Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome.

Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.

Chemigenetic Far-Red Labels and Ca2+ Indicators Optimized for Photoacoustic Imaging.

Photoacoustic imaging is an emerging modality with significant promise for biomedical applications such as neuroimaging, owing to its capability to capture large fields of view deep inside complex scattering tissue. However, widespread adoption of this technique has been hindered by a lack of suitable molecular reporters for this modality. In this work, we introduce chemigenetic labels and calcium sensors specifically tailored for photoacoustic imaging, using a combination of synthetic dyes and HaloTag-based self-labeling proteins. We rationally design and engineer far-red "acoustogenic" dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. These first-generation photoacoustic reporters show excellent performance in tissue-mimicking phantoms, with the best variants outperforming existing sensors in terms of signal intensity, sensitivity, and photostability. We demonstrate the application of these ligands for labeling HaloTag-expressing neurons in mouse brain tissue, producing strong, specifically targeted photoacoustic signal, and provide a first example of in vivo labeling with these chemigenetic photoacoustic probes. Together, this work establishes a new approach for the design of photoacoustic reporters, paving the way toward deep tissue functional imaging.

Mechanistic Insights into Curvature Formation in Synthetic Vesicles.

The mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to dynamically induce asymmetry in an artificially bilayer membrane. The molecules were designed around an extended π-conjugated hydrophobic core with tertiary amine terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life-like materials.

Exposure to elevated glucocorticoid during development primes altered transcriptional responses to acute stress in adulthood.

Early life stress (ELS) is a major risk factor for developing psychiatric disorders, with glucocorticoids (GCs) implicated in mediating its effects in shaping adult phenotypes. In this process, exposure to high levels of developmental GC (hdGC) is thought to induce molecular changes that prime differential adult responses. However, identities of molecules targeted by hdGC exposure are not completely known. Here, we describe lifelong molecular consequences of hdGC exposure using a newly developed zebrafish double-hit stress model, which shows altered behaviors and stress hypersensitivity in adulthood. We identify a set of primed genes displaying altered expression only upon acute stress in hdGC-exposed adult fish brains. Interestingly, this gene set is enriched in risk factors for psychiatric disorders in humans. Lastly, we identify altered epigenetic regulatory elements following hdGC exposure. Thus, our study provides comprehensive datasets delineating potential molecular targets mediating the impact of hdGC exposure on adult responses.

Clinical usability study of a home-based self-administration transcranial direct current stimulation for primary dysmenorrhea: A randomized controlled trial.

This study tested the usability of a home-based self-administration transcranial direct current stimulation (tDCS) device designed specifically for women's health needs. This is a single center triple blinded clinical usability study for a new wireless, Bluetooth-controlled wearable tDCS device for women's health. The study aims to evaluate the usability and effective blinding of a home-based tDCS system. A total of forty-nine women of reproductive age were randomly allocated (1:1) to receive one session of active tDCS (n = 24) or sham tDCS (n = 25) over the motor and dorsolateral prefrontal cortex. Each participant self-administered one 20-minute session without supervision following guidance on a software application alone. The System Usability Scale (SUS) and the Patient Global Impression of Change (PGIC) were used to evaluate the usability of the system. Regardless of sham or active conditions, all users found the system easy to use without the support of researchers. Usability scores were considered to be "excellent" in both groups and no significant difference was found between sham and active groups showing effective blinding of the device (Active group: 93.7 (83.1-97.5); Sham group 90 (86.2-95) p = 0.79) and PGIC (Active group: 2 (1-2.75); Sham group 2 (1-2) p = 0.99) using an unpaired t-test or non-parametric statistical tests accordingly. The new Bluetooth-controlled wearable tDCS device is easy, safe to use and completely controlled by a smartphone app. This device is focused on women's health and will be tested as an alternative treatment for chronic pelvic pain and mood disturbance associated with menstrual cycles in further research.

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Tuning of Cationic Polymer Functionality in Complex Coacervate Artificial Cells for Optimized Enzyme Activity.

Complex coacervates are a versatile platform to mimic the structure of living cells. In both living systems and artificial cells, a macromolecularly crowded condensate phase has been shown to be able to modulate enzyme activity. Yet, how enzyme activity is affected by interactions (particularly with cationic charges) inside coacervates is not well studied. Here, we synthesized a series of amino-functional polymers to investigate the effect of the type of amine and charge density on coacervate formation, stability, protein partitioning, and enzyme function. The polymers were prepared by RAFT polymerization using as monomers aminoethyl methacrylate (AEAM), 2-(dimethylamino)ethyl methacrylate (DMAEMA), imidazolepropyl methacrylamide (IPMAm), and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (TMAEMA). Membranized complex coacervate artificial cells were formed with these polycations and an anionic amylose derivative. Results show that polycations with reduced charge density result in higher protein mobility in the condensates and also higher enzyme activity. Insights described here could help guide the use of coacervate artificial cells in applications such as sensing, catalysis, and therapeutic formulations.

Matrix metalloproteinase responsive hydrogel microplates for programmed killing of invasive tumour cells.

Interactive materials are an emerging class of systems that can offer control over response and adaptivity in polymer structures towards the meso- and macroscale. Here, we use enzyme regulated cleavage of peptide crosslinkers in polymer hydrogels to release a cytotoxic therapeutic nanoparticle with an adaptable mechanism. Hydrogel microplates were formed through polyethylene glycol/peptide photoinitiated thiol-ene chemistry in a soft-lithography process to give square plates of 20 by 20 µm with a height of 10 µm. The peptide was chosen to be degradable in the presence of matrix metalloproteinase 2/9 (MMP-2/9). The hydrogel material's mechanical properties, swelling, and protease degradation were characterised. The microfabricated hydrogels were loaded with docetaxel (DTXL) containing poly(dl-lactide-co-glycolide) (PLGA) nanoparticles, and characterised for enzyme responsivity, and toxicity to MMP-2/9 overexpressing brain cancer cell line U87-MG. A 5-fold decrease in EC50 was seen compared to free DTXL, and a 20-fold decrease was seen for the MMP responsive microplates versus a non-degradable control microplate. Potential applications of this system in post-resection glioblastoma treatment are envisioned.

Trypanosomes and complement: more than one way to die?

African trypanosomes show a remarkable ability to survive as extracellular parasites in the blood and tissue spaces of an infected mammal. Throughout the infection they are exposed to the molecules and cells of the immune system, including complement. In this opinion piece, we review decades-worth of evidence about how complement affects African trypanosomes. We highlight the discovery of a trypanosome receptor for complement C3 and we critically assess three recent studies which attempt to provide a structural and mechanistic view of how this receptor helps trypanosomes to survive in the presence of complement.

Acute Stress Modulates Social Approach and Social Maintenance in Adult Zebrafish.

Stress alters social functioning in a complex manner. An important variable determining the final effects of stress is stressor intensity. However, the precise relationship between stressor intensity and social behavior is not well understood. Here, we investigate the effects of varying acute stressor intensity exposure on social behavior using adult zebrafish. We first establish a novel test using adult zebrafish that allows distinguishing fish's drive to approach a social cue and its ability to engage and maintain social interaction within the same behavioral paradigm. Next, we combined this test with a new method to deliver an acute stress stimulus of varying intensities. Our results show that both social approach and social maintenance are reduced in adult zebrafish on acute stress exposure in an intensity-dependent manner. Interestingly, lower stress intensity reduces social maintenance without affecting the social approach, while a higher stress level is required to alter social approach. These results provide evidence for a direct correlation between acute stressor intensity and social functioning and suggest that distinct steps in social behavior are modulated differentially by the acute stress level.

Complex Coacervate Materials as Artificial Cells.

Cells have evolved to be self-sustaining compartmentalized systems that consist of many thousands of biomolecules and metabolites interacting in complex cycles and reaction networks. Numerous subtle intricacies of these self-assembled structures are still largely unknown. The importance of liquid-liquid phase separation (both membraneless and membrane bound) is, however, recognized as playing an important role in achieving biological function that is controlled in time and space. Reconstituting biochemical reactions in vitro has been a success of the last decades, for example, establishment of the minimal set of enzymes and nutrients able to replicate cellular activities like the in vitro transcription translation of genes to proteins. Further than this though, artificial cell research has the aim of combining synthetic materials and nonliving macromolecules into ordered assemblies with the ability to carry out more complex and ambitious cell-like functions. These activities can provide insights into fundamental cell processes in simplified and idealized systems but could also have an applied impact in synthetic biology and biotechnology in the future. To date, strategies for the bottom-up fabrication of micrometer scale life-like artificial cells have included stabilized water-in-oil droplets, giant unilamellar vesicles (GUV's), hydrogels, and complex coacervates. Water-in-oil droplets are a valuable and easy to produce model system for studying cell-like processes; however, the lack of a crowded interior can limit these artificial cells in mimicking life more closely. Similarly membrane stabilized vesicles, such as GUV's, have the additional membrane feature of cells but still lack a macromolecularly crowded cytoplasm. Hydrogel-based artificial cells have a macromolecularly dense interior (although cross-linked) that better mimics cells, in addition to mechanical properties more similar to the viscoelasticity seen in cells but could be seen as being not dynamic in nature and limiting to the diffusion of biomolecules. On the other hand, liquid-liquid phase separated complex coacervates are an ideal platform for artificial cells as they can most accurately mimic the crowded, viscous, highly charged nature of the eukaryotic cytoplasm. Other important key features that researchers in the field target include stabilizing semipermeable membranes, compartmentalization, information transfer/communication, motility, and metabolism/growth. In this Account, we will briefly cover aspects of coacervation theory and then outline key cases of synthetic coacervate materials used as artificial cells (ranging from polypeptides, modified polysaccharides, polyacrylates, and polymethacrylates, and allyl polymers), finishing with envisioned opportunities and potential applications for coacervate artificial cells moving forward.

HaloTag-Based Reporters for Fluorescence Imaging and Biosensing.

Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self-labelling proteins, which combine a genetically encoded tag with a small-molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell-permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission.

Plasmodium species cause malaria and kill hundreds of thousands annually. The microtubule-based motor kinesin-8B is required for development of the flagellated Plasmodium male gamete, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B's essential role, we characterised the in vitro properties of kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined these motors' microtubule-bound structures using cryo-electron microscopy, which showed very similar modes of microtubule interaction in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is required for motility and depolymerisation activities of these motors. These data suggest that the mechanochemistry of Plasmodium kinesin-8Bs is functionally tuned to support flagella formation.

Invariant surface glycoprotein 65 of Trypanosoma brucei is a complement C3 receptor.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

Enhancing Diabetes Surveillance Across Alberta by Adding Laboratory and Pharmacy Data to the National Diabetes Surveillance System Methods.

OBJECTIVES: The National Diabetes Surveillance System (NDSS) case definition, which identifies a case of diabetes using administrative health records as "two physician claims or one hospital discharge abstract record, within a 2-year period for a diagnosis bearing International Classification of Disease codes for diabetes," was compared with expanded case definitions, including pharmacy (PHARM) and laboratory (LAB) data. The PHARM definition included any therapeutic antihyperglycemic agents, and the LAB definition included thresholds of ≥1 glycated hemoglobin measurement of ≥6.5%, or 2 instances of random glucose ≥11.1 mmol/L or fasting glucose ≥7.0 mmol/L. METHODS: In this retrospective study we used administrative data from the Diabetes Infrastructure for Surveillance, Evaluation, and Research project. Descriptive statistics were used to characterize participants by several subgroups. RESULTS: The NDSS identified 291,242 diabetes cases, indicating a provincial prevalence of 6.83%. Using LAB plus PHARM identified 52,040 additional cases, so the combination of NDSS or LAB or PHARM identified the largest number of cases (n=343,282), increasing the diabetes prevalence estimate to 8.06%. These 3 sources resulted in 7 unique subsets: NDSS only (n=42,606), PHARM only (n=16,310), LAB only (n=32,202), NDSS+LAB (n=32,582), NDSS+PHARM (n=22,503), LAB+PHARM (n=3,528) and NDSS+LAB+PHARM (n=193,551). Refinement using demographic and clinical characteristics allowed presumptive cases of polycystic ovarian syndrome to be excluded. CONCLUSIONS: The widely used NDSS case definition can be enhanced by the addition of LAB and PHARM data. Including PHARM and LAB data identified subsets of the diabetes population, which can maximize the yield for detection of diabetes cases in Alberta and provide a richer understanding of this population to target interventions to improve health outcomes.

Shape-specific microfabricated particles for biomedical applications: a review.

The storied history of controlled the release systems has evolved over time; from degradable drug-loaded sutures to monolithic zero-ordered release devices and nano-sized drug delivery formulations. Scientists have tuned the physico-chemical properties of these drug carriers to optimize their performance in biomedical/pharmaceutical applications. In particular, particle drug delivery systems at the micron size regime have been used since the 1980s. Recent advances in micro and nanofabrication techniques have enabled precise control of particle size and geometry-here we review the utility of microplates and discoidal polymeric particles for a range of pharmaceutical applications. Microplates are defined as micrometer scale polymeric local depot devices in cuboid form, while discoidal polymeric nanoconstructs are disk-shaped polymeric particles having a cross-sectional diameter in the micrometer range and a thickness in the hundreds of nanometer range. These versatile particles can be used to treat several pathologies such as cancer, inflammatory diseases and vascular diseases, by leveraging their size, shape, physical properties (e.g., stiffness), and component materials, to tune their functionality. This review highlights design and fabrication strategies for these particles, discusses their applications, and elaborates on emerging trends for their use in formulations.

Myosin VI regulates the spatial organisation of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

Inhibiting parasite proliferation using a rationally designed anti-tubulin agent.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.