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PURPOSE Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship with NF2 loss. Given the predominance of NF2 mutations in meningiomas, we evaluated the efficacy of GSK2256098, a FAK inhibitor, as part of the first genomically driven phase II study in recurrent or progressive grade 1-3 meningiomas. PATIENTS AND METHODS Eligible patients whose tumors screened positively for NF2 mutations were treated with GSK2256098, 750 mg orally twice daily, until progressive disease. Efficacy was evaluated using two coprimary end points: progression-free survival at 6 months (PFS6) and response rate by Macdonald criteria, where PFS6 was evaluated separately within grade-based subgroups: grade 1 versus 2/3 meningiomas. Per study design, the FAK inhibitor would be considered promising in this patient population if either end point met the corresponding decision criteria for efficacy. RESULTS Of 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients with NF2 mutations were enrolled and treated: 12 grade 1 and 24 grade 2/3 patients. Across all grades, one patient had a partial response and 24 had stable disease as their best response to treatment. In grade 1 patients, the observed PFS6 rate was 83% (10/12 patients; 95% CI, 52 to 98). In grade 2/3 patients, the observed PFS6 rate was 33% (8/24 patients; 95% CI, 16 to 55). The study met the PFS6 efficacy end point both for the grade 1 and the grade 2/3 cohorts. Treatment was well tolerated; seven patients had a maximum grade 3 adverse event that was at least possibly related to treatment with no grade 4 or 5 events. CONCLUSION GSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive NF2-mutated meningiomas, compared with historical controls. The criteria for promising activity were met, and FAK inhibition warrants further evaluation for this patient population.

Manganese (Mn) is key to oxygenic photosynthesis as it catalyzes the splitting of water in photosystem II and functions as cofactor of multiple enzymes. A single ABC-type transporter, MntCAB, is so far established for the uptake of the metal under limiting conditions in cyanobacteria. It is unknown, how Mn is imported under replete conditions. We identified two proteins in the model cyanobacteriumSynechocystissp. PCC 6803, which are homologous to the unknown protein family 0016 (UPF0016) member manganese exporter (Mnx). In contrast to Mnx, which consists of six transmembrane domains, the new candidate proteins contain only three transmembrane domains. Hence, we named them hemi manganese exporter (Hmx) 1 and 2. Knock-out mutants inhmx1and/orhmx2showed sensitivity toward low Mn supplementation, and reduced intracellular Mn pools. Additional deletion ofmntChindered the cells to thrive unless external Mn was added and enhanced the depletion of their intracellular Mn pool. In accordance with the observed localization of Hmx1 and Hmx2 in the plasma membrane, we postulate a Mn uptake function for a heteromeric Hmx1/2 across the plasma membrane under a wide range of Mn concentrations and a supporting role for the MntCAB system under Mn-limiting conditions. On the basis of their phylogenies, we propose that Hmx1 and Hmx2 are the ancestral progenitors of eukaryote-type UPF0016 proteins with six transmembrane domains. The Mn transport function of Hmx1/2 underscores this as fundamental ancient feature of the UPF0016 family. Likely, Hmx1 and Hmx2 coevolved with the internalization of the oxygen-evolving complex.

BACKGROUND: Accelerated partial breast irradiation (APBI) has emerged as an alternative to whole breast irradiation (WBI) following lumpectomy for select patients. However, the challenges to post-operative APBI include targeting inaccuracy and the inability to measure tumor response to radiation. We hypothesized that preoperative APBI (pAPBI) could reliably target the tumor using MRI-guidance that is acquired prone in combination with our established prone breast radiation delivery. We developed methodology for prone CT simulation to establish radiation position, MRI acquisition in treatment position, registration of MRI-CT data for treatment planning, and treatment delivery with daily cone-beam CT using the same immobilization platform. This study aimed to assess the reproducibility, toxicity and local control associated with MRI/CT-directed prone pAPBI. METHODS: This was a prospective, single arm study enrolling patients >50 yo with clinical (c) Stage IA ER+/PR+/HER2- breast cancer intending lumpectomy. Axillary US and MRI imaging to confirm clinical node negative disease was required. A planning CT in the prone position was followed by MRI using the same prone immobilization platform. Rigid registration of MRI-CT data was used for radiation planning. pAPBI of 3850 cGy was delivered in 10 fractions BID with the same prone immobilization technique. Another MRI was obtained 4 weeks post-APBI to assess tumor response. The intensity, kinetics and volume of the lesion on MRI was quantitatively assessed and an experienced reader evaluated MRI volume and kinetic changes in the tumor post-APBI. Lumpectomy was performed 4-6 weeks after APBI. Simon 2 stage design required assessment after accrual of 19 patients for assessment of feasibility. RESULTS: Nineteen cStage IA ER+/PR+/HER2- breast cancer patients with a median age of 65 (range 51-78) were enrolled on the study, completed APBI, lumpectomy, and adjuvant AI. Median follow up was 3.4 years. Mean clinical tumor size was 1.1 cm ± 0.4 and mean path tumor size was 0.94 cm ± 0.6. There was complete pathologic response in 10.5% (n=2) and an additional 36.8% (n=7) were downstaged from clinical stage (measured by mammogram/US) to pathologic stage, resulting in a total response of 47.4% (n=9). Six (31.6%) patients had stable disease after APBI. Four (21.0%) were upstaged from clinical stage to pathological stage. Tumor response detected by MRI significantly correlated with tumor response based on clinical to pathologic stage (p=0.03). Cosmesis was rated as excellent/good in 89.5% (n=17) patients post-APBI. Cosmesis worsened to fair in 2 patients post-APBI, one of which required adjuvant WBI after focal triple negative breast cancer was detected on pathology. Three patients had positive macrometastatic lymph nodes on final pathology despite clinically negative nodes on imaging. One in-breast recurrence outside the RT field was detected by MRI at 14 months, resulting in a locoregional recurrence rate of 5.4% at 3.4 years. Another patient developed metastases at 20 months. CONCLUSIONS: Using the same prone platform, all patients successfully underwent CT simulation, MRI acquisition, and completed pAPBI. Nearly half of enrolled patients had a measurable tumor response to pAPBI based on MRI and pathologic response, confirming the accuracy and reproducibility of defining tumor targets with our MRI/CT-directed pAPBI approach. While this methodology for prone pAPBI resulted in good cosmesis and local control and remains a promising approach for select patients, the challenge of excluding patients with subclinical lymph node positive disease remains. Correlative studies will determine whether Ki-67 and OncotypeDx pre- and post-APBI can help predict response to pAPBI. Citation Format: Sasha Beyer, Tamara Smith, Ashley Sekhon, Jose Bazan, Sachin Jhawar, Erin Healy, Lai Wei, Vedat Yildiz, Mohamed Mohamed, Michael Knopp, Julia White. Preliminary results of a feasibility study assessing radiation response with MRI/CT directed preoperative accelerated partial breast irradiation in the prone position for hormone responsive early stage breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-19-03.

Drug combinations present a powerful strategy to tackle antimicrobial resistance, but have not been systematically tested in many bacterial species. Here, we used an automated high-throughput setup to profile ∼ 8000 combinations between 65 antibacterial drugs in three Gram-positive species: the model species,Bacillus subtilisand two prominent pathogens,Staphylococcus aureusandStreptococcus pneumoniae. Thereby, we recapitulate previously known drug interactions, but also identify ten times more interactions than previously reported in the pathogenS. aureus, including two synergies that were also effective in multi-drug resistant clinicalS. aureusisolatesin vitroandin vivo. Interactions were largely species-specific and mostly synergistic for drugs targeting the same cellular process, as observed also for Gram-negative species1. Yet, the dominating synergies are clearly distinct between Gram-negative and Gram-positive species, and are driven by different bottlenecks in drug uptake and vulnerabilities of their cell surface structures. To further explore interactions of commonly prescribed non-antibiotic drugs with antibiotics, we tested 2728 of such combinations inS. aureus, detecting a plethora of unexpected antagonisms that could compromise the efficacy of antimicrobial treatments in the age of polypharmacy. We uncovered even more synergies than antagonisms, some of which we could demonstrate as effective combinations in vivo against multi-drug resistant clinical isolates. Among them, we showed that the antiaggregant ticagrelor interferes with purine metabolism and changes the surface charge ofS. aureus,leading to strong synergies with cationic antibiotics. Overall, this exemplifies the untapped potential of approved non-antibacterial drugs to be repurposed as antibiotic adjuvants. All data can be browsed through an interactive interface (https://apps.embl.de/combact/).

Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship withEligible patients whose tumors screened positively forOf 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients withGSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive

Clinical trials in radiation oncology have improved our translational science and patient care. All patients referred to departments of radiation oncology can be invited to participate in a clinical trial with multiple venues. Study endpoints can include intradepartmental endpoints to improve workflow and patient access as well as interdepartmental clinical translational trials that include the National Clinical Trials Network (NCTN) and industry. The quality of the trial is important to trial outcome and influences interpretation of the results of the study and how the results can be applied to patient care moving forward. Clinical trials in radiation oncology to date have accomplished much, however many important questions remain as patient care matures and systemic therapies become more sophisticated and associated with specific biomarkers and cellular expression products. In this chapter we review the history of clinical trials in radiation oncology and review the current status of the structure of quality assurance in clinical trials. We will review unanswered questions and areas to study in each disease area and how to design strategy for trials to address modern unmet needs in our discipline.

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a "ticket canceller" for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene.

Objectives: Patients with anterior cruciate ligament (ACL) injury are at high risk for the development of post-traumatic osteoarthritis (PTOA), despite ACL reconstruction (ACLR). ACL injuries are frequently associated with damage of other structures within the knee, such as the meniscus. The meniscus is an important structure that provides protection for articular cartilage and stabilization of the joint. Long-term studies of PTOA after ACLR mainly used radiographs. Conventional magnetic resonance imaging (MRI) has been used in a limited number of studies to evaluate structural damages, but this only provides information on morphologic changes that occur at relatively late stages of the disease. In this study, we aim to use quantitative MRI (qMRI) to evaluate cartilage and meniscus degeneration in patients at 10 years after ACLR. Methods: This is a multi-site multi-vendor study that involves three sites and two MR platforms (Siemens 3T and Philips 3T). MRI protocols have been harmonized between sites and cross validation data were collected using phantoms. The patients are from a nested cohort within Multicenter Orthopaedic Outcomes Network (MOON) Onsite Cohort at 10 years after ACLR. Inclusion/Exclusion criteria were: 22-50 years old; ACL tear during a sport; no previous knee injury; no graft rupture during follow-up. In this preliminary report, 51 patients (age 32.8 ± 6.4 years; 25 females; body mass index [BMI] 25.7 ± 5.7 kg/m2; 40 hamstring autograft, 9 bone-patellar tendon-bone autograft, and 2 allograft) and 17 healthy control participants (age 30.8 ± 7.8 years; 10 females; BMI 23.8 ± 5.6 kg/m2) were studied. The MRI protocol included high-resolution Dual-Echo Steady State (DESS), and combined gradient echo MAPSS T1ρ and T2 mapping. Cartilage and meniscus were automatically segmented on DESS images using an in-house developed deep-learning model into medial/lateral femoral condyle (MFC/LFC), medial/lateral tibia (MT/LT), trochlear (TRO), and patellar cartilage (PAT), and medial and lateral menisci (MM/LM). Each cartilage compartment was further divided into sub-regions based on a modified MRI Osteoarthritis Knee Score (MOAKS) definition: central and posterior for MFC/LMC (cMFC/cLMC, pMFC/pLMC); anterior, central, and posterior for MT/LT (aMT/aLT, cMT/cLT, pMT/pLT); medial, central, and lateral for PAT/TRO (mPAT/mTRO, cPAT/cTRO, lPAT/lTRO). Menisci were further divided into anterior horn (aMM, aLM), central (body) (cMM, cLM), and posterior horn (pMM, pLM) subregions. These cartilage and menisci subregions were then transformed and overlaid onto the T1ρ and T2 parameter maps after co-registering the DESS image to the first echo of the 3D MAPSS sequence using the Elastix toolbox. T1ρ and T2 parameter maps were obtained by a voxel-wise two-parameter monoexponential fitting. The mean and standard deviation for each subregion was recorded and compared between three knee groups: operated and contralateral knees from patients, and control knees from healthy controls, using a mixed-effects regression model, adjusted for age, sex, and BMI. Results: For cartilage, compared to contralateral knees, operated knees in patients had significantly higher T1ρ and T2 values in MFC, MT, and TRO compartments. Looking into subcompartments, for MFC, MT, and TRO, most of the subcompartments (cMFC, pMFC; cMT, pMT; mTRO, cTRO) showed significantly higher T1ρ and T2 values compared to contralateral knees. For LFC and LT, only the posterior subcompartments showed significantly higher T1ρ and T2 values compared to contralateral knees. For PAT, no significant differences were observed between operated and contralateral knees. Compared to healthy control knees, operated knees in patients had significantly higher T1ρ and T2 values in all the six compartments. Besides, contralateral knees also showed higher T1ρ and T2 values in LFC, LT and PAT compartments compared to healthy control knees (Figure 1 for T1ρ, T2 with similar trend was not shown). For meniscus, no significant differences in T1ρ and T2 values were observed between injured and contralateral knees. Compared to healthy control knees, both operated and contralateral knees in patients had significantly higher T1ρ values in LM and significantly higher T2 values in MM (Figure 2). Conclusions: Cartilage T1ρ and T2 values were higher in operated knees compared to contralateral knees at 10 years after ACLR, except for patellar compartment. In patellar cartilage, no significant differences were observed between sides in patients, but both sides were significantly higher than control knees. Our data showed that contralateral knees after ACLR may not represent ‘healthy controls’ as there might be compensatory changes and early degeneration in contralateral knees as a result of injury and surgery to their other knee. Although we observed this general trend of higher cartilage T1ρ and T2 values in the operated knees compared to contralateral knees, no significant differences were observed in meniscus T1ρ and T2 values between sides in patients, suggesting the timing of cartilage and meniscus degeneration may be different for patients after ACLR. Meniscus T1ρ and T2 values in both sides are higher than control knees, suggesting early degeneration in meniscus in patients in both sides. The results will be confirmed with more patient data being collected in the ongoing study. The relationship between qMRI, morphological tissue changes, and patient-reported outcomes after ACLR will also be evaluated in future work. [Figure: see text][Figure: see text]

Objectives: Anterior closing wedge proximal tibia osteotomy (ACWPTO) is an effective treatment for patients with elevated posterior tibial slope (PTS) and recurrent anterior cruciate ligament (ACL) injuries. However, the preoperative planning and surgical execution can be challenging, especially with limited surgeon experience and training. The purpose of this study was to evaluate the initial design of a 3D printed patient-specific cutting guide (PSG) for performing ACWPTO in an animal model. It was hypothesized that the PSG for ACWPTO will more accurately achieve the desired PTS correction compared to a traditional free-hand (FH) technique. Methods: Thirty cadaveric porcine knees underwent lateral radiographs and computed tomography (CT) scans, and were randomly assigned to ACWPTO by PSG or FH technique. Three knees within each group were then assigned to a 10°,12°,14°,16° or 18° correction. Knees assigned to the PSG group then underwent 3D modeling and a PSG was created in SolidWorks to complete the desired PTS correction (Figure 1). Knees assigned to the FH group were templated by an experienced orthopedic surgeon for performing ACWPTO by measuring a pre-determined wedge of bone to be removed from the anterior tibia. Surgeries were then performed by an experienced (attending physician), intermediate (resident physician), and novice surgeon (medical student). Each surgeon performed ACWPTO using the PSG and FH technique for each of the intended angles of correction (10°,12°,14°,16°,18°). Following ACWPTO, post-operative radiographs and CT scans were taken to measure the PTS and the accuracy was determined as the difference between the planned and actual PTS correction (Figure 2). The accuracy, surgical time, and radiation exposure were compared between surgeons using ANOVA. Further analyses to compare the variables of interest between the FH and PSG techniques were conducted using independent samples t-tests. Results: All data are summarized in Tables 1 and 2. Overall, we found the PSG to have significantly faster surgical time (6.4 +/- 2.2 minutes versus 12.0 +/- 3.9 minutes, p < 0.001) and less radiation exposure (8.7 +/- 4.9 mGy versus 58.7 +/- 28.1 mGy, p < 0.001). While the PSG trended towards increased accuracy with radiographic measurements of PTS, this did not reach statistical significance (1.6 +/- 1.6 degrees versus 2.9 +/- 2.0 degrees, p = 0.059). The highest accuracy was found for the resident (intermediate) surgeon using the PSG (0.5 +/- 0.5 degrees), which was significantly more accurate than FH technique for this surgeon (3.6 +/- 2.5 degrees, p = 0.026). Based on CT measurements of medial and lateral PTS we found the FH technique to be as accurate as the PSG (Table 2). Conclusions: One reason for the discrepancy between radiographic and CT measurements may be the lack of standardized methods for measuring PTS in porcine knees. The proximal tibia articular surface has a more complex geometry as compared to human knees, which did make measuring consistent landmarks challenging and is one of the primary limitations of this study. Other limitations of this study include the margin of error in the design of the 3D printed PSG, and accurate placement of the PSG on the proximal tibia. Guide placement was challenging if any cartilage remained around the tibial tubercle as this was not templated in the CT design. Also, PTS measurements can be highly dependent on limb rotation and observer experience. Despite these limitations, this study demonstrated the feasibility of a 3D printed PSG for performing ACWPTO in an animal model. This PSG was as accurate as the free-hand technique based on CT measurements and trended towards improved accuracy based on radiographic measurement of PTS. Future studies are planned to investigate this design in human cadaveric specimens. [Table: see text][Figure: see text][Table: see text][Figure: see text]

The identification of the asgard archaea has fueled speculations regarding the nature of the archaeal host in eukaryogenesis and its level of complexity prior to endosymbiosis. Here, we analyzed the coding capacity of 150 eukaryotes, 1,000 bacteria, and 226 archaea, including the only cultured member of the asgard archaea. Clustering methods that consistently recover endosymbiotic contributions to eukaryotic genomes recover an asgard archaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that this group’s diversity rivals that of all other archaea combined. The number of homologs shared exclusively between asgard archaea and eukaryotes is only 27 on average. This tiny asgard archaeal-unique contribution to the root of eukaryotic protein families questions claims that archaea evolved complexity prior to eukaryogenesis. Genomic and cellular complexity remains a eukaryote-specific feature and is best understood as the archaeal host’s solution to housing an endosymbiont.

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA’s bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host’s chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.

Significance StatementEver since the first report of a new archaeal lineage, the asgardarchaea, their metagenome analyses have encouraged continued speculations on a type of cell biology ranging between that of prokaryotes and eukaryotes. While it appears a tempting notion, recent microscopic images of an asgardarchaeon suggest otherwise. We inspected the origin of eukaryotic protein families with respect to their distribution across bacteria and archaea. This reveals that the protein families shared exclusively between asgardarchaea and eukaryotes amounts to only 0.3% of the protein families conserved across all eukaryotes. Asgardarchaeal diversity is likely unrivaled across archaea, but their cell biology remains prokaryotic in nature and lends support for the importance of endosymbiosis in evolving eukaryotic traits.SummaryThe difference between pro- and eukaryotic biology is evident in their genomes, cell biology, and evolution of complex and macroscopic body plans. The lack of intermediates between the two types of cells places the endosymbiotic acquisition of the mitochondrion through an archaeal host at the event horizon of eukaryote origin. The identification of eukaryote specific proteins in a new archaeal phylum, the asgardarchaea, has fueled speculations about their cellular complexity, suggesting they could be eukaryote-like. Here we analyzed the coding capacity of 150 eukaryotes, 1000 bacteria, and 226 archaea, including the only cultured member of the asgardarchaea, CandidatusPrometheoarchaeon syntrophicumMK-D1. Established clustering methods that recover endosymbiotic contributions to eukaryotic genomes, recover an asgardarchaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that asgardarchaeal diversity rivals that of all other archaea combined. Furthermore, we show that the number of homologs shared exclusively between asgardarchaea and eukaryotes is only 27 on average. Genomic and in particular cellular complexity remains a eukaryote-specific feature and, we conclude, is best understood as the archaeal host’s solution to housing an endosymbiont and not as a preparation for obtaining one.

Antibiotic resistance is a rapidly increasing medical problem that severely limits the success of antibiotic treatments, and the identification of resistance determinants is key for surveillance and control of resistance dissemination. Horizontal transfer is the dominant mechanism for spread of resistance genes between bacteria but little is known about the original emergence of resistance genes. Here, we examined experimentally if random sequences can generate novel antibiotic resistance determinants de novo. By utilizing highly diverse expression libraries encoding random sequences to select for open reading frames that confer resistance to the last-resort antibiotic colistin in Escherichia coli, six de novo colistin resistance conferring peptides (Dcr) were identified. The peptides act via direct interactions with the sensor kinase PmrB (also termed BasS in E. coli), causing an activation of the PmrAB two-component system (TCS), modification of the lipid A domain of lipopolysaccharide and subsequent colistin resistance. This kinase-activation was extended to other TCS by generation of chimeric sensor kinases. Our results demonstrate that peptides with novel activities mediated via specific peptide-protein interactions in the transmembrane domain of a sensory transducer can be selected de novo, suggesting that the origination of such peptides from non-coding regions is conceivable. In addition, we identified a novel class of resistance determinants for a key antibiotic that is used as a last resort treatment for several significant pathogens. The high-level resistance provided at low expression levels, absence of significant growth defects and the functionality of Dcr peptides across different genera suggest that this class of peptides could potentially evolve as bona fide resistance determinants in natura., Author summary We expressed over 100 million randomly generated DNA sequences in Escherichia coli and selected 6 variants that encode peptides that provide resistance to the last-resort antibiotic colistin. We show that the selected peptides are auxiliary activators of the two-component system PmrAB, and that resistance is mediated via modifications of the cell envelope causing decreased antibiotic uptake. This is the first example where random expression libraries have been employed to select for peptides that perform an activating function by direct peptide-protein interactions in vivo, adding support to the idea that non-coding DNA can serve as a substrate for de novo gene evolution. Additionally, the described peptides expand the narrow list of colistin resistance genes and further analyses of clinical isolates will be necessary to determine if similar resistance determinants have evolved in natura.

Most mitochondrial proteins are synthesized in the cytosol as precursors that carry N-terminal presequences. After import into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase MPP, giving rise to shorter mature proteins. Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into the mitochondrial matrix and subsequently separated into two distinct enzymes that function in arginine biogenesis. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor both at its N-terminus and at an internal site between the Arg5 and Arg6 parts. The peculiar organization and biogenesis of Arg5,6 is conserved across fungi and might preserve the mode of co-translational subunit association of the arginine biosynthesis complex of the polycistronic arginine operon in prokaryotic mitochondrial ancestors. Putative MPP cleavage sites are also present at the junctions in other mitochondrial fusion proteins from fungi, plants and animals. Our data suggest that, in addition to its role as “ticket canceller” for the removal of presequences, MPP exhibits a second, widely conserved activity as internal processing peptidase for complex mitochondrial precursor proteins.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process.

Experimental evolution under controlled laboratory conditions is becoming increasingly important to address various evolutionary questions, including, for example, the dynamics and mechanisms of genetic adaptation to different growth and stress conditions. In such experiments, mutations typically appear that increase the fitness under the conditions tested (medium adaptation), but that are not necessarily of interest for the specific research question. Here, we have identified mutations that appeared during serial passage of E. coli and S. enterica in four different and commonly used laboratory media and measured the relative competitive fitness and maximum growth rate of 111 genetically re-constituted strains, carrying different single and multiple mutations. Little overlap was found between the mutations that were selected in the two species and the different media, implying that adaptation occurs via different genetic pathways. Furthermore, we show that commonly occurring adaptive mutations can generate undesired genetic variation in a population and reduce the accuracy of competition experiments. However, by introducing media adaptation mutations with large effects into the parental strain that was used for the evolution experiment, the variation (standard deviation) was decreased 10-fold, and it was possible to measure fitness differences between two competitors as small as |s| < 0.001. Michael Knopp, Lisa M. Albrecht, Erik Lundin and Ulrika Lustig contributed equally to this work.

One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.