Aquaponics in schools: Hands-on learning about healthy eating and a healthy planet.

Our food system is giving rise to a growing social, health and environmental crisis. Much of the food consumed in the United Kingdom is cheap, nutrient-poor and highly processed, leading to under-consumption of essential foods such as grains, beans, vegetables and fruit. This has contributed to a rise in diet-related diseases, with approximately 22% of primary school leavers being overweight or obese. Food production is unsustainable with agriculture responsible for 10% of the UK's greenhouse gas emissions and intensive farming practices have led to a significant loss of soil carbon and a decline in biodiversity. COVID-19 increased inequalities in our food system. Therefore, there is an urgent need for interventions to counteract these adverse social, health and environmental impacts. Education can play a crucial role as an intervention to address challenges in the food system. We tested an innovative school initiative using portable aquaponic pods and aligned to the national curriculum, to engage pupils in food production and foster learning about sustainability, climate change and healthy eating. The evaluation, based on teacher surveys, aquapod chart data, student blogs and postcards and feedback from the development team, revealed positive impacts on students' environmental awareness, as well as sustainability and practical food production knowledge. However, the programme encountered logistical challenges and we therefore highlight future improvements to produce a curriculum programme that can be delivered at scale to enhance food education and empower pupils to drive the agenda on tackling food sustainability and climate change.

[Palliative Treatment of Metastatic Rectal Cancer]. / Palliative Systemtherapie des metastasierten Rektumkarzinoms.

Palliative treatment of metastatic rectal cancer (mCRC) has developed considerably in recent years, with new therapeutic strategies such as induction and maintenance therapies, the establishment of targeted therapies and molecularly defined strategies in defined subgroups such as MSI-H-patients. The following article presents evidence based therapeutic options and algorithms.

Efficacy of ramucirumab combination chemotherapy as second-line treatment in patients with advanced adenocarcinoma of the stomach or gastroesophageal junction after exposure to checkpoint inhibitors and chemotherapy as first-line therapy.

FOLFOX plus nivolumab represents a standard of care for first-line therapy of advanced gastroesophageal cancer (aGEC) with positive PD-L1 expression. The efficacy of second-line VEGFR-2 inhibition with ramucirumab (RAM) plus chemotherapy after progression to immunochemotherapy remains unclear. Medical records of patients with aGEC enrolled in the randomized phase II AIO-STO-0417 trial after treatment failure to first-line FOLFOX plus nivolumab and ipilimumab were retrospectively analyzed. Patients were divided into two groups based on second-line therapy: RAM plus chemotherapy (RAM group) or treatment without RAM (control group). Eighty three patients were included. In the overall population, progression-free survival (PFS) in the RAM group was superior to the control (4.5 vs 2.9 months). Responders (CR/PR) to first-line immunochemotherapy receiving RAM containing second-line therapy had prolonged OS from start of first-line therapy (28.9 vs 16.5 months), as well as second-line OS (9.6 vs 7.5 months), PFS (5.6 vs 2.9 months) and DCR (53% vs 29%) compared to the control. PD-L1 CPS ≥1 was 42% and 44% for the RAM and the control, respectively. Patients with CPS ≥1 in the RAM group showed better tumor control (ORR 25% vs 10%) and improved survival (total OS 11.5 vs 8.0 months; second-line OS 6.5 vs 3.9 months; PFS 4.5 vs 1.6 months) compared to the control. Prior exposure to first-line FOLFOX plus dual checkpoint inhibition followed by RAM plus chemotherapy shows favorable response and survival rates especially in patients with initial response and positive PD-L1 expression and has the potential to advance the treatment paradigm in aGEC.

Perioperative Atezolizumab Plus Fluorouracil, Leucovorin, Oxaliplatin, and Docetaxel for Resectable Esophagogastric Cancer: Interim Results From the Randomized, Multicenter, Phase II/III DANTE/IKF-s633 Trial.

PURPOSE: This trial evaluates the addition of the PD-L1 antibody atezolizumab (ATZ) to standard-of-care fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) as a perioperative treatment for patients with resectable esophagogastric adenocarcinoma (EGA). METHODS: DANTE started as multicenter, randomized phase II trial, which was subsequently converted to a phase III trial. Here, we present the results of the phase II proportion, focusing on surgical pathology and safety outcomes on an exploratory basis. Patients with resectable EGA (≥cT2 or cN+) were assigned to either four preoperative and postoperative cycles of FLOT combined with ATZ, followed by eight cycles of ATZ maintenance (arm A) or FLOT alone (arm B). RESULTS: Two hundred ninety-five patients were randomly assigned (A, 146; B, 149) with balanced baseline characteristics between arms. Twenty-three patients (8%) had tumors with microsatellite instability (MSI), and 58% patients had tumors with a PD-L1 combined positive score (CPS) of ≥1. Surgical morbidity (A, 45%; B, 42%) and 60-day mortality (A, 3%; B, 2%) were comparable between arms. Downstaging favored arm A versus arm B (ypT0, 23% v 15% [one-sided P = .044]; ypT0-T2, 61% v 48% [one-sided P = .015]; ypN0, 68% v 54% [one-sided P = .012]). Histopathologic complete regression rates (pathologic complete response or TRG1a) were higher after FLOT plus ATZ (A, 24%; B, 15%; one-sided P = .032), and the difference was more pronounced in the PD-L1 CPS ≥10 (A, 33%; B, 12%) and MSI (A, 63%; B, 27%) subpopulations. Complete margin-free (R0) resection rates were relatively high in both arms (A, 96%; B, 95%). The incidence and severity of adverse events were similar in both groups. CONCLUSION: Within the limitations of the exploratory nature of the data, the addition of ATZ to perioperative FLOT is safe and improved postoperative stage and histopathologic regression.

Utility of a three-gene transcriptomic signature in the diagnosis of tuberculosis in a low-endemic hospital setting.

BACKGROUND: Host transcriptomic blood signatures have demonstrated diagnostic potential for tuberculosis (TB), requiring further validation across different geographical settings. Discriminating TB from other diseases with similar clinical manifestations is crucial for the development of an accurate immunodiagnostic tool. In this exploratory cohort study, we evaluated the performance of potential blood-based transcriptomic signatures in distinguishing TB disease from non-TB lower respiratory tract infections in hospitalised patients in a TB low-endemic country. METHOD: Quantitative real-time polymerase chain reaction qPCR) was used to evaluate 26 previously published genes in blood from 31 patients (14 TB and 17 lower respiratory tract infection cases) admitted to Oslo University Hospital in Norway. The diagnostic accuracies of differentially expressed genes were determined by receiver operating characteristic curves. RESULTS: A significant difference (p < .01) in the age distribution was observed between patients with TB (mean age, 40 ± 15 years) and lower respiratory tract infection (mean age 59 ± 12 years). Following adjustment for age, ETV7, GBP1, GBP5, P2RY14 and BLK were significantly differentially expressed between patients with TB and those with LRI. A general discriminant analysis generated a three-gene signature (BAFT2, ETV7 and CD1C), which diagnosed TB with an area under the receiver operating characteristic curve (AUC) of 0.86 (95% CI, 0.69 - 1.00), sensitivity of 69.23% (95% CI, 38.57%-90.91%) and specificity of 94.12% (95% CI, 71.31%-99.85%). CONCLUSION: The three-genes signature may have potential to improve diagnosis of TB in a hospitalised low-burden setting. However, the influence of confounding variables or covariates such as age requires further evaluation in larger studies.

Atezolizumab Plus Bevacizumab in Patients with Advanced and Progressing Hepatocellular Carcinoma: Retrospective Multicenter Experience.

Atezolizumab plus bevacizumab is the standard of care for first-line systemic therapy for advanced hepatocellular carcinoma (aHCC). Data on the efficacy and safety of atezolizumab plus bevacizumab in patients with aHCC who have received prior systemic therapy are not available. Methods: Patients with aHCC who received atezolizumab plus bevacizumab after at least one systemic treatment between December 2018 and March 2022 were retrospectively identified in 13 centers in Germany and Austria. Patient characteristics, tumor response rates, progression-free survival (PFS), overall survival (OS), and adverse events (AE) were analyzed. Results: A total of 50 patients were identified; 41 (82%) were male. The median age at initiation of treatment with atezolizumab plus bevacizumab was 65 years, 41 (82%) patients had cirrhosis, 30 (73%) Child A, 9 (22%) B, and 2 (5%) C. A total of 34 patients (68%) received atezolizumab plus bevacizumab in the second-line setting and 16 (32%) in later lines. The best radiologic tumor responses were complete remission (2%), partial remission (30%), stable disease (36%), and progressive disease (18%), resulting in an objective response rate of 32% and a disease control rate of 68%. Median OS was 16.0 months (95% confidence interval 5.6-26.4 months), and median PFS was 7.1 months (95% confidence interval 4.4-9.8 months). AE grades 3-4 were observed in seven (14%) and resulted in death in three patients (6%). There were five (10%) bleeding events with a grade ≥ 3, including one (2%) with a fatal outcome. Conclusions: Atezolizumab plus bevacizumab is effective in patients with aHCC who did not have access to this option as first-line therapy. The safety profile was consistent with previous reports.

Gallbladder Cancer: Current Multimodality Treatment Concepts and Future Directions.

Gallbladder cancer (GBC) is the most common primary tumor site of biliary tract cancer (BTC), accounting for 0.6% of newly diagnosed cancers and 0.9% of cancer-related deaths. Risk factors, including female sex, age, ethnic background, and chronic inflammation of the gallbladder, have been identified. Surgery is the only curative option for early-stage GBC, but only 10% of patients are primary eligible for curative treatment. After neoadjuvant treatment, up to one-third of locally advanced GBC patients could benefit from secondary surgical treatment. After surgery, only a high-risk subset of patients benefits from adjuvant treatment. For advanced-stage GBC, palliative chemotherapy with gemcitabine and cisplatin is the current standard of care in line with other BTCs. After the failure of gemcitabine and cisplatin, data for second-line treatment in non-resectable GBC is poor, and the only recommended chemotherapy regimen is FOLFOX (5-FU/folinic acid and oxaliplatin). Recent advances with the PD-L1 inhibitor durvalumab open the therapy landscape for immune checkpoint inhibition in GBC. Meanwhile, targeted therapy approaches are a cornerstone of GBC therapy based on molecular profiling and new evidence of molecular differences between different BTC forms and might further improve the prognosis of GBC patients.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.

MrParse: finding homologues in the PDB and the EBI AlphaFold database for molecular replacement and more.

Crystallographers have an array of search-model options for structure solution by molecular replacement (MR). The well established options of homologous experimental structures and regular secondary-structure elements or motifs are increasingly supplemented by computational modelling. Such modelling may be carried out locally or may use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help to streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any experimental homologues found using eLLG, which indicates the likelihood that a given search model will work in MR. Inbuilt displays of predicted secondary structure, coiled-coil and transmembrane regions further inform the choice of MR protocol. MrParse can also identify and rank homologues in the EBI AlphaFold database, a function that will also interest other structural biologists and bioinformaticians.

Blood-Based Targeted Metabolomics Discriminate Patients with Alcoholic Liver Cirrhosis from Those with Non-Cirrhotic Liver Damage: An Explorative Study.

BACKGROUND: Early detection of liver cirrhosis is crucial for secondary prevention of complications. However, noninvasive blood-based patient monitoring tools are lacking. In this explorative study, we conducted a targeted metabolomic analysis in order to identify possible serum markers indicating alcoholic liver cirrhosis (aLiC) with or without hepatocellular carcinoma (HCC). METHODS: Venous blood of 30 individuals was collected: healthy controls ("Con", n = 12), patients with aLiC without and with HCC ("aLiC": n = 6 and "aLiC + HCC": n = 6), and patients with other liver diseases ("oLiD": n = 6). A targeted metabolomic analysis was conducted using the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences®, Innsbruck, Austria). Statistical analysis was performed by applying a one-way ANOVA on all subgroups followed by a t test for pairwise comparison of subgroups and logistic regression analysis. RESULTS: ANOVA revealed 29 metabolites that significantly discriminate between the different cohorts. Among these analytes, 25 were significantly altered in Con versus aLiC, as indicated by t test, most importantly SM C18:1 (p < 0.001), SM C20:2 (p = 0.001), SM (OH) C22:2 (p < 0.001), lysoPC a C20:4 (p < 0.001), and PC aa C36:5 (p < 0.001). To a similar extent, the metabolites discriminated also between the oLiD and aLiC but less between the Con or oLiD and aLiC + HCC cohorts. Most of these analytes were either lyso- and phosphatidylcholines or sphingomyelins. Results were not significant for comparison of Con versus oLiD and aLiC versus aLiC + HCC. CONCLUSION: Decreased lyso- and phosphatidylcholine as well as sphingomyelin species in venous blood could help to detect liver cirrhosis in patients with non-cirrhotic liver disease.

Shaping convex edges in borosilicate glass by single pass perforation with an Airy beam.

We demonstrate curved modifications with lengths of up to 2 mm within borosilicate glass produced by single 1030 nm picosecond laser shots with an Airy beam profile. Plasma ignition in the sidelobes of the beam as well as surface damage prove to be the crucial limitations for confined bulk energy deposition on a curved trajectory. A combined experimental and numerical analysis reveals optimum laser parameters for confined bulk energy deposition. This way, we achieved single pass perforation of a 525 µm thick glass sheet and separation by a subsequent etching step, resulting in a well-defined convex edge down to a radius of curvature of 774 µm.

Diagnostic accuracy of plasma kynurenine/tryptophan ratio, measured by enzyme-linked immunosorbent assay, for pulmonary tuberculosis.

INTRODUCTION: The World Health Organization has identified the need for a non-sputum-based test capable of detecting active tuberculosis (TB) as a priority. The plasma kynurenine-to-tryptophan (K/T) ratio, largely mediated by activity of the enzyme indoleamine 2,3-dioxygenase, may have potential as a suitable biomarker for active TB. METHOD: We evaluated a commercial enzyme-linked immunosorbent assay (ELISA) in comparison to mass spectrometry for measuring the K/T ratio. We also used ELISA to determine the K/T ratio in plasma from patients with active TB compared to latently infected controls, with and without HIV. RESULTS: The two methods showed good agreement, with a mean bias of 0.01 (limit of agreement from -0.06 to 0.10). Using ELISA, it was found that HIV-infected patients with active TB disease had higher K/T ratios than those without TB (median, 0.101 [interquartile range (IQR), 0.091-0.140] versus 0.061 [IQR, 0.034-0.077], P<0.0001). At a cutoff of 0.080, the K/T ratio produced a sensitivity of 90%, a specificity of 80%, a positive predictive value (PPV) of 82%, and a negative predictive value (NPV) of 90%. In a receiver operating characteristics analysis, the K/T ratio had an area under the curve of 0.93. HIV-uninfected patients with active TB also had higher K/T ratios than those with latent TB infections (median, 0.064 [IQR, 0.040-0.088] versus 0.022 [IQR, 0.016-0.027], P<0.0001). A cutoff of 0.040 gave a sensitivity of 85%, a specificity of 92%, a PPV of 91%, and an NPV of 84%. CONCLUSION: The plasma K/T ratio is a sensitive biomarker for active TB. The K/T ratio can be measured from blood using ELISA. The K/T ratio should be evaluated as an initial test for TB.

Extending the scope of coiled-coil crystal structure solution by AMPLE through improved ab initio modelling.

The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.

Using Phaser and ensembles to improve the performance of SIMBAD.

The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.

Molecular replacement using structure predictions from databases.

Molecular replacement (MR) is the predominant route to solution of the phase problem in macromolecular crystallography. Where the lack of a suitable homologue precludes conventional MR, one option is to predict the target structure using bioinformatics. Such modelling, in the absence of homologous templates, is called ab initio or de novo modelling. Recently, the accuracy of such models has improved significantly as a result of the availability, in many cases, of residue-contact predictions derived from evolutionary covariance analysis. Covariance-assisted ab initio models representing structurally uncharacterized Pfam families are now available on a large scale in databases, potentially representing a valuable and easily accessible supplement to the PDB as a source of search models. Here, the unconventional MR pipeline AMPLE is employed to explore the value of structure predictions in the GREMLIN and PconsFam databases. It was tested whether these deposited predictions, processed in various ways, could solve the structures of PDB entries that were subsequently deposited. The results were encouraging: nine of 27 GREMLIN cases were solved, covering target lengths of 109-355 residues and a resolution range of 1.4-2.9 Å, and with target-model shared sequence identity as low as 20%. The cluster-and-truncate approach in AMPLE proved to be essential for most successes. For the overall lower quality structure predictions in the PconsFam database, remodelling with Rosetta within the AMPLE pipeline proved to be the best approach, generating ensemble search models from single-structure deposits. Finally, it is shown that the AMPLE-obtained search models deriving from GREMLIN deposits are of sufficiently high quality to be selected by the sequence-independent MR pipeline SIMBAD. Overall, the results help to point the way towards the optimal use of the expanding databases of ab initio structure predictions.

SIMBAD: a sequence-independent molecular-replacement pipeline.

The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.

Recent developments in MrBUMP: better search-model preparation, graphical interaction with search models, and solution improvement and assessment.

Increasing sophistication in molecular-replacement (MR) software and the rapid expansion of the PDB in recent years have allowed the technique to become the dominant method for determining the phases of a target structure in macromolecular X-ray crystallography. In addition, improvements in bioinformatic techniques for finding suitable homologous structures for use as MR search models, combined with developments in refinement and model-building techniques, have pushed the applicability of MR to lower sequence identities and made weak MR solutions more amenable to refinement and improvement. MrBUMP is a CCP4 pipeline which automates all stages of the MR procedure. Its scope covers everything from the sourcing and preparation of suitable search models right through to rebuilding of the positioned search model. Recent improvements to the pipeline include the adoption of more sensitive bioinformatic tools for sourcing search models, enhanced model-preparation techniques including better ensembling of homologues, and the use of phase improvement and model building on the resulting solution. The pipeline has also been deployed as an online service through CCP4 online, which allows its users to exploit large bioinformatic databases and coarse-grained parallelism to speed up the determination of a possible solution. Finally, the molecular-graphics application CCP4mg has been combined with MrBUMP to provide an interactive visual aid to the user during the process of selecting and manipulating search models for use in MR. Here, these developments in MrBUMP are described with a case study to explore how some of the enhancements to the pipeline and to CCP4mg can help to solve a difficult case.

Ensembles generated from crystal structures of single distant homologues solve challenging molecular-replacement cases in AMPLE.

Molecular replacement (MR) is the predominant route to solution of the phase problem in macromolecular crystallography. Although routine in many cases, it becomes more effortful and often impossible when the available experimental structures typically used as search models are only distantly homologous to the target. Nevertheless, with current powerful MR software, relatively small core structures shared between the target and known structure, of 20-40% of the overall structure for example, can succeed as search models where they can be isolated. Manual sculpting of such small structural cores is rarely attempted and is dependent on the crystallographer's expertise and understanding of the protein family in question. Automated search-model editing has previously been performed on the basis of sequence alignment, in order to eliminate, for example, side chains or loops that are not present in the target, or on the basis of structural features (e.g. solvent accessibility) or crystallographic parameters (e.g. B factors). Here, based on recent work demonstrating a correlation between evolutionary conservation and protein rigidity/packing, novel automated ways to derive edited search models from a given distant homologue over a range of sizes are presented. A variety of structure-based metrics, many readily obtained from online webservers, can be fed to the MR pipeline AMPLE to produce search models that succeed with a set of test cases where expertly manually edited comparators, further processed in diverse ways with MrBUMP, fail. Further significant performance gains result when the structure-based distance geometry method CONCOORD is used to generate ensembles from the distant homologue. To our knowledge, this is the first such approach whereby a single structure is meaningfully transformed into an ensemble for the purposes of MR. Additional cases further demonstrate the advantages of the approach. CONCOORD is freely available and computationally inexpensive, so these novel methods offer readily available new routes to solve difficult MR cases.

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds.

Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form ß-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αß folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αßß fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.

Approaches to ab initio molecular replacement of α-helical transmembrane proteins.

α-Helical transmembrane proteins are a ubiquitous and important class of proteins, but present difficulties for crystallographic structure solution. Here, the effectiveness of the AMPLE molecular replacement pipeline in solving α-helical transmembrane-protein structures is assessed using a small library of eight ideal helices, as well as search models derived from ab initio models generated both with and without evolutionary contact information. The ideal helices prove to be surprisingly effective at solving higher resolution structures, but ab initio-derived search models are able to solve structures that could not be solved with the ideal helices. The addition of evolutionary contact information results in a marked improvement in the modelling and makes additional solutions possible.