Understanding 'error' in the forensic sciences: A primer.

This paper distils seven key lessons about 'error' from a collaborative webinar series between practitioners at Victoria Police Forensic Services Department and academics. It aims to provide the common understanding of error necessary to foster interdisciplinary dialogue, collaboration and research. The lessons underscore the inevitability, complexity and subjectivity of error, as well as opportunities for learning and growth. Ultimately, we argue that error can be a potent tool for continuous improvement and accountability, enhancing the reliability of forensic sciences and public trust. It is hoped the shared understanding provided by this paper will support future initiatives and funding for collaborative developments in this vital domain.

Phytotherapeutic potential against MRSA: mechanisms, synergy, and therapeutic prospects.

BACKGROUND: Rising resistance to antimicrobials, particularly in the case of methicillin-resistant Staphylococcus aureus (MRSA), represents a formidable global health challenge. Consequently, it is imperative to develop new antimicrobial solutions. This study evaluated 68 Chinese medicinal plants renowned for their historical applications in treating infectious diseases. METHODS: The antimicrobial efficacy of medicinal plants were evaluated by determining their minimum inhibitory concentration (MIC) against MRSA. Safety profiles were assessed on human colorectal adenocarcinoma (Caco-2) and hepatocellular carcinoma (HepG2) cells. Mechanistic insights were obtained through fluorescence and transmission electron microscopy (FM and TEM). Synergistic effects with vancomycin were investigated using the Fractional Inhibitory Concentration Index (FICI). RESULTS: Rheum palmatum L., Arctium lappa L. and Paeonia suffructicosaas Andr. have emerged as potential candidates with potent anti-MRSA properties, with an impressive low MIC of 7.8 µg/mL, comparable to the 2 µg/mL MIC of vancomycin served as the antibiotic control. Crucially, these candidates demonstrated significant safety profiles when evaluated on Caco-2 and HepG2 cells. Even at 16 times the MIC, the cell viability ranged from 83.3% to 95.7%, highlighting their potential safety. FM and TEM revealed a diverse array of actions against MRSA, such as disrupting the cell wall and membrane, interference with nucleoids, and inducing morphological alterations resembling pseudo-multicellular structures in MRSA. Additionally, the synergy between vancomycin and these three plant extracts was evident against MRSA (FICI < 0.5). Notably, aqueous extract of R. palmatum at 1/4 MIC significantly reduced the vancomycin MIC from 2 µg/mL to 0.03 µg/mL, making a remarkable 67-fold decrease. CONCLUSIONS: This study unveil new insights into the mechanistic actions and pleiotropic antibacterial effectiveness of these medicinal plants against resistant bacteria, providing robust evidence for their potential use as standalone or in conjunction with antibiotics, to effectively combat antimicrobial resistance, particularly against MRSA.

Conductive block copolymer elastomers and psychophysical thresholding for accurate haptic effects.

Electrotactile stimulus is a form of sensory substitution in which an electrical signal is perceived as a mechanical sensation. The electrotactile effect could, in principle, recapitulate a range of tactile experience by selective activation of nerve endings. However, the method has been plagued by inconsistency, galvanic reactions, pain and desensitization, and unwanted stimulation of nontactile nerves. Here, we describe how a soft conductive block copolymer, a stretchable layout, and concentric electrodes, along with psychophysical thresholding, can circumvent these shortcomings. These purpose-designed materials, device layouts, and calibration techniques make it possible to generate accurate and reproducible sensations across a cohort of 10 human participants and to do so at ultralow currents (≥6 microamperes) without pain or desensitization. This material, form factor, and psychophysical approach could be useful for haptic devices and as a tool for activation of the peripheral nervous system.

Capturing acyl-enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate identification.

Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl-enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36USP) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12-13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.

Intraoperative Pedal Pressure Changes Offers Another Quantitative Assessment for Revascularization.

BACKGROUND: Lower extremity angiography is one of the most prevalent vascular procedures performed, generally via the contralateral common femoral artery. The use of retrograde pedal artery access to perform angiography has long been reserved as a "bail-out" technique to help cross chronic total occlusions that were not amenable from an antegrade approach. Recently, there have been reports and discussions involving increased utilization of pedal access for primary revascularization. The purpose of this study is to describe the outcomes of pedal access as a primary approach and to propose a novel evaluation of distal perfusion changes associated with interventions using direct pressure measurements. METHODS: A retrospective observational study evaluating all patients who underwent lower extremity angiography via retrograde pedal access between December 1, 2020, and June 30, 2021, within a single health-care system spanning 3 hospitals was performed. Demographics, comorbidities, procedural indications, and details were all recorded. Hemodynamic measurements were obtained and recorded upon initial pedal access and post intervention with a pressure transducer connected directly to the access sheath. Outcomes were analyzed with paired t-test. RESULTS: Twenty-eight angiograms using primary pedal access for endovascular intervention were performed during the study period. Most patients were African American (75%) females (57.1%) with hypertension (89.3%), hyperlipidemia (78.6%), diabetes (85.7%), coronary artery disease (64.3%), and current tobacco users (57.1%). The most prevalent indication for angiography was nonhealing wounds (67.9%). Pedal access was mostly achieved via the anterior tibial artery (79%). Sixty-three vessels were treated during the 28 angiograms (averaging 2.3 vessels per angiogram), most commonly the superficial femoral (27%), anterior tibial (25%), and popliteal (22%) arteries. Balloon angioplasty with or without stenting (98.5%) was predominately performed with an overall technical success rate of 94%. The mean preintervention and postintervention pressures were 36.5 mm Hg (standard deviation [SD] 25.7) and 83.4 mm Hg (SD 19.5), respectively. The mean change in pressure after intervention was 46.9 mm Hg (SD 23.3) (Table 3). There was a statistically significant difference detected between preintervention and postintervention pressure (P < 0.001) (Figure 1). There were no major amputations or adverse cardiovascular events at a mean first follow-up duration of 89 days. Six of the total 28 patients (21.4%) underwent repeat endovascular intervention on the ipsilateral extremity within a median of 45 (interquartile range 22.5-62.3) days. CONCLUSIONS: Primary pedal access is a viable option for performing lower extremity angiographic interventions. A significant increase in pedal artery pressure can be observed after angiographic intervention from retrograde pedal artery access. Further studies are necessary to define the clinical prognostic importance of these findings in relation to wound healing rates.

Global diversity, recurrent evolution, and recent selection on amylase structural haplotypes in humans.

The adoption of agriculture, first documented ~12,000 years ago in the Fertile Crescent, triggered a rapid shift toward starch-rich diets in human populations. Amylase genes facilitate starch digestion and increased salivary amylase copy number has been observed in some modern human populations with high starch intake, though evidence of recent selection is lacking. Here, using 52 long-read diploid assemblies and short read data from ~5,600 contemporary and ancient humans, we resolve the diversity, evolutionary history, and selective impact of structural variation at the amylase locus. We find that amylase genes have higher copy numbers in populations with agricultural subsistence compared to fishing, hunting, and pastoral groups. We identify 28 distinct amylase structural architectures and demonstrate that nearly identical structures have arisen recurrently on different haplotype backgrounds throughout recent human history. AMY1 and AMY2A genes each exhibit multiple duplications/deletions with mutation rates >10,000-fold the SNP mutation rate, whereas AMY2B gene duplications share a single origin. Using a pangenome graph-based approach to infer structural haplotypes across thousands of humans, we identify extensively duplicated haplotypes present at higher frequencies in modern day populations with traditionally agricultural diets. Leveraging 533 ancient human genomes we find that duplication-containing haplotypes (i.e. haplotypes with more amylase gene copies than the ancestral haplotype) have increased in frequency more than seven-fold over the last 12,000 years providing evidence for recent selection in West Eurasians. Together, our study highlights the potential impacts of the agricultural revolution on human genomes and the importance of long-read sequencing in identifying signatures of selection at structurally complex loci.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli.

The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and is not copied by host polymerases but is selectively copied by an orthogonal DNA polymerase (O-DNAP), which does not copy the genome. We designed mutant O-DNAPs that selectively increase the mutation rate of the orthogonal replicon by two to four orders of magnitude. We demonstrate the utility of our system for accelerated continuous evolution by evolving a 150-fold increase in resistance to tigecycline in 12 days. And, starting from a GFP variant, we evolved a 1000-fold increase in cellular fluorescence in 5 days.

The Microbial Degradation of Natural and Anthropogenic Phosphonates.

Phosphonates are compounds containing a direct carbon-phosphorus (C-P) bond, which is particularly resistant to chemical and enzymatic degradation. They are environmentally ubiquitous: some of them are produced by microorganisms and invertebrates, whereas others derive from anthropogenic activities. Because of their chemical stability and potential toxicity, man-made phosphonates pose pollution problems, and many studies have tried to identify biocompatible systems for their elimination. On the other hand, phosphonates are a resource for microorganisms living in environments where the availability of phosphate is limited; thus, bacteria in particular have evolved systems to uptake and catabolize phosphonates. Such systems can be either selective for a narrow subset of compounds or show a broader specificity. The role, distribution, and evolution of microbial genes and enzymes dedicated to phosphonate degradation, as well as their regulation, have been the subjects of substantial studies. At least three enzyme systems have been identified so far, schematically distinguished based on the mechanism by which the C-P bond is ultimately cleaved-i.e., through either a hydrolytic, radical, or oxidative reaction. This review summarizes our current understanding of the molecular systems and pathways that serve to catabolize phosphonates, as well as the regulatory mechanisms that govern their activity.

Deciphering the role of recurrent FAD-dependent enzymes in bacterial phosphonate catabolism.

Phosphonates-compounds containing a direct C-P bond-represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized "hydrolytic" pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.

The transparency of quantitative empirical legal research published in highly ranked law journals (2018-2020): an observational study.

Background: Scientists are increasingly concerned with making their work easy to verify and build upon. Associated practices include sharing data, materials, and analytic scripts, and preregistering protocols. This shift towards increased transparency and rigor has been referred to as a "credibility revolution." The credibility of empirical legal research has been questioned in the past due to its distinctive peer review system and because the legal background of its researchers means that many often are not trained in study design or statistics. Still, there has been no systematic study of transparency and credibility-related characteristics of published empirical legal research. Methods: To fill this gap and provide an estimate of current practices that can be tracked as the field evolves, we assessed 300 empirical articles from highly ranked law journals including both faculty-edited journals and student-edited journals. Results: We found high levels of article accessibility (86%, 95% CI = [82%, 90%]), especially among student-edited journals (100%). Few articles stated that a study's data are available (19%, 95% CI = [15%, 23%]). Statements of preregistration (3%, 95% CI = [1%, 5%]) and availability of analytic scripts (6%, 95% CI = [4%, 9%]) were very uncommon. (i.e., they collected new data using the study's reported methods, but found results inconsistent or not as strong as the original). Conclusion: We suggest that empirical legal researchers and the journals that publish their work cultivate norms and practices to encourage research credibility. Our estimates may be revisited to track the field's progress in the coming years.

On the potential roles of phosphorus in the early evolution of energy metabolism.

Energy metabolism in extant life is centered around phosphate and the energy-dense phosphoanhydride bonds of adenosine triphosphate (ATP), a deeply conserved and ancient bioenergetic system. Yet, ATP synthesis relies on numerous complex enzymes and has an autocatalytic requirement for ATP itself. This implies the existence of evolutionarily simpler bioenergetic pathways and potentially primordial alternatives to ATP. The centrality of phosphate in modern bioenergetics, coupled with the energetic properties of phosphorylated compounds, may suggest that primordial precursors to ATP also utilized phosphate in compounds such as pyrophosphate, acetyl phosphate and polyphosphate. However, bioavailable phosphate may have been notably scarce on the early Earth, raising doubts about the roles that phosphorylated molecules might have played in the early evolution of life. A largely overlooked phosphorus redox cycle on the ancient Earth might have provided phosphorus and energy, with reduced phosphorus compounds potentially playing a key role in the early evolution of energy metabolism. Here, we speculate on the biological phosphorus compounds that may have acted as primordial energy currencies, sources of environmental energy, or sources of phosphorus for the synthesis of phosphorylated energy currencies. This review encompasses discussions on the evolutionary history of modern bioenergetics, and specifically those pathways with primordial relevance, and the geochemistry of bioavailable phosphorus on the ancient Earth. We highlight the importance of phosphorus, not only in the form of phosphate, to early biology and suggest future directions of study that may improve our understanding of the early evolution of bioenergetics.

Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs.

Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNAPyl) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNAPyl sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNAPyl pairs (the existing classes +N, A and B, and newly defined classes C and S). Most of the PylRS clusters belong to classes that were unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyrrolysyl-tRNAs with unusual structures, we resolve 80% of the pairwise specificities required to make quintuply orthogonal PylRS/tRNAPyl pairs; we control the remaining specificities by engineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS/tRNAPyl pairs, 1,324 triply orthogonal pairs, 128 quadruply orthogonal pairs and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.

Astrocytic control of extracellular GABA drives circadian timekeeping in the suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity, which in turn control circadian behavior. Intercellular signals, mediated by neuropeptides, synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level timekeeping is unclear. How can a GABAergic circuit sustain circadian cycles of electrical activity, when such increased neuronal firing should become inhibitory to the network? To explore this paradox, we show that SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counterintuitively, runs in antiphase to neuronal activity, with a prolonged peak in circadian night and a pronounced trough in circadian day. Resolving this unexpected relationship, we found that [GABA]e is regulated by GABA transporters (GATs), with uptake peaking during circadian day, hence the daytime trough and nighttime peak. This uptake is mediated by the astrocytically expressed transporter GAT3 (Slc6a11), expression of which is circadian-regulated, being elevated in daytime. Clearance of [GABA]e in circadian day facilitates neuronal firing and is necessary for circadian release of the neuropeptide vasoactive intestinal peptide, a critical regulator of TTFL and circuit-level rhythmicity. Finally, we show that genetic complementation of the astrocytic TTFL alone, in otherwise clockless SCN, is sufficient to drive [GABA]e rhythms and control network timekeeping. Thus, astrocytic clocks maintain the SCN circadian clockwork by temporally controlling GABAergic inhibition of SCN neurons.

Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Cell-of-origin-specific proteomics of extracellular vesicles.

The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native purification of intact EVs from a target cell, via a bioorthogonal tetrazine ligation, leading to copurification of the largely unlabeled EV proteome from the same cell. SIEVE enables capture of EV proteins at levels comparable with those obtained by antibody-based methods, which capture all EVs regardless of cellular origin, and at levels 20× higher than direct capture of SORT-labeled proteins. Using proteomic analysis, we analyze nonlabeled cargo proteins of EVs and show that the enhanced sensitivity of SIEVE allows for unbiased and comprehensive analysis of EV proteins from subpopulations of cells as well as for cell-specific EV proteomics in complex coculture systems. SIEVE can be applied with high efficiency in a diverse range of existing model systems for cell-cell communication and has direct applications for cell-of-origin EV analysis and for protein biomarker discovery.

Bilateral common carotid artery mycotic aneurysms in the setting of intravenous drug abuse.

Carotid mycotic aneurysms are rare, and fewer than five case reports have described carotid mycotic aneurysms due to intravenous drug abuse. Rare bilateral intracranial mycotic carotid aneurysms have been reported, although a review of literature revealed no cases of bilateral extracranial carotid aneurysms. We have reported the case of a 41-year-old man who had presented with intermittent fevers, headaches, and myalgias of 2 weeks' duration. He was found to have bilateral carotid artery mycotic aneurysms after intravenous drug abuse with neck injections. We used a management strategy entailing unilateral endovascular balloon control with open surgical resection followed by placement of a saphenous vein graft. The contralateral aneurysm was managed nonoperatively with antibiotics.

Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles.

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.