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IntroductionLameness is a major welfare challenge facing the dairy industry worldwide. Monitoring herd lameness prevalence, and early detection and therapeutic intervention are important aspects of lameness control in dairy herds. The objective of this study was to evaluate the performance of a commercially available video surveillance system for automatic detection of dairy cattle lameness (CattleEye Ltd).MethodsThis was achieved by first measuring mobility score agreement between CattleEye and two veterinarians (Assessor 1 and Assessor 2), and second, by investigating the ability of the CattleEye system to detect cows with potentially painful foot lesions. We analysed 6,040 mobility scores collected from three dairy farms. Inter-rate agreement was estimated by calculating percentage agreement (PA), Cohen’s kappa (κ) and Gwet’s agreement coefficient (AC). Data regarding the presence of foot lesions were also available for a subset of this dataset. The ability of the system to predict the presence of potentially painful foot lesions was tested against that of Assessor 1 by calculating measures of accuracy, using lesion records during the foot trimming sessions as reference.ResultsIn general, inter-rater agreement between CattleEye and either human assessor was strong and similar to that between the human assessors, with PA and AC being consistently above 80% and 0.80, respectively. Kappa agreement between CattleEye and the human scorers was in line with previous studies (investigating agreement between human assessors) and within the fair to moderate agreement range. The system was more sensitive than Assessor 1 in identifying cows with potentially painful lesions, with 0.52 sensitivity and 0.81 specificity compared to the Assessor’s 0.29 and 0.89 respectively.DiscussionThis pilot study showed that the CattleEye system achieved scores comparable to that of two experienced veterinarians and was more sensitive than a trained veterinarian in detecting painful foot lesions.

Lameness represents a significant challenge for the dairy cattle industry, resulting in economic losses and reduced animal health and welfare. The existence of underlying genomic variation for lameness associated traits has the potential to improve selection strategies by using genomic markers. Therefore, the aim of this study was to identify genomic regions and potential candidate genes associated with lameness traits. Lameness related lesions and digital cushion thickness were studied using records collected by our research team, farm records, and a combination of both. Genome-wide analyses were performed to identify significant genomic effects, and a combination of single SNP association analysis and regional heritability mapping was used to identify associated genomic regions. Significant genomic effects were identified for several lameness related traits: Two genomic regions were identified on chromosome 3 associated with digital dermatitis and interdigital hyperplasia, one genomic region on chromosome 23 associated with interdigital hyperplasia, and one genomic region on chromosome 2 associated with sole haemorrhage. Candidate genes in those regions are mainly related to immune response and fibroblast proliferation. Quantitative trait loci (QTL) identified in this study could enlighten the understanding of lameness pathogenesis, providing an opportunity to improve health and welfare in dairy cattle with the addition of these regions into selection programs.

Stream insects are essential components of aquatic and terrestrial ecosystem structure and function. Terrestrial stages are important components of terrestrial food webs, and flight-capable individuals are responsible for long-distance dispersal. Horizontal migrations by flying or crawling adults away from stream channels that link insects to riparian food webs and movements across catchment boundaries are well established through empirical research, but studies examining vertical migration of adult stream insects into forest canopies are generally lacking. This study focused on differences in adult Plecoptera and Trichoptera abundance at ground level versus the riparian canopy and differences in abundances among summer and autumn sampling periods to empirically demonstrate use of canopy ecosystems by stream insects. Malaise traps at ground level and canopy traps placed 8 to 10 m above the stream at four sites in the Mosquito Creek watershed (Pennsylvania) were used to examine vertical migration. Larval assemblages were collected and compared to adult assemblage to investigate patterns of local migration in the catchment. We found significantly more stream insects at ground level than in the forest canopy for Trichoptera, Plecoptera, and all individual plecopteran families, but a meaningful number of individuals were found in the riparian canopy. Canopy abundances were similar to abundances captured in adjacent ground-level habitats in other studies. Comparisons of adult and larval abundances among sites, taxa, and stages indicated site- and taxon-specific patterns for vertical movement into riparian canopies. Demonstrating that adult stream insects utilize riparian forest canopies indicates that riparian forest conservation should be prioritized over reforestation and that several potential research questions exist to inform riparian management.

In the face of a steady decline in dairy cow fertility over several decades, using hormones to assist reproduction has become common. In the European Union, hormones are prescription-only medicines, giving veterinary practitioners a central role in their deployment. This study explored the clinical and ethical beliefs of practitioners, and provides data on their current prescribing practices. During 2011, 93 practitioners working in England completed a questionnaire (95% response rate). Of the 714 non-organic farms they attended, only 4 farms (0.6%) never used hormones to assist the insemination of lactating dairy cows. Practitioners agreed (>80%) that hormones improve fertility and farm businesses profitability. They also agreed (>80%) that if farmers are able to tackle management issues contributing to poor oestrus expression, then over a five year period these outcomes would both improve, relative to using hormones instead. If management issues are addressed instead of prescribing hormones, practitioners envisaged a less favourable outcome for veterinary practices profitability (p

Neospora caninum is an intracellular protozoan parasite which is a major cause of abortion in cattle worldwide. It forms persistent infections which recrudesce during pregnancy leading to foetal infection and in a proportion of cases, abortion. The mechanisms underlying abortion are not understood. In this study, recrudescence of a persistent infection in eight naturally infected cows occurred between 20 and 33 weeks of gestation. Animals were killed at the time of recrudescence and parasites were detected in the placentae and foetuses. An active maternal immune response consisting of an infiltration of CD4+ and CD8+ T cells and a 46-49 fold increase in interferon-γ and interleukin-4 mRNA was detected. Other cytokines, notably interleukin-12 p40, interleukin-10 and tumour necrosis factor-α were also significantly increased and Major Histocompatibility Class II antigen was expressed on maternal and foetal epithelial and stromal fibroblastoid cells. Significantly, despite the presence of an active maternal immune response in the placenta, all the foetuses were alive at the time of maternal euthanasia. There was evidence of parasites within foetal tissues; their distribution was restricted to the central nervous system and skeletal muscle and their presence was associated with tissue necrosis and a non-suppurative inflammatory response involving lymphocytes and macrophages, irrespective of the gestational age of the foetus. Whilst an active maternal immune response to a pathogen in the placenta is generally considered to be damaging to the foetal trophoblast, our findings suggest that the presence of a parasite-induced maternal immune response in the placenta is not detrimental to foetal survival but may contribute to the control of placental parasitosis.

Chemical permeation enhancers (CPEs) represent a prevalent and safe strategy to enable noninvasive drug delivery across skin-like biological barriers such as the tympanic membrane (TM). While most existing CPEs interact strongly with the lipid bilayers in the stratum corneum to create defects as diffusion paths, their interactions with the delivery system, such as polymers forming a hydrogel, can compromise gelation, formulation stability, and drug diffusion. To overcome this challenge, differing interactions between CPEs and the hydrogel system are explored, especially those with sodium dodecyl sulfate (SDS), an ionic surfactant and a common CPE, and those with methyl laurate (ML), a nonionic counterpart with a similar length alkyl chain. Notably, the use of ML effectively decouples permeation enhancement from gelation, enabling sustained delivery across TMs to treat acute otitis media (AOM), which is not possible with the use of SDS. Ciprofloxacin and ML are shown to form a pseudo-surfactant that significantly boosts transtympanic permeation. The middle ear ciprofloxacin concentration is increased by 70-fold in vivo in a chinchilla AOM model, yielding superior efficacy and biocompatibility than the previous highest-performing formulation. Beyond improved efficacy and biocompatibility, this single-CPE formulation significantly accelerates its progression toward clinical deployment., (© 2024 Wiley‐VCH GmbH.)

Colloidal and nanoparticle self-assembly enables the creation of ordered structures with a variety of electronic and photonic functionalities. The outcomes of the self-assembly processes used to synthesize such structures, however, strongly depend on the uniformity of the individual nanoparticles. Here, we explore the simplest form of particle size dispersity-bidispersity-and its impact on the self-assembly process. We investigate the robustness of self-assembling bcc-type crystals via isotropic interaction potentials in binary systems with increasingly disparate particle sizes by determining their terminal size ratio-the most extreme size ratio at which a mixed binary bcc crystal forms. Our findings show that two-well pair potentials produce bcc crystals that are more robust with respect to particle size ratio than one-well pair potentials. This suggests that an improved self-assembly process is accomplished with a second attractive length scale encoded in the particle-particle interaction, which stabilizes the second-nearest neighbor shell. In addition, we document qualitative differences in the process of ordering and disordering: in bidisperse systems of particles interacting via one-well potentials, we observe a breakdown of order prior to demixing, while in systems interacting via two-well potentials, demixing occurs first and bcc continues to form in parts of the droplet down to low size ratios., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. Stabilizing improperly ubiquitinated proteins via target-specific deubiquitination is thus a critical therapeutic goal. Building off the major advances in targeted protein degradation (TPD) using bifunctional small-molecule degraders, targeted protein stabilization (TPS) modalities have been described recently. However, these rely on a limited set of chemical linkers and warheads, which are difficult to generate de novo for new targets and do not exist for classically "undruggable" targets. To address the limited reach of small molecule-based degraders, we previously engineered ubiquibodies (uAbs) by fusing computationally-designed "guide" peptides to E3 ubiquitin ligase domains for modular, CRISPR-analogous TPD. Here, we expand the TPS target space by engineering "deubiquibodies" (duAbs) via fusion of computationally-designed guides to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. To demonstrate duAb modularity, we swap in new target-binding peptides designed via our generative language models to stabilize diverse target proteins, including key tumor suppressor proteins such as p53 and WEE1, as well as heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. In total, our duAb system represents a simple, programmable, CRISPR-analogous strategy for TPS., Competing Interests: Competing Interests Statement P.C., L.H., and M.P.D. are listed as inventors on US Patent Application 63/541,921: “Peptide-Guided Protein Stabilizers and Uses Thereof”. P.C. and M.D. are co-founded of UbiquiTx, Inc., which commercializes genetically-encoded proteome editing technologies, and are co-inventors of duAb patents. P.C.’s interests are reviewed and managed by Duke University in accordance with their conflict-of-interest policies. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict-of-interest policies.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Pep tide Pr ioritization via CLIP ( PepPrCLIP ) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro . Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins., Competing Interests: Competing Interests P.C., K.P, and S.B. are listed as inventors for U.S. Provisional Application No. 63/344,820, entitled: “Contrastive Learning for Peptide Based Degrader Design and Uses Thereof.” P.C. is listed as an inventor for U.S. Provisional Application No. 63/032,513, entitled: “Minimal Peptide Fusions for Targeted Intracellular Degradation.” P.C. and M.P.D. are co-founders of and have financial interests in UbiquiTx, Inc. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict-of-interest policies. P.C.’s interests are reviewed and managed by Duke University in accordance with their conflict-of-interest policies. S.B. is a current paid consultant for UbiquiTx, Inc, and K.P. is a former paid consultant for UbiquiTx, Inc.

Hydrogen sulfide (H 2 S) is an endogenously produced gasotransmitter involved in many physiological processes that are integral to proper cellular functioning. Due to its profound anti-inflammatory and antioxidant properties, H 2 S plays important roles in preventing inflammatory skin disorders and improving wound healing. Transdermal H 2 S delivery is a therapeutically viable option for the management of such disorders. However, current small-molecule H 2 S donors are not optimally suited for transdermal delivery and typically generate electrophilic byproducts that may lead to undesired toxicity. Here, we demonstrate that H 2 S release from metal-organic frameworks (MOFs) bearing coordinatively unsaturated metal centers is a promising alternative for controlled transdermal delivery of H 2 S. Gas sorption measurements and powder X-ray diffraction (PXRD) studies of 11 MOFs support that the Mg-based framework Mg 2 (dobdc) (dobdc 4- = 2,5-dioxidobenzene-1,4-dicarboxylate) is uniquely well-suited for transdermal H 2 S delivery due to its strong yet reversible binding of H 2 S, high capacity (14.7 mmol/g at 1 bar and 25 °C), and lack of toxicity. In addition, Rietveld refinement of synchrotron PXRD data from H 2 S-dosed Mg 2 (dobdc) supports that the high H 2 S capacity of this framework arises due to the presence of three distinct binding sites. Last, we demonstrate that transdermal delivery of H 2 S from Mg 2 (dobdc) is sustained over a 24 h period through porcine skin. Not only is this significantly longer than sodium sulfide but this represents the first example of controlled transdermal delivery of pure H 2 S gas. Overall, H 2 S-loaded Mg 2 (dobdc) is an easily accessible, solid-state source of H 2 S, enabling safe storage and transdermal delivery of this therapeutically relevant gas.

Plant natural products (PNPs) play important roles in plant physiology and have been applied across diverse fields of human society. Understanding their biosynthetic pathways informs plant evolution and meanwhile enables sustainable production through metabolic engineering. However, the discovery of PNP biosynthetic pathways remains challenging due to the diversity of enzymes involved and limitations in traditional gene mining approaches. In this review, we will summarize state-of-the-art strategies and recent examples for predicting and characterizing PNP biosynthetic pathways, respectively, with multiomics-guided tools and heterologous host systems and share our perspectives on the systematic pipelines integrating these various bioinformatic and biochemical approaches., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic β-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Controllable synthesis of homochiral nano/micromaterials has been a constant challenge for fabricating various stimuli-responsive chiral sensors. To provide an avenue to this goal, we report electrospinning as a simple and economical strategy to form continuous homochiral microfibers with strain-sensitive chiroptical properties. First, electrospun homochiral microfibers from self-assembled cadmium sulfide (CdS) quantum dot magic-sized clusters (MSCs) are produced. Highly sensitive and reversible strain sensors are then fabricated by embedding these chiroptically active fibers into elastomeric films. The chiroptical response on stretching is indicated quantitatively as reversible changes in magnitude, spectral position (wavelength), and sign in circular dichroism (CD) and linear dichroism (LD) signals and qualitatively as a prominent change in the birefringence features under cross-polarizers. The observed periodic twisted helical fibrils at the surface of fibers provide insights into the origin of the fibers' chirality. The measurable shifts in CD and LD are caused by elastic deformations of these helical fibrillar structures of the fiber. To elucidate the origin of these chiroptical properties, we used field emission-electron microscopy (FE-SEM), atomic force microscopy (AFM), synchrotron X-ray analysis, polarized optical microscopy, as well as measurements to isolate the true CD, and contributions from photoelastic modulators (PEM) and LD. Our findings thus offer a promising strategy to fabricate chiroptical strain-sensing devices with multiple measurables/observables using electric-field-assisted spinning of homochiral nano/microfibers.

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As membrane-mediated antibiotic resistance continues to evolve in Gram-positive bacteria, the development of new approaches to elucidate the membrane properties involved in antibiotic resistance has become critical. Membrane vesicles (MVs) secreted by the cytoplasmic membrane of Gram-positive bacteria contain native components, preserving lipid and protein diversity, nucleic acids, and sometimes virulence factors. Thus, MV-derived membrane platforms present a great model for Gram-positive bacterial membranes. In this work, we report the development of a planar bacterial cytoplasmic membrane-based biosensor using MVs isolated from the Bacillus subtilis WT strain that can be coated on multiple surface types such as glass, quartz crystals, and polymeric electrodes, fostering the multimodal assessment of drug-membrane interactions. Retention of native membrane components such as lipoteichoic acids, lipids, and proteins is verified. This biosensor replicates known interaction patterns of the antimicrobial compound, daptomycin, with the Gram-positive bacterial membrane, establishing the applicability of this platform for carrying out biophysical characterization of the interactions of membrane-acting antibiotic compounds with the bacterial cytoplasmic membrane. We report changes in membrane viscoelasticity and permeability that correspond to partial membrane disruption when calcium ions are present with daptomycin but not when these ions are chelated. This biomembrane biosensing platform enables an assessment of membrane biophysical characteristics during exposure to antibiotic drug candidates to aid in identifying compounds that target membrane disruption as a mechanism of action.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Creating the next generation of advanced materials will require controlling molecular architecture to a degree typically achieved only in biopolymers. Sequence-defined polymers take inspiration from biology by using chain length and monomer sequence as handles for tuning structure and function. These sequence-defined polymers can assemble into discrete structures, such as molecular duplexes, via reversible interactions between functional groups. Selectivity can be attained by tuning the monomer sequence, thereby creating the need for chemical platforms that can produce sequence-defined polymers at scale. Developing sequence-defined polymers that are specific for their complementary sequence and achieve their desired binding strengths is critical for producing increasingly complex structures for new functional materials. In this Review Article, we discuss synthetic platforms that produce sequence-defined, duplex-forming oligomers of varying length, strength and association mode, and highlight several analytical techniques used to characterize their hybridization., (© 2023. Springer Nature Limited.)

Arranging semiconducting nanocrystals into ordered superstructures is a promising platform to study fundamental light-matter interactions and develop programmable optical metamaterials. We investigated how the geometrical arrangement of CdS nanocrystals in hierarchical assemblies affects chiroptical properties. To create these structures, we controlled the evaporation of a colloidal CdS nanocrystal solution between two parallel plates. We combined in situ microscopy and computational modeling to establish a formation mechanism involving the shear-induced alignment of nanocrystal fibers and the subsequent mechanical relaxation of the stretched fibers to form Raman noodle-type band textures. The high linear anisotropy in these films shares many similarities with cholesteric liquid crystals. The films deposited on top and bottom surfaces exhibit opposite chirality. The mechanistic insights from this study are consequential to enable future advances in the design and fabrication of programmable optical metamaterials for further development of polarization-based optics toward applications in sensing, hyperspectral imaging, and quantum information technology.

Vaccines remain one of the most important pillars in preventative medicine, providing protection against a wide array of diseases by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of all living cells, viruses, and parasites as well as their known interactions with both innate and adaptive immune cells. Indeed, several licensed vaccines leverage bacterial cell-surface carbohydrates as antigens for inducing antigen-specific plasma cells secreting protective antibodies and the development of memory T and B cells. Carbohydrates have also garnered attention in other aspects of vaccine development, for example, as adjuvants that enhance the immune response by either activating innate immune responses or targeting specific immune cells. Additionally, carbohydrates can function as immunomodulators that dampen undesired humoral immune responses to entire protein antigens or specific, conserved regions on antigenic proteins. In this review, we highlight how the interplay between carbohydrates and the adaptive and innate arms of the immune response is guiding the development of glycans as vaccine components that act as antigens, adjuvants, and immunomodulators. We also discuss how advances in the field of synthetic glycobiology are enabling the design, engineering, and production of this new generation of carbohydrate-containing vaccine formulations with the potential to prevent infectious diseases, malignancies, and complex immune disorders., Competing Interests: Declaration of Competing Interest M.P.D. and M.C.J. have financial interests in Gauntlet, Inc. and Resilience, Inc. M.P.D. also has financial interests in Glycobia, Inc., MacImmune, Inc., UbiquiTX, Inc., and Versatope Therapeutics, Inc. M.P.D.'s and M.C.J. interests are reviewed and managed by Cornell University and Stanford University, respectively, in accordance with their conflict-of-interest policies. All other authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous β-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation., (© 2023. Springer Nature Limited.)

Tumor-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real-time, noninvasive monitoring of TEV-induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple-negative breast cancer cell line MDA-MB-231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine-day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV-treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV-induced EMT. Together, these results demonstrate the utility of the platform for TEV-targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Microbe-semiconductor biohybrids, which integrate microbial enzymatic synthesis with the light-harvesting capabilities of inorganic semiconductors, have emerged as promising solar-to-chemical conversion systems. Improving the electron transport at the nano-bio interface and inside cells is important for boosting conversion efficiencies, yet the underlying mechanism is challenging to study by bulk measurements owing to the heterogeneities of both constituents. Here we develop a generalizable, quantitative multimodal microscopy platform that combines multi-channel optical imaging and photocurrent mapping to probe such biohybrids down to single- to sub-cell/particle levels. We uncover and differentiate the critical roles of different hydrogenases in the lithoautotrophic bacterium Ralstonia eutropha for bioplastic formation, discover this bacterium's surprisingly large nanoampere-level electron-uptake capability, and dissect the cross-membrane electron-transport pathways. This imaging platform, and the associated analytical framework, can uncover electron-transport mechanisms in various types of biohybrid, and potentially offers a means to use and engineer R. eutropha for efficient chemical production coupled with photocatalytic materials., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Discovering natural product biosynthetic pathways of medicinal plants is challenging and laborious. Capturing the coregulation patterns of pathway enzymes, particularly transcriptomic regulation, has proven an effective method to accelerate pathway identification. In this study, we developed a yeast-based screening method to capture the protein-protein interactions (PPI) between plant enzymes, which is another useful pattern to complement the prevalent approach. Combining this method with plant multiomics analysis, we discovered four enzyme complexes and their organized pathways from kratom, an alkaloid-producing plant. The four pathway branches involved six enzymes, including a strictosidine synthase, a strictosidine β-D-glucosidase (MsSGD), and four medium-chain dehydrogenase/reductases (MsMDRs). PPI screening selected six MsMDRs interacting with MsSGD from 20 candidates predicted by multiomics analysis. Four of the six MsMDRs were then characterized as functional, indicating the high selectivity of the PPI screening method. This study highlights the opportunity of leveraging post-translational regulation features to discover novel plant natural product biosynthetic pathways., (© 2023 Wiley-VCH GmbH.)

Cyclodextrin molecules are increasingly being used in biological research and as therapeutic agents to alter membrane cholesterol content, yet there is much to learn about their interactions with cell membranes. We present a biomembrane-based organic electronic platform capable of detecting interactions of cell membrane constituents with methyl-β-cyclodextrin (MβCD). This approach enables label-free sensing and quantification of changes in membrane integrity resulting from such interactions. In this work, we employ cholesterol-containing supported lipid bilayers (SLBs) formed on conducting polymer-coated electrodes to investigate how MβCD impacts membrane resistance. By examining the outcomes of MβCD interactions with SLBs of varying cholesterol content, we demonstrate that changes in membrane permeability or resistance can be used as a functional measure for predicting cyclodextrin-mediated cholesterol extraction from cellular membranes. Furthermore, we use the SLB platforms to electronically monitor cholesterol delivery to membranes following exposure to MβCD pre-loaded with cholesterol, observing that cholesterol enrichment is commensurate with an increase in resistance. This biomembrane-based bioelectronic sensing system offers a tool to quantify the modulation of membrane cholesterol content using membrane resistance and provides information regarding MβCD-mediated changes in membrane integrity. Given the importance of membrane integrity for barrier function in cells, such knowledge is essential for our fundamental understanding of MβCD as a membrane cholesterol modulator and therapeutic delivery vehicle.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery., Competing Interests: The authors declare the following competing financial interest(s): M.P.D. has a financial interest in Gauntlet, Inc., Glycobia, Inc., MacImmune, Inc., SwiftScale, Inc., UbiquiTX, Inc., and Versatope Therapeutics, Inc. M.P.D.'s interests are reviewed and managed by Cornell University in accordance with their conflict of interest policies. All other authors declare no competing interests., (© 2023 The Authors. Published by American Chemical Society.)

Lichen secondary metabolites are well explored medicinal agents with diverse pharmacological properties. One of the important antibiotic lichen secondary metabolites is usnic acid. Its diverse medicinal profiles prompted us to explore it as a potential antitubercular molecule. Towards this direction, continuing our efforts on the discovery and development of new analogs with potent antitubercular properties we designed, synthesized, and evaluated a set of 37 usnic acid enaminone-coupled aryl-n-hexanamides (3-39). The study yielded a 3,4-dimethoxyphenyl compound (13, 5.3 µM) as the most active anti-TB molecule. The docking studies were performed on 7 different enzymes to better understand the binding modes, where it was observed that compound 13 bound strongly with glucose dehydrogenase (Gscore: - 9.03). Further antibacterial investigations revealed compound 2 with potent inhibition on Salmonella typhi and Bacillus subtilis (MIC 3 µM) and MIC values of 7 and 14 µM on Streptococcus mutans and Escherichia coli respectively. Compound 19 (3-F-5-CF 3 -phenyl) displayed encouraging antibacterial profiles against E. coli, S. typhi and S. mutans with MIC values of 10 µM respectively. Interestingly, compound 20 (2,6-difluorophenyl) also displayed good antibacterial activity against E. coli with an MIC value of 6 µM. These encouraging pharmacological results will help for better designing and developing usnic acid-based semi-synthetic derivatives as potential antimicrobial agents. A set of 37 new usnic acid enaminone-coupled aryl-n-hexanamides were synthesized and evaluated as potential antimicrobial agents. Compound 13 was identified as the most active antitubercular molecule. 13 was further docked against 7 different enzymes of tuberculosis. The molecule displayed maximum binding energy with the enzyme Glucose dehydrogenase (Gscore: - 9.03), indicating that these hexanamides possibly act by inhibiting the glucose metabolic pathway of the bacterium. Surprisingly, the intermediate hexanoic acid 2 was identified as potent antibacterial agent, acting on both gram-positive and gram-negative bacterial strains (3-14 μM). The active compounds may be subjected to structural iterations to develop further leads., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Mechano-bactericidal (MB) nanopatterns have the ability to inactivate bacterial cells by rupturing cellular envelopes. Such biocide-free, physicomechanical mechanisms may confer lasting biofilm mitigation capability to various materials encountered in food processing, packaging, and food preparation environments. In this review, we first discuss recent progress on elucidating MB mechanisms, unraveling property-activity relationships, and developing cost-effective and scalable nanofabrication technologies. Next, we evaluate the potential challenges that MB surfaces may face in food-related applications and provide our perspective on the critical research needs and opportunities to facilitate their adoption in the food industry.

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing., Competing Interests: MD and MJ have financial interests in Gauntlet, Inc. and Resilience, Inc. MD has financial interests in Glycobia, Inc, MacImmune, Inc, UbiquiTx, Inc, and Versatope Therapeutics, Inc. MD’s and MJ’s interests are reviewed and managed by Cornell University and Northwestern University, respectively, in accordance with their conflict-of-interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Williams, Warfel, Desai, Li, Lee, Wong, Nguyen, Qin, Sobol, Jewett, Chang and DeLisa.)

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Berberine is an extensively used pharmaceutical benzylisoquinoline alkaloid (BIA) derived from plants. Microbial manufacturing has emerged as a promising approach to source valuable BIAs. Here, we demonstrated the complete biosynthesis of berberine in Saccharomyces cerevisiae by engineering 19 genes including 12 heterologous genes from plants and bacteria. Overexpressing bottleneck enzymes, fermentation scale-up, and heating treatment after fermentation increased berberine titer by 643-fold to 1.08 mg L-1. This pathway also showed high efficiency to incorporate halogenated tyrosine for the synthesis of unnatural BIA derivatives that have higher therapeutical potentials. We firstly demonstrate the in vivo biosynthesis of 11-fluoro-tetrahydrocolumbamine via nine enzymatic reactions. The efficiency and promiscuity of our pathway also allow for the simultaneous incorporation of two fluorine-substituted tyrosine derivatives to 8, 3'-di-fluoro-coclaurine. This work highlights the potential of yeast as a versatile microbial biosynthetic platform to strengthen current pharmaceutical supply chain and to advance drug development., (© 2023. The Author(s).)

Plant biosynthetic gene clusters (BGCs) contain multiple physically clustered non-homologous genes that encode enzymes catalyzing diverse reactions in one plant natural product biosynthetic pathway. A growing number of plant BGCs have emerged as an underlying resource for understanding plant specialized metabolism and evolution, but the characterization remains challenging. Recent studies have demonstrated that baker's yeast can serve as a versatile platform for the characterization of plant BGCs, from single-gene characterization to multiple genes and hitherto unknown putative BGC validation and elucidation. In this review, we will summarize the strategies and examples of the applications of yeast in plant BGC characterization and share our perspective on the development of a systematic pipeline to fully leverage yeast to advance the understanding of plant BGCs and plant natural product biomanufacturing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats., (© 2023. The Author(s).)

Otitis media (OM) is the main cause of pediatric antibiotic prescriptions. Nontypeable Haemophilus influenzae (NTHi) is a major OM pathogen, which forms a biofilm that resists conventional antimicrobials and immune clearance. Thus, novel treatments that are effective against NTHi and its biofilm are urgently required. Nanozymes (often inorganic nanoparticles) mimic natural enzymes' catalytic activities to generate strong antimicrobials at the site of infection, and thus represent one of the emerging solutions to the crisis of antimicrobial resistance. They mimic natural enzymes' activities, such as generating strong antimicrobials catalytically at the site of infection, to minimize overexposure. However, that in situ generation often relies on Reactive Oxygen Species (ROS) as precursors, a prerequisite that limits the broad deployment of nanozymes. To address this challenge, we designed a cascade nanozyme that generates an antiseptic, HOBr, from a ubiquitous non-ROS, i.e. , O 2 , which successfully eradicates NTHi. The cascade nanozyme simultaneously exhibits glucose oxidase (GOx)-like activity from gold nanoparticles (AuNPs) and haloperoxidase (HPO)-mimicking activity from vanadium pentoxide nanowires (V 2 O 5 NWs) connected using dopamine (DPA). The cascade nanozyme demonstrated strong antimicrobial efficacy against NTHi and its biofilm, while showing improved biocompatibility compared to the nanozyme of V 2 O 5 NWs alone. The cascade nanozyme thus points to a material-oriented infectious disease treatment strategy, where small-molecule antimicrobials are generated in real time at the site of infection for the benefit of autonomous dosing. This strategy potentially mitigates the development of antimicrobial resistance and reduces side effects.

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked ( N -linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N -glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N -glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N -glycoproteins with advantageous biological and biophysical properties.

Otitis media (OM) is the main reason for pediatric antibiotic prescriptions. The current treatment mandates a rigorous regimen of multidose antibiotics over 5-10 days. The systemic antibiotic exposure and often prematurely terminated treatment due to the challenge of drug administration to young patients are believed to breed antibiotic resistance. To address these challenges, we designed a local treatment that converted a metabolic product (H 2 O 2 ) of an OM pathogen ( Streptococcus pneumoniae ) into a potent antiseptic (HOBr), a reaction catalyzed by locally administered vanadium pentoxide nanowires. The therapeutic, HOBr, was only synthesized in the presence of the pathogen, enabling on-demand generation of therapeutics for OM treatment. Hypohalous acids are broad-spectrum and have a long history in general disinfection applications without breeding substantial drug resistance. A single dose of the nanowire formulation eradicated OM in a standard chinchilla model in 7 days with no observable tissue toxicity or negative impact on hearing sensitivity., Competing Interests: 8Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os 2 (μ-N)(NH 3 ) 8 Cl 2 ] 3+ ) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os 2 (μ-N)(NH 3 ) 8 (OH 2 ) 2 ] 5+ ) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 ( k obs = 4.92 × 10 -3 s -1 ), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower ( k obs = 1.63 × 10 -5 s -1 ) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC 50 for MCU inhibition = 103 nM; Os245' IC 50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg -1 ), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors., Competing Interests: The authors have no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

The mitochondrial calcium uniporter (MCU) is a transmembrane protein that resides on the inner membrane of the mitochondria and mediates calcium uptake into this organelle. Given the critical role of mitochondrial calcium trafficking in cellular function, inhibitors of this channel have arisen as tools for studying the biological relevance of this process and as potential therapeutic agents. In this study, four new analogues of the previously reported Ru-based MCU inhibitor [ClRu(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 Cl]Cl 3 (Ru265) are reported. These compounds, which bear axial carboxylate ligands, are of the general formula [(RCO 2 )Ru(NH 3 ) 4 (μ-N)Ru(NH 3 ) 4 (O 2 CR)]X 3 , where X = NO 3 - or CF 3 SO 3 - and R = H ( 1 ), CH 3 ( 2 ), CH 2 CH 3 ( 3 ), and (CH 2 ) 2 CH 3 ( 4 ). These complexes were fully characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis. X-ray crystal structures of 1 and 3 were obtained, revealing the expected presence of both the linear Ru(μ-N)Ru core and axial formate and propionate ligands. The axial carboxylate ligands of complexes 1 - 4 are displaced by water in buffered aqueous solution to give the aquated compound Ru265'. The kinetics of these processes were measured by 1 H NMR spectroscopy, revealing half-lives that span 5.9-9.9 h at 37 °C. Complex 1 with axial formate ligands underwent aquation approximately twice as fast as the other compounds. In vitro cytotoxicity and mitochondrial membrane potential measurements carried out in HeLa and HEK293T cells demonstrated that none of these four complexes negatively affects cell viability or mitochondrial function. The abilities of 1 - 4 to inhibit mitochondrial calcium uptake in permeabilized HEK293T cells were assessed and compared to that of Ru265. Fresh solutions of 1 - 4 are approximately 2-fold less potent than Ru265 with IC 50 values in the range of 14.7-19.1 nM. Preincubating 1 - 4 in aqueous buffers for longer time periods to allow for the aquation reactions to proceed increases their potency of mitochondrial uptake inhibition to match that of Ru265. This result indicates that 1 - 4 are aquation-activated prodrugs of Ru265'. Finally, 1 - 4 were shown to inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules., (© 2022. The Author(s).)

Plant specialized metabolites occupy unique therapeutic niches in human medicine. A large family of plant specialized metabolites, namely plant polyketides, exhibit diverse and remarkable pharmaceutical properties and thereby great biomanufacturing potential. A growing body of studies has focused on plant polyketide synthesis using plant type III polyketide synthases (PKSs), such as flavonoids, stilbenes, benzalacetones, curcuminoids, chromones, acridones, xanthones, and pyrones. Microbial expression of plant type III PKSs and related biosynthetic pathways in workhorse microorganisms, such as Saccharomyces cerevisiae , Escherichia coli , and Yarrowia lipolytica , have led to the complete biosynthesis of multiple plant polyketides, such as flavonoids and stilbenes, from simple carbohydrates using different metabolic engineering approaches. Additionally, advanced biosynthesis techniques led to the biosynthesis of novel and complex plant polyketides synthesized by diversified type III PKSs. This review will summarize efforts in the past 10 years in type III PKS-catalyzed natural product biosynthesis in microorganisms, especially the complete biosynthesis strategies and achievements., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu and Li.)

RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Glycans, including oligosaccharides and glycoconjugates, play an integral role in modulating the biological functions of macromolecules. Many physiological and pathological processes are mediated by interactions between glycans, which has led to the use of glycans as biosensors for pathogen and biomarker detection. Elucidating the relationship between glycan structure and biological function is critical for advancing our understanding of the impact glycans have on human health and disease and for expanding the repertoire of glycans available for bioanalysis, especially for diagnostics. Such efforts have been limited by the difficulty in obtaining sufficient quantities of homogenous glycan samples needed to resolve the exact relationships between glycan structure and their structural or modulatory functions on a given glycoconjugate. Synthetic strategies offer a viable route for overcoming these technical hurdles. In recent years, microfluidics have emerged as powerful tools for realizing high-throughput and reproducible syntheses of homogenous glycans for the potential use in functional studies. This critical review provides readers with an overview of the microfluidic technologies that have been developed for chemical and enzymatic glycan synthesis. The advantages and limitations associated with using microreactor platforms to improve the scalability, productivity, and selectivity of glycosylation reactions will be discussed, as well as suggested future work that can address certain pitfalls., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)