensity. As explained in detail in Reference Figure Azz′ is a spectroscopic determinant of the polarity of the molecular environment.Azz′ decreases. Therefore, the point in Figure Azz′ is at its minimum can be associated with the molten globule state.Azz′ to the MG state is that the minimum of Azz′ also denotes the maximum rotational mobility of the spin probes. This stems from the fact that for fast and isotropic rotations of nitroxide spin probes, the apparent spectral separation of the outermost extrema (Azz′) converges towards its minimum value and approach the typical three‐line pattern. Hence, when rotational mobility increases at the MG state, these spectral features move closer together, as it can be seen in Figure On the other hand, the value of apparent hyperfine coupling constant Azz′, with a broad minimum centered at around pH 11.80 ± 0.2 and a noticeable increase after that. It should be noted, though, that the drop in the apparent hyperfine coupling when increasing pH is much shallower than that observed for the dilute BSA solutions (ΔAzz′ ~ 2.0 G in gels vs. ~3.0 G in dilute solution). These results suggest there could be a molten globule like state in the hydrogels. This further suggests that the functionality of the protein is mostly preserved during the electrostatically triggered method of the BSA gelation and even though the binding, configurations and even the macroscopic phase of the whole system is changed, the delicate state of the molten globule survived the process  and the second stage was the repetition of the same measurements after approximately 4 hr (t = 4 hr). The difference between these spectra revealed an unexpected characteristic  minimal state in these rather harsh conditions (pH > 11), seems robust enough to even persist the regime of strong intermolecular interaction posed by the gel state.To further substantiate this remarkable EPR‐derived conclusion, we expanded our research portfolio. As stated above, in literature there are different ways to verify the MG state in proteins. However, most of them are not applicable in this research work. IR spectroscopy cannot be used, as the pH range needed (above 10) is higher than the allowed pH limit of ATR‐IR spectrometers' crystals. Moreover, viscosity measurements and rheological characterizations in general cannot be employed. Here, we are working with very high protein concentration and pH ranges. Accordingly, the gelation is a matter of seconds even at low temperatures . Therefore, with these standard rheological methods it is almost impossible to get meaningful data of the initial stages of the gelation process. Furthermore, the data collected for low concentration media  show no We aimed at exploring transmission electron microscopy (TEM) on lower concentration samples at pH 11.8 but the images are not conclusive and do not give additional information .Azz′ in EPR spectroscopy, we can conclude that BSA molecules in the gel with 16‐DSA at pH ~11.65 and in the gel without 16‐DSA at pH ~11.4 are in the MG state. This would mean that the MG state does not only survive the gel‐formation process and the gel state but it also seems to dominate the properties of the gel compared to non‐MG state gels.It may sound trivial, but one may also emphasize the importance of simple visual inspection of samples. In this case, we observed that the gelation process for all the samples  state. Figure Azz′~ is larger again, indicating stronger immobilization) which may again stabilize secondary structure elements and such, indirectly, the gel. Amphiphilic FAs may also form hydrophobic bridges from the less exposed hydrophobic areas between BSA molecules and consequently stabilize these gels as shown in Figure One may further conclude that when BSA is not in the MG state, residual classical binding sites for FAs are again available  conformational state.As depicted schematically in Figure Being compacted, the intermolecular network formation between individual albumins can take place despite the large negative charge that the molecules have at this pH , these hydrogels are water soluble before the leaching process3In conclusion, the existence of basic molten globule states for BSA hydrogels (and the non‐existence in the acidic range hydrogels) has been shown with the help of CW EPR. This method takes advantage of the fact that there are more exposed hydrophobic patches in the MG state in comparison to the native or unfolded states that can then be detected by CW EPR spectroscopy on amphiphilic fatty acid derivatives (16‐DSA). The MG state hydrogel without this fatty acid (pH 11.4) is the maximum stability point for the gel state Figures  and S4 tTo the best of our knowledge, the presence of the molten globule‐state in the high‐crowding environment of a hydrogel is proposed in the present research for the first time. Furthermore, we made the observation (in independent measurements by different experimenters) of enhanced gel stability at the special pH value of the MG‐state. These results indicate a new path for experimental studies of MG states in proteins  and given the broad spectrum of dynamic protein structures, reaching from globally folded via MG to intrinsically disordered states it may be of interest to test the persistence of many MG state proteins at high crowding conditions. By addition of equimolar amounts of other amphiphilic molecules like long‐chain fatty acids, one may even apparently fine‐tune of intermolecular interactions and thus the macroscopic gel properties. Even though the pH values needed for BSA MG hydrogels are too high to be of direct practical use, one may envisage new biocompatible materials (probably from proteins in MG states that are induced at less harsh conditions) with unique properties which can have practical use in biomaterials science such as in drug delivery applications and beyond. From a fundamental protein biophysical point of view, it is remarkable that the enigmatic molten globule state can apparently be very robust and not only persist in the high concentration environment of a hydrogel but even dominate the gels nanoscopic and macroscopic properties.4Our experimental strategy is based on co‐dissolution of BSA and hydrophobic 16‐DOXYL‐stearic acid spin probes  with an accuracy of ∼0.2°C. The exact frequency was recorded using a frequency counter .The parameter used for EPR measurements included microwave frequency of approximately S. Arabi: Data curation; investigation; methodology; visualization; writing‐original draft; writing‐review and editing. Behdad Aghelnejad: Data curation; formal analysis; investigation; validation; visualization; writing‐original draft; writing‐review and editing. Jonas Volmer: Data curation; formal analysis; investigation; validation; writing‐original draft. Dariush Hinderberger: Conceptualization; formal analysis; funding acquisition; investigation; methodology; project administration; resources; supervision; visualization; writing‐original draft; writing‐review and editing.Data S1 Additional EPR experiments at high and low pH values, TEM micrograph and viscosity measurements.Click here for additional data file.Video S1 Short video showing the robustness of the MG‐state hydrogel.Click here for additional data file.


AbDesign, to exploit the natural modularity of many protein families and computationally assemble a large number of new backbones by combining naturally occurring modular fragments. This strategy yielded large, atomically accurate, and highly efficient proteins, including antibodies and enzymes exhibiting dozens of mutations from any natural protein. The combinatorial backbone‐conformation space that can be accessed by AbDesign even for a modestly sized family of homologs may exceed the diversity in the entire PDB, providing the sub‐Ångstrom level of control over the positioning of active‐site groups that is necessary for obtaining highly active proteins. This manuscript describes how to implement the pipeline using code that is freely available at https://github.com/Fleishman‐Lab/AbDesign_for_enzymes.The functional sites of many protein families are dominated by diverse backbone regions that lack secondary structure (loops) but fold stably into their functionally competent state. Nevertheless, the design of structured loop regions from scratch, especially in functional sites, has met with great difficulty. We therefore developed an approach, called  Mutations that are rarely observed in the homologs are eliminated from the design options, thus guiding the atomistic design calculations towards implementing potentially stabilizing long‐range interactions, such as those between the CDRs and the framework. Furthermore, by eliminating rarely observed mutations, AbDesign reduces the chances of protein misfolding which has been a major obstacle in protein engineering and design.AbDesign approach thus generates a potentially very large number of backbones through the combination of naturally occurring ones followed by sequence design calculations to stabilize the entire protein including its active site. By controlling the fine details of the active‐site backbone, this approach provides the accuracy that is essential for encoding precise molecular recognition of substrates.Inspired by this natural approach to generate backbone diversity through the assembly of modular parts, we developed AbDesign relative to fixed‐backbone design strategies: (a) the combinatorial expansion of backbone‐conformation space. Consider a family of homologous proteins comprising 20 nonredundant structures that can be segmented into four modular parts. Through modular assembly, we can obtain 204 = 160,000 unique backbone conformations, exceeding the number of nonredundant structures in the PDB; and (b) optimizing the sequence using a constrained space of amino acids commonly observed in homologs. The resultant sequence subspace is likely to fold stably into the target conformation. AbDesign produced new atomically accurate antibodies.To recapitulate, there are two main methodological advances in AbDesign can be extended,AbDesign's generality by using it to automatically design new backbones for two unrelated TIM‐barrel fold family enzymes: glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase‐like lactonases (PLL). Some designs exhibited strikingly different substrate specificities and high activity levels comparable to natural enzymes while differing from any known protein by more than 100 mutations, insertions and deletions. Remarkably, these enzymes were thermally stable and highly active without requiring iterative experimental mutagenesis and screening as has been the norm for protein‐design methodology.AbDesign thus opens the way to the application of protein design methodology to outstanding challenges in the design of protein function where sub‐Ångstrom accuracy is often a requirement. In this Tools contribution, we outline the steps that are required to apply AbDesign to a target enzyme family, noting that the same steps can be used, with necessary changes, to design binders and other proteins.2In the sections below, we describe the steps needed to design a repertoire of enzymes exhibiting a diversity of backbones using the TIM‐barrel fold glycoside hydrolase 10 (GH10) xylanase family as a representative example Figure . We note2.1We select a protein family of interest and download all available structures from freely available resources, such as the Pfam database of protein families.2.2AbDesign are carried out in torsion space only; thus, bond lengths and angles are not optimized during any of the calculations. Experimentally determined structures often exhibit deviations from ideal bond lengths and angles, and while some such deviations may be important structurally or functionally, these deviations are often due to the uncertainty of structure determination from low‐to‐medium resolution X‐ray crystallographic data. To reduce the noise in energy evaluations due to such deviations from ideal bond lengths and angles, we start by subjecting all of the structures to the Rosetta idealize procedure,All of the design calculations in 2.3Next, we generate backbone‐conformation databases for each of the segments by segmenting the original structure files (PDB format) into fragments at the segmentation points. One of the structures of the protein family is arbitrarily designated to be the template (PDB entry 3w24 for GH10s)—the one according to which all other structures will be segmented. For that, we first align all structures to the template; then we extract from each structure all of the fragments.To further refine the backbone database and promote accurate backbone assembly, a custom PyMOL script aligns all fragments belonging to the same segment such that the ends of the fragments are positioned identically, improving the seamless assembly of fragments into full‐length backbones. We note that for protein families with many representatives in the PDB, it may be beneficial to cluster the fragments and select a single representative for each cluster, thus reducing computational cost without compromising the backbone diversity Figure . For the2.4AbDesign approach uses position‐specific sequence constraints (based on PSSMs) for each backbone fragment based on the sequences observed in structurally homologous fragments. PSSM generation starts by assembling a multiple sequence alignment (MSA) based on the homologs. Sequences are typically extracted from the nr sequence databaseThe 2.5AbDesign, is computationally very demanding. Modular assembly enables precomputing a relaxed conformation for each fragment, saving these conformations in a database, and quickly combining them during backbone assembly. The precomputation step enables the sequence design and ranking of hundreds of thousands of unique backbones. During precomputation, each segment of the template protein is removed one at a time, and each of the structurally aligned fragments from each homolog is relaxed using cyclic‐coordinate descent (CCD) backbone relaxationAbDesign allows precomputing these tails with only one constraint. To facilitate refinement, we enable sequence optimization (constrained by the PSSMs) in the modeled fragment and its spatial vicinity. At the end of relaxation, we automatically verify that the backbone mainchain is continuous and that the root‐mean‐square deviation (rmsd) to the segment as observed in the source structure is low (<0.2 Å) before entering the segment's backbone torsion angles in the database.Relaxing large backbone fragments, such as the ones used in 2.6AbDesign automatically changes the PSSM constraints to reflect the newly assembled structure. Then, the new fragment and its spatial vicinity are sequence‐optimized for the backbone structure subject to these PSSMs. For GH10 xylanases, 3,000 backbones were generated, but in cases where the number of fragments in each segment is modest, it may be best to enumerate all possible backbones to ensure that the most optimal ones are selected for further analysis. When backbone design is complete and all segments are replaced with fragments from the database, we use PROSS stability designThe design process starts with the template structure, then replaces the different segments with fragments from the precomputed fragment databases. Whenever a fragment is replaced, 2.7The backbone design algorithm described above may generate hundreds of thousands of unique designs, and a subset is chosen for experimental characterization according to the experimental screening capacity. In the designed enzyme set described in Reference x is the value of the criterion  and μ and σ are the mean and standard deviation of the criterion in the design ensemble, respectively. The fitness, f, asymptotically approaches 1 at negative x values and 0 at positive x values. Undesirable features, such as binding energy to off‐target substrates, may be encoded by simply negating x. We can further combine several such fitness scores, each expressing a different feature, into an optimization objective function by multiplying the individual fitness terms:When applying several criteria in design selection, strict cutoffs are likely to drastically decrease the pool of designs. Instead, one may combine the different criteria using a “fuzzy‐logic” expression.Odesign=∏x∈featuresfxDesigns with objective‐function scores close to 1 find a favorable compromise among all of the features, whereas designs with scores close to 0 exhibit at least one poor feature. Examples for using fuzzy‐logic design are discussed in References 3Until recently, the control that computational design methods exercised over the protein backbone degrees of freedom was quite limited. The recent breakthroughs in the application of de novo protein design methodology to increasingly complex problems are very encouraging,AbDesign algorithm leverages the fact that many functionally versatile protein folds are modular—likely because modularity facilitates the evolution of new activitiesAbDesign to recombine fragments from non‐homologous proteins to generate new backbones and polar interaction networks in binding pairs.AbDesign provides a practical route to designing large‐scale changes in natural enzymes and binding proteins.The de novo enzyme design has so far resulted in enzymes that exhibited low efficiency and required further optimization by directed evolution.Backbone design in catalytic pockets and binding surfaces paves the way to address long‐standing challenges in protein design. For instance, 4https://github.com/Fleishman-Lab/AbDesign_for_enzymes. Backbone clustering was performed using MaxCluster,.All design calculations were performed using Rosetta, freely available for academic and non‐profit users. Scripts, files, and command lines to generate a backbone repertoire can be found at Rosalie Lipsh‐Sokolik: Conceptualization; data curation; formal analysis; investigation; methodology; software; validation; visualization; writing‐original draft; writing‐review and editing. Dina Listov: Data curation; formal analysis; investigation; methodology; validation. Sarel Fleishman: Conceptualization; funding acquisition; investigation; methodology; project administration; supervision; validation; writing‐original draft; writing‐review and editing.


A range of immunomodulatory therapies have been proposed as adjuncts to conventional antivirals to suppress harmful inflammation during severe influenza infection. We conducted a systematic review to assess available data of the effect of adjunctive non‐corticosteroid immunomodulatory therapy and potential adverse effects.We searched MEDLINE, Embase, Web of Science and clinical trial databases for published and unpublished studies, and screened the references of included articles. We included RCTs, quasi‐RCTs and observational studies of virologically confirmed influenza infections in hospitalised patients. We did not restrict studies by language of publication, influenza type/subtype or age of participants. Where possible, we pooled estimates of effect using random‐effects meta‐analysis models.We identified 11 eligible studies for inclusion: five studies  of passive immune therapy; four studies  of macrolides and/or non‐steroidal anti‐inflammatory drugs (NSAIDs), one RCT of mTOR inhibitors , and one RCT of statin therapy . Meta‐analysis of RCTs of passive immune therapy indicated no significant reduction in mortality , but better clinical outcomes at Day 7 . There was a significant reduction in mortality associated with macrolides and/or NSAIDs .Passive immune therapy is unlikely to offer substantial mortality benefit in treatment of severe seasonal influenza, but may improve clinical outcomes. The effect of other immunomodulatory agents is uncertain, but promising. There is a need for high‐quality RCTs with sufficient statistical power to address this evidence gap. It is estimated to cause more than a billion infections annually, with three to five million severe illnesses and 250 000‐650 000 deaths.Recommended antiviral treatments of severe seasonal influenza are currently limited to the neuraminidase inhibitors (NAIs).An effective immune response to the influenza virus following infection is necessary for viral clearance and recovery from infection. Viral shedding is prolonged in immunocompromised patients with influenza, associated with an increased risk of emergent NAI resistance, and secondary bacterial infections.But in a delicate balance, this immune response to an infection can also be harmful to the host. For example, an excessively pro‐inflammatory cytokine and chemokine environment has been cited as the key explanation for the severity of human infections with highly pathogenic avian influenza, and the 1918 H1N1 “Spanish flu” pandemic.A range of immunomodulators for severe influenza have been proposed,I2 = 68%; 15 studies).A Cochrane review in 2017 found moderate‐quality evidence that corticosteroids also reduce mortality when used in severe community‐acquired pneumonia .The recent 2018 Infectious Diseases Society of America (IDSA) seasonal influenza guidelines do not recommend any immunomodulatory therapies as adjunctive treatments.This systematic review was commissioned by the World Health Organization (WHO) to inform the development of clinical practice guidelines for severe influenza. It aims to provide a comprehensive and up‐to‐date assessment of the available data investigating the clinical effectiveness and safety of non‐corticosteroid immunomodulatory therapy adjunctive to conventional antiviral medication for the treatment of severe influenza.2This systematic review and meta‐analysis was conducted in accordance with the PRISMA Statement 2009.Only studies conducted in humans with a virologically confirmed influenza infection  were included, but without restriction on age or sex. There was no restriction on date or language of publication. We included randomised trials, quasi‐experimental and observational studies published in academic, peer‐reviewed literature. Population studies and studies with fewer than 10 participants were excluded. For studies with an observational design, only studies that attempted to adjust for differences between groups in disease severity and/or propensity to receive the immunomodulatory therapy were included.Clinicaltrials.gov and the WHOs International Clinical Trials Registry Platform (ICTRP) were also searched for ongoing clinical trials, and data from these studies were included if the study was completed and results were available from online sources such as the clinical study report. Web of Science was used for citation searching by collating the bibliographies and citations of included studies to identify additional studies which may be eligible.We included studies of any of passive immune therapy, macrolides, mTOR inhibitors, non‐steroid anti‐inflammatory drugs (NSAIDs) or statins. Comparator groups were those who received antiviral therapy or supportive care alone.Our primary outcome of interest was mortality, and secondary outcomes were severity of illness , duration of hospital admission, serious adverse events, duration of viral shedding and emergence of resistance.Studies were selected in two stages: first review of study title and abstract, followed by analysis of the full text of the article. Each study was independently reviewed by two authors, and disagreements were resolved by discussion . Data from studies to be included in the review were then independently extracted by these two review authors using a standardised data collection form developed and piloted for this systematic review (Appendix ).Two review authors independently assessed the methodological quality of included studies . RCTs were assessed using the Cochrane Risk of Bias (RoB 2.0) tool, while non‐randomised studies were assessed using a Newcastle‐Ottawa Scale (NOS) modified for the purposes of this review (Appendix ).Mortality data from individual studies were tabulated and odds ratios (OR) with 95% confidence intervals (CI) calculated using Review Manager 5.3.I2 statistic was used to assess heterogeneity across experimental and observational studies. An I2 value >75% was inferred to reflect substantial heterogeneity between the findings from the studies.The Outcome data from observational studies were aggregated using a random‐effects meta‐analysis model to pool data to reflect expected differences in the measured effectiveness of adjunctive therapies—due to differing patient characteristics, interventions and outcome definitions. Data from different therapies and study designs  were aggregated separately and combined when considered appropriate.The five Grading of Recommendations, Assessment, Development and Evaluation (GRADE) considerations  were used to assess the quality of evidence from the studies that contribute data to the meta‐analyses for the pre‐specified outcomes. Reasons for the decision to downgrade or upgrade the quality of studies were provided.3The search strategy was implemented on 25 January 2019, and identified 5928 articles after removal of duplicates   and after last hospital discharge (P = .029). This analysis was based on a 6‐point ordinal scale with outcomes scaled categorically from death, to ICU admission, to hospitalised with or without supplementary oxygen, to not hospitalised with or without resumption of normal activities. Using these clinical outcomes reported by Beigel et alP = .019) using proportional odds logistic regression, where an OR >1.0 indicates a more favourable outcome with passive immune therapy.Beigel et alP = .33) and 1.22 , respectively. A post hoc meta‐analysis of Day 7 clinical outcomes from these 3 studies indicated overall significant clinical benefit from passive therapy . No transfusion‐associated side effects were described. The incidence of SAEs was similar in the studies from Beigel et alIn the study by Beigel et al,3.2Two studies investigated the effect of macrolide therapy alone: one prospective cohort studyThe cohort study enrolled adults admitted to the ICU with primary viral pneumonia and pandemic A/H1N1‐2009 influenza. Macrolides administered were clarithromycin , azithromycin  and erythromycin . Experimental and control groups were similar in age and gender, but were more likely to be pregnant, immunosuppressed or have COPD. They were also more likely to receive adjunctive corticosteroid therapy, but had lower APACHE II scores and were less likely to have a haematological disease.Lee et alP = .5).There were no deaths recorded by Lee et al.Subgroup analysis by Martin‐Loeches et alP = .009) and had shorter hospital stays (P < .0001) compared to the group who received standard care. Virus titre and the number of patients with a proportion of NAI‐resistant quasispecies viruses >5% were also lower in the intervention group.In the study by Hung et al,Lee et alP = .04). Admission to ICU and length of hospitalisation was not significantly different between treatment groups, and no cardiac, renal or gastrointestinal side effects were reported.Hung et al3.3One open‐label RCT studied the effect of the mTOR inhibitor sirolimus in the treatment of patients critically ill with influenza in ICU Table .34 ThirtP < .05).In the study by Wang et al,P = .45).Wang et al3.4One unpublished blinded, placebo‐controlled RCT of adjunctive statin therapy was identified.P = .611). However, a significant improvement in symptom score over this time period was described in the atorvastatin treatment group (P = .029). Incidence of AEs was similar between treatment groups (6.8% vs. 8.8%), and no SAEs occurred.No deaths were reported in this study in either treatment group. For the study primary endpoint, no significant difference between experimental and control groups in the change in IL‐6 levels from baseline to 72 hours was reported .GRADE assessments of the certainty of evidence were conducted based on summary effect data from RCTs only. Certainty of evidence was graded as low for passive immune therapy, due to imprecision and uncertainty surrounding the applicability of evidence in individuals with severe influenza at highest risk of mortality and imprecision. The two largest clinical trials . One additional RCT of the macrolide clarithromycin was also identified but at the time of review had not yet been initiated.4The systematic review identified eleven completed studies of non‐corticosteroid immunomodulatory therapies, including some high‐quality RCTs. The results of these studies indicate there is unlikely to be a substantial mortality benefit from passive immune therapy as an adjunct to conventional antiviral therapy for the treatment of severe seasonal influenza. Currently, there is insufficient evidence to recommend routine administration of any of the reviewed adjunctive therapies; however, the results from study of macrolides and NSAIDs warrant further study in well‐designed RCTs.The highest quality evidence uncovered through this systematic review was for the effect of passive immune therapy on severe influenza mortality. Our pooled estimate after including two recently completed and relatively large RCTs indicated it is unlikely to be of benefit in reducing mortality in most situations. This conclusion is different from previous reviews of passive immune therapy, but exclusion of non‐randomised and uncontrolled historical studies conducted during the 1918 pandemic suggests our summary effect estimate is more likely relevant to treatment of influenza today. One further study of passive immune therapy is expected to report results within the next year. This is a phase 2 dose‐ranging study with pharmacologic and safety primary endpoints, which recruited 65 individuals, and its results are unlikely to substantially alter the conclusions of this review.There was statistically significant evidence of clinical benefit from passive immune therapy in the post hoc meta‐analysis of Day 7 clinical outcomes. Outcomes in this analysis are ordered on an 6‐point categorical scale from death to “not hospitalised with resumption of normal activities.” This endpoint has been adopted for a number of ongoing phase III clinical trials of influenza antivirals,The summary OR from the meta‐analysis indicates the magnitude of any benefit is likely to be small, while the relevance of desirable but less clinically significant endpoints in this scale is questionable for passive immune therapy. The cost and difficulty of obtaining significant volumes of convalescent plasma/serum currently constrain its accessibility as part of routine care for severe seasonal influenza. Further, as highlighted by Beigel et alIn terms of other immunomodulatory agents, despite the apparent mortality benefit with adjunctive mTOR inhibitors observed in the small RCT by Wang et alThe immunomodulatory effects of statins have been well described, and this may be the mechanism of action for some of their observed cardiovascular benefits.Finally, what is the role of macrolides and NSAIDs in the management of severe influenza? Both are frequently administered, either because of diagnostic uncertainty as to whether there is a concomitant bacterial infection or for symptom relief. The effects of these agents are complex, involving multiple inflammatory pathways. For example, changes in the respiratory tract microbiome from macrolide antibiotics could affect susceptibility to secondary bacterial infection or the inflammatory response itself,As low‐cost therapies with a favourable adverse effect profile, these are both attractive adjunctive therapies. The significant reduction in mortality observed in the two RCTs published by Hung et alThis systematic review has several limitations. Focusing on the treatment of influenza in hospitalised patients, and the effects of immunomodulatory therapy on mortality overlooks their potential benefit/harm in the treatment of milder infections. The design of the review did not allow us to systematically assess published evidence as to whether immunomodulatory therapy may be able to reduce hospital admissions, speed symptom recovery or prevent influenza‐associated complications. Additional limitations include not searching for grey literature and the small number of abstracts with potentially eligible studies for inclusion, but where we were unable to locate full‐text copies of articles. We also included data from a number of unpublished studies, and it is possible published results may change—though this is unlikely for mortality.The decision to exclude observational studies, which did not perform multivariate adjustment for confounding, is not likely to alter the overall conclusions of our review, as the majority of excluded studies that were included in other systematic reviews were of small size. Incorporating these studies into the review would ideally require an individual patient data meta‐analysis.The influenza treatment landscape may also change significantly over the coming years, if any of the antivirals currently in clinical development demonstrate superiority to current standard of care with NAI. If so, a re‐assessment of the role of immunomodulatory therapy will be required.There is insufficient evidence currently for any of the assessed immunomodulatory therapies to be routinely used in clinical practice for the treatment of influenza infection. The results of studies of macrolides, NSAIDs and mTOR inhibitors are encouraging, however, and warrant further study in RCTs. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Mycobacterium tuberculosis have been studied with great interest since their discovery. Named after the conserved proline (P) and glutamic acid (E) residues in their N‐terminal domains, these proteins are postulated to perform wide‐ranging roles in virulence and immune modulation. However, technical challenges in studying these proteins and their encoding genes have hampered the elucidation of molecular mechanisms and leave many open questions regarding the biological functions mediated by these proteins. Here, I review the shared and unique characteristics of PE and PPE proteins from a molecular perspective linking this information to their functions in mycobacterial virulence. I discuss how the different subgroups  are defined and why this classification of paramount importance to understand the PE and PPE proteins as individuals and or groups. The goal of this MicroReview is to summarize and structure the existing information on this gene family into a simplified framework of thinking about PE and PPE proteins and genes. Thereby, I hope to provide helpful starting points in studying these genes and proteins for researchers with different backgrounds. This has particular implications for the design and monitoring of novel vaccine candidates and in understanding the evolution of the M. tuberculosis complex.The PE and PPE proteins of  et al., 2012b; Korotkova et al., 2014; Phan et al., 2017). The rest of the PPE‐protein interacts with its PE partner  via hydrophobic interactions. The conserved WxG residues of the PPE (Pink) and YxxxD/E of the PE (Red) are closely associated and may form a composite TypeVII secretion signal. The C‐terminus of the PPE (Green) extends toward the C‐terminus of the PE (Red), suggesting that PE/PPE protein pairs with C‐terminal extensions may have further PE/PPE interactions between those specific domains.Structure of the EspG5‐PPE41‐PE25 complex (4KXR). The EspG chaperone (Gold) binds to the EspG‐binding domain of the PPE protein (Light blue), thereby conferring TypeVII secretion system specificity (Daleke  Mycobacterium tuberculosis genome sequencing project was the identification of a family of genes, called pe and ppe genes, covering up to 10% of the M. tuberculosis genome .In spite of all these methodological challenges, perhaps the biggest hurdle for researchers attempting to study PE and PPE proteins is to make sense of the vast body of literature. In this review, I aim to provide a framework to study PE and PPE proteins from a molecular biology perspective by establishing their common characteristics while highlighting the importance of classifying PE and PPE proteins within subgroups  proteins such as the EspA/C and EspE/F heterodimers and EspB  are all present in the inner membrane and their dimensions do not allow for a one‐step secretion process over both the inner and outer mycobacterial membrane , the EspG chaperones or the cytosolic EccA ATPases, which have clearly characterized other functions and localization  and LxG (PF04740) family of TypeVII secretion substrates , which define 65 of the 99 PE proteins present in the M. tuberculosis reference genome H37Rv , the PE_PGRS subgroup is the most difficult to study. The rest of the PE proteins cannot be reliably subgrouped in further functional categories based on C‐terminal sequences. However, more information can be obtained from the sequences of the PE domains and their genetic environment  and ESX‐3 (PE5) are defined as sublineage I and II, respectively. Sublineage III may be the most loosely defined sublineage and includes the esx2‐localized PE36 as well as the ESX‐5 substrate PE25. Sublineage IV is a group of ESX‐5 secreted PE proteins, whose genes are often transcribed as bicistronic transcripts together with PPE‐SVP’s (discussed below)  amino acid sequence present in their C‐terminal domain (PFAM: PF12484). PPE50 is likely a truncated protein and PPE9 is not a true PPE‐SVP, because it consists of only a PPE domain of 180 amino acids, but was classified as a PPE‐SVP protein based on phylogenetic analysis ‐containing PPE‐MPTR proteins  and 5::tnM. marinum espG was similarly not hypervirulent in zebrafish embryos  protection of this recombinant strain was seen in two different mouse models , PE13‐PPE18 ), PE5 (ML_2534c), PPE1 (ML_1991), PPE2 (ML_1828c) and PPE68  have all obtained independent RD1‐like deletions inactivating the ESX‐1 secretion system, which should theoretically lead to complete attenuation 5, have occurred independently in Box 1ppe38‐containing genomic locus is one of the most variable regions in the M. tuberculosis genome . While the reference genome only contains a single ppe38 gene, most M. tuberculosis genomes contain two copies of the ppe  that together flank two esx genes, esxXY ) when assessing polymorphisms in this region , but may also be aided by using more refined reference genomes and mapping algorithms . Click here for additional data file.


Many estuarine ecosystems and the fish communities that inhabit them have undergone substantial changes in the past several decades, largely due to multiple interacting stressors that are often of anthropogenic origin. Few are more impactful than droughts, which are predicted to increase in both frequency and severity with climate change. In this study, we examined over five decades of fish monitoring data from the San Francisco Estuary, California, USA, to evaluate the resistance and resilience of fish communities to disturbance from prolonged drought events. High resistance was defined by the lack of decline in species occurrence from a wet to a subsequent drought period, while high resilience was defined by the increase in species occurrence from a drought to a subsequent wet period. We found some unifying themes connecting the multiple drought events over the 50‐yr period. Pelagic fishes consistently declined during droughts (low resistance), but exhibit a considerable amount of resiliency and often rebound in the subsequent wet years. However, full recovery does not occur in all wet years following droughts, leading to permanently lower baseline numbers for some pelagic fishes over time. In contrast, littoral fishes seem to be more resistant to drought and may even increase in occurrence during dry years. Based on the consistent detrimental effects of drought on pelagic fishes within the San Francisco Estuary and the inability of these fish populations to recover in some years, we conclude that freshwater flow remains a crucial but not sufficient management tool for the conservation of estuarine biodiversity. Climate change models forecast increased frequency and intensity of drought both in the western United States and globally  of California have been conducted continuously for up to five decades and provide an opportunity to study species and community data over multiple drought cycles. The SFE has experienced major ecological change, including the decline of a suite of pelagic fishes  How do we define drought cycles from 1967 to 2017 in the SFE? (2) Do fish species exhibit drought resistance? (3) Do fish species recover from drought when wet conditions return ? The fishes of the SFE watershed and estuary have evolved in a Mediterranean climate characterized by periodic floods and droughts and have life histories with high resistance or resilience to drought .We identified drought periods using California Department of Water Resources (CDWR) water year hydrologic classification index (Morone saxatilis); however, over the years it has provided valuable information on the endangered delta smelt (Hypomesus transpacificus) and other species of management interest  monitoring program and littoral fish data were obtained from the United States Fish and Wildlife Service's Delta Juvenile Fish Monitoring Program (DJFMP). The FMWT has conducted monitoring for juvenile fishes in the open water  habitat of the SFE since 1967 (Stevens and Miller Oncorhynchus tshawytscha) and various resident fish species within the SFE , threadfin shad (Dorosoma petenense), and American shad , Mississippi silverside (Menidia audens), largemouth bass , Sacramento sucker , bluegill (Lepomis macrochirus), redear sunfish (Lepomis microlophus), and Chinook salmon .To ensure the validity of our drought and wet period classifications and better understand how they differ, we conducted an ANOVA to test for significant differences between wet and dry periods using hydrologic variables  or absence (y = 0) of the species in sample i at station s in month m and year r within period d of drought cycle c and pc,d,r,m,s is the rate of occurrence. For the first drought cycle this rate is modeled asr, αm, and αs are random intercepts corresponding to year, month, and station; and Δ1,c=1 and Δ2,c=1 are the resistance and resilience coefficients for the first drought cycle. The binary indicator variable Id>1 takes the value 1 in the second (drought) period and after in the time series and 0 otherwise; similarly, Id>2 takes the value 1 in the third (post‐drought) period and after in the time series and 0 otherwise. Note that the first four terms on the right side of Eq. logitpc=1,d=1,r,m,s=α+αr+αm+αs, which serves as the reference level for periods two and three.Let m = 1) is identical to the previous cycle's post‐drought wet period  between wet and drought periods for the majority of the hydrologic variables tested  and habitat  of fishes that we selected for our analysis Table .Drought periods can be characterized as having less flow, as well as shorter duration and lower magnitude of peak flows relative to recovery periods. During droughts, there is also a shift in the relative contributions of the major water sources for the estuary, which could contribute to changes in water quality. The weight of evidence from our models suggests that the occurrence of young‐of‐year and annual pelagic fishes in the SFE consistently decline during droughts, regardless of whether they are native or nonnative. Pelagic fish species also demonstrated resilience in most cases, where occurrence levels returned to pre‐drought values. Yet full recoveries do not occur in every drought cycle, leading to permanently lower baseline numbers over the –50‐yr study period, a finding consistent with earlier studies  generally exhibited low drought resistance and high resilience during our study period. However, these nonnative pelagic fish species did not demonstrate synchronous decline and rebound throughout every drought cycle Appendix : Fig. S6In addition to interspecific variation in drought response, we observed notable differences in the general response of fishes to some drought cycles. The post‐drought recovery period of 2003–2006 was particularly striking as many species continued to decline after the end of the drought period. This post‐drought period took place after 2002, when the SFE saw an abrupt decline of multiple pelagic fish species, likely due to multiple interacting stressors such as low food availability and predation pressure (Mac Nally et al. The management implications of our study are relevant to the SFE and are also instructive for other estuaries, where many similar ecological changes are occurring (Matthews and Marsh‐Matthews Appendix S1Click here for additional data file.Data S1Click here for additional data file.Metadta S1Click here for additional data file.


This study seeks to document the imaging series used in contemporary Australian practice for imaging the augmented breast, with a secondary focus on differences in practice and opinion between BreastScreen Australia and diagnostic imaging services.ASMIRT) and was assessable during December 2017 and January 2018. The questionnaire investigated: years of experience, facility type and location, image acquisition systems, appointment times, patients imaged per week, technique and imaging series used, use of limited compression views, rationale for variation in imaging series and the use of ultrasound. Descriptive statistics were produced for all variables with chi‐squared tests used for comparisons between categorical variables.A SurveyMonkey link was distributed through the Australian Society of Medical Imaging and Radiation Therapy (ID technique 64% and 59% (submuscular) and 68% and 58% (subglandular) for BSA and diagnostic services, respectively. Eighteen different combinations of projections were reported with eight combinations common to both subglandular and submuscular imaging. The majority of participants attributed imaging series preferences to dose reduction and radiologist preference.The most frequently used series was the eight‐image Eklund This research has demonstrated varied approaches to the routine imaging of women with breast implants and identified the need for the establishment of dedicated evidence‐based imaging protocols to ensure that regardless of which setting a woman attends that they receive standardised imaging with minimal dose and maximum breast coverage. This is a reassurance that is not applicable to current practice. ID technique. The results demonstrate varied approaches to the routine imaging of women with breast implants identifying the need for the establishment of dedicated evidence‐based protocols for imaging of the augmented breast.This study documents the imaging series used in contemporary Australian practice for imaging the augmented breast. Eighteen different combinations of projections were reported to be used for routine imaging with eight combinations common to both subglandular and submuscular imaging; the most frequently cited series was the eight‐image Eklund  Mammography is the gold standard for the detection, analysis and diagnosis of breast abnormalities including breast cancer. In 2016, breast augmentation was ranked the number one most commonly undertaken cosmetic surgical procedures worldwide with 1.6 million surgeries reported. To improve imaging of the augmented breast, a dedicated method of implant imaging was devised in 1988, by Eklund et al., and is most commonly referred to as the implant displacement (ID) technique .The most common complication of breast augmentation is capsular contraction caused by an inflammatory reaction and resulting in fibrosis. In 1992, Rickard et al. published on a new imaging series based upon the ID technique to provide a more thorough examination of the augmented breast.In addition to an increase in radiation dose due to the increased number of routine projections when utilising the ID technique, Smathers et al. (2007) identified that patients also receive higher doses of radiation due to the increased size of their breasts.There is a lack of guidelines for the imaging of breast implant patients in Australia. While the ID method is encouraged, no specific implant imaging protocol  is listed in BreastScreen Australia's National Accreditation Standards (BSA NAS) or in the Royal Australian and New Zealand College of Radiologist (RANZCR) Mammography Quality Control Manual.This study investigated the current practice with respect to imaging of augmented breasts in the diagnostic and BreastScreen Australia (BSA) setting. It is unknown if the approach to imaging the augmented breast in each setting is in common with each other.® (https://www.surveymonkey.com), used to manage the questionnaire and collect responses. The questionnaire was designed to gather information on routine imaging practices in diagnostic and BreastScreen settings. Questions investigated were as follows: years of experience, facility type and location, image acquisition systems, appointment times, patients imaged per week, the technique and imaging series used, use of limited compression views, rationale for variation in imaging series and the use of ultrasound.An online questionnaire was developed to gather information related to current practices for imaging of augmented breasts with the online platform, SurveyMonkeyTo understand imaging behaviours, respondents were asked to report use of one or more of the following when performing routine imaging of those with implants, those with submuscular implants, and when using tomosynthesis. The views were as follows: non‐compressed CC and MLO, implant displaced CC – lateral, nipple back or medial; implant displaced MLO – superior, nipple back or inferior/inframammary angle (IMA). In addition for submuscular implants, an option ‘routine 4 views with full compression’ was included.The specific questions were devised based on the literature and 27 years of clinical experience in both diagnostic and screening mammography of one of the authors (KS). The questionnaire was edited for clarity through the Charles Sturt University (CSU) Spatial Data Analysis Network (SPAN).A SurveyMonkey link was distributed through the Australian Society of Medical Imaging and Radiation Therapy (ASMIRT) e‐newsletter and was accessible during December 2017 and January 2018. The ASMIRT has 8091 members comprised of radiographers, radiation therapists, sonographers and mammographic technologists.The absolute number of medical radiation science professionals who perform mammographic imaging in Australia is unknown as there is no official monitoring of this statistic by the Australian Health Practitioner Regulation Agency (AHPRA) or the professional body, ASMIRT. Some insight into population size may be gained considering the number of ASMIRT members who hold a Certificate of Clinical Proficiency in Mammography (CCPM); however, medical radiation science professionals practicing mammography in Australia are not required by AHPRA to acquire the CCPM. Currently, 712 ASMIRT members hold the CCPM; however, there have been 1532 CCPM awarded since 1998 . As the survey encouraged participation for members with mammography experience past or present, 1532 could be considered closer to the true population of Australian medical radiation science professionals who have undertaken mammography. An ideal sample size of 308 was calculated, with a confidence level of 95%.P < 0.05 deemed to be statistically significant. Free‐text responses were analysed thematically.Data obtained through the questionnaire were analysed using SPSS, Version 23 . Descriptive statistics were produced for all variables with chi‐squared tests used for comparisons between categorical variables with Ethics approval for this study was granted by the CSU Human Research Ethics Committee (HREC) .One hundred and eleven (111) responses were received during the collection period with 21 responses subsequently deemed incomplete and excluded from further analysis.n = 36) worked for a diagnostic service, 37% (n = 33) worked for BSA and 23% (n = 21) worked in both settings. Overall the highest portion of respondents was from Queensland (30%) and New South Wales (26%) with smaller percentages from the other States/Territories, except the Northern Territory where there were no respondents. The majority of respondents (69%) worked in metropolitan settings with populations greater than 100,000Of the 90 completed surveys, 40%  or FFDM and tomosynthesis (41%); the remaining 2 respondents had used either a combination of film screen, computed radiography (CR) and FFDM, or CR and tomosynthesis. In contrast for those working in diagnostic settings, the most frequent system, or combination of systems, being used were FFDM and tomosynthesis (46%), FFDM only or tomosynthesis only (both 22%); remaining respondents used a mix of film screen, CR, FFDM and tomosynthesis.n = 6, 11%) reported imaging >6 women per week. Chi‐squared analysis showed that there was a statistically significant difference between the two groups .For those working in BSA settings, most (51%) imaged 2–4 women with implants per week; 23% reported imaging 0 or 1 woman per week and 13% reported imaging each of 4–6 or greater than 6 women per week. Those working in diagnostic settings reported imaging fewer women with implants per week with 55% reporting imaging 0–1 woman and 32% imaging 2–4 women. In this group, a small number (Most respondents (95%) working in BSA settings reported that the average appointment time for women without implants was 15 min or less; however, for women with implants there was an increase in average appointment time with 39% of respondents reporting an average time of 20 min and 26% reporting an average time of 30 min or more. In contrast, those in diagnostic settings reported longer average appointment times for women with or without implants with the most frequent response being 30 min .The Eklund ID technique was reported as always used by 57% of BSA respondents; 15% reports use sometimes, 2% rarely and 11% did not use this technique. Similarly, 50% of those in diagnostic settings always used the Eklund ID technique, 19% sometimes, 8% rarely and 10% did not use the technique. The remaining respondents in each setting  were unsure whether they used this technique. When asked why the Eklund technique was not used, the highest frequency single response for those at BSA was ‘unknown’ (23%) followed by ‘dose reduction’ (13%). The remaining respondents selected a combination of the item response options which included radiologist preference, dose reduction, not needed due to new technology (FFDM) and not needed due to new technology (tomosynthesis) with combinations which included radiologist preference and or dose reduction selected by 51% of respondents. Similarly, for those in diagnostic setting ‘unknown’ (16%) was the highest frequency single response with remaining responses being a combination of the available response options. The majority (73%) were combinations which included radiologist preference &/or dose reduction. The routine use of non‐compressed views prior to ID imaging was reported by the majority of respondents; 85% for BSA and 83% for diagnostic.Reflective of the differing purposes of breast screening and diagnostic imaging, the routine use of ultrasound as an adjunct to routine implant imagining proved rare amongst BSA respondents with 80% indicating it is never used in their service; whilst the majority of diagnostic respondents (89%) indicated ultrasound is always or usually scheduled for patients.Respondents were asked on a range of 4–16 how many images are routinely taken for patients with breast implants. For BSA, 91% of respondents reported taking 8 images, while 6% took 10 images and 4% ‘other’. While the majority (79%) of those in diagnostic settings also took 8 images, there was greater variability with 10% taking 10 images, 6% taken 4 images and 2% each taking 6, 12 or 16 images.Respondents were asked which of 8 projections were used when imaging patients with breast implants; this resulted in a total of 18 different combinations , 50% used a combination of implant displaced CC and MLO nipple back views. For those in diagnostic settings, 11 different combinations were used with 31% using the eight‐image Eklund series. Other combinations were used by 4 or fewer respondents.In response to a question about the standard views for a patient with breast implants when using tomosynthesis, 78% of those in BSA settings and 41% of those in diagnostic settings indicated that they did not use tomosynthesis. Of the remaining respondents, again there were 5 different combinations of views used. While the number of respondents using tomosynthesis in BSA settings was small  including non‐compressed CC, non‐compressed MLO, ID CC and ID MLO was the most frequently selected image series for both subglandular and submuscular implant imaging . However, of great concern is that the remaining respondents reported 18 and 17 different combinations of views forming a variety of imaging series being used to image subglandular and submuscular implants, respectively.Protocols need to demonstrate some flexibility to accommodate for variability in patient presentation, such as encapsulation or minimal tissue volume. However, the wide variation in the imaging series reported cannot be explained by the need to accommodate patient presentation alone. Protocols such as those developed for the NHSBSP, which take into account patient history, implant type  and position, provide a contemporary framework on which to base any future Australian protocols.In terms of imaging technique, the order of projections should be pre‐set; the NHSBSP advocates initial use of the non‐compressed MLO projection to show implant position and assist in the planning of subsequent projections.The results of this study reflect standard DBT use in the assessment setting only for BSA facilities and routinely, where available, for diagnostic sites (59%). The imaging series put forward by BSA respondents appears adjunct to the routine imaging series and by diagnostic respondents favouring the standard eight‐image Eklund series (31%). Similar to the results for 2D imaging, the remaining respondents reported a variety of different combinations of views, 5 and 11, for BSA and diagnostic imaging, respectively. The benefits of DBT in terms of lesion detection and the imaging of dense breasts are undeniable The development protocols for imaging the augmented breast should consider the image acquisition system used and the benefits of separate imaging series for 2D and DBT technology. Further research into comparisons between dose and mammographic coverage of breast tissue between FFDM and DBT may contribute to a revised and standardised imaging series for use with this technology.The standardisation of imaging techniques for both subglandular and submuscular breast implants, both of which have been shown to benefit from ID technique n = 90). The method of survey distribution resulted in the exclusion of mammographers who are not members of the ASMIRT. Response rates also varied between questions, with a considerably smaller response rate to questions relating to DBT practice. This may be explained by its limited use in Australian diagnostic facilities and use only for assessment at BSA facilities.The study had several limitations, including a small number of respondents (This study documents for the first time the imaging series used in contemporary imaging of the augmented breast in Australia. In this snapshot of current practice, the research has revealed that the eight‐view ID technique is the primary imaging series for both FFDM and DBT imaging of patients with breast implants The results of the study however also demonstrated varied additional imaging series used for routine imaging of patients with breast implants both between BSA and diagnostic imaging and within these providers. Little difference was demonstrated between imaging of breasts with submuscular and subglandular breast implants; imaging protocols are likely applied to all patients with breast implants rather than utilising a specific protocol based on implant position. Based on data obtained, current imaging is reportedly driven by dose reduction and radiologist preference within both facilities, possibly indicating an acceptance that sufficient mammographic coverage of the breast for breast cancer detection. The absolute amount of tissue imaged and that not included on the image during implant imaging remains unknown, as there is currently no evidence base upon which to draw. Looking forward the adoption of new technology such as coned beam breast CT may resolve many of these issues.This research has identified the need for the establishment of dedicated evidence‐based imaging protocols for women with breast implants to ensure that regardless of which setting a woman attends that they will consistently receive an appropriate standard imagining series and imaging that results in sufficient mammographic coverage of their breast tissue for breast cancer detection and a dose that is ALARA. This is a reassurance that is not applicable to current practice. There is an urgent need for the development of evidence‐based standards to ensure that no woman is disadvantaged by imaging practice in Australia as a result of breast augmentation.The authors declare no conflict of interest.