Synthetic extremophiles via species-specific formulations improve microbial therapeutics.

Microorganisms typically used to produce food and pharmaceuticals are now being explored as medicines and agricultural supplements. However, maintaining high viability from manufacturing until use remains an important challenge, requiring sophisticated cold chains and packaging. Here we report synthetic extremophiles of industrially relevant gram-negative bacteria (Escherichia coli Nissle 1917, Ensifer meliloti), gram-positive bacteria (Lactobacillus plantarum) and yeast (Saccharomyces boulardii). We develop a high-throughput pipeline to define species-specific materials that enable survival through drying, elevated temperatures, organic solvents and ionizing radiation. Using this pipeline, we enhance the stability of E. coli Nissle 1917 by more than four orders of magnitude over commercial formulations and demonstrate its capacity to remain viable while undergoing tableting and pharmaceutical processing. We further show, in live animals and plants, that synthetic extremophiles remain functional against enteric pathogens and as nitrogen-fixing plant supplements even after exposure to elevated temperatures. This synthetic, material-based stabilization enhances our capacity to apply microorganisms in extreme environments on Earth and potentially during exploratory space travel.

Drinkable in situ-forming tough hydrogels for gastrointestinal therapeutics.

Pills are a cornerstone of medicine but can be challenging to swallow. While liquid formulations are easier to ingest, they lack the capacity to localize therapeutics with excipients nor act as controlled release devices. Here we describe drug formulations based on liquid in situ-forming tough (LIFT) hydrogels that bridge the advantages of solid and liquid dosage forms. LIFT hydrogels form directly in the stomach through sequential ingestion of a crosslinker solution of calcium and dithiol crosslinkers, followed by a drug-containing polymer solution of alginate and four-arm poly(ethylene glycol)-maleimide. We show that LIFT hydrogels robustly form in the stomachs of live rats and pigs, and are mechanically tough, biocompatible and safely cleared after 24 h. LIFT hydrogels deliver a total drug dose comparable to unencapsulated drug in a controlled manner, and protect encapsulated therapeutic enzymes and bacteria from gastric acid-mediated deactivation. Overall, LIFT hydrogels may expand access to advanced therapeutics for patients with difficulty swallowing.

Deep learning-guided discovery of an antibiotic targeting Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial Gram-negative pathogen that often displays multidrug resistance. Discovering new antibiotics against A. baumannii has proven challenging through conventional screening approaches. Fortunately, machine learning methods allow for the rapid exploration of chemical space, increasing the probability of discovering new antibacterial molecules. Here we screened ~7,500 molecules for those that inhibited the growth of A. baumannii in vitro. We trained a neural network with this growth inhibition dataset and performed in silico predictions for structurally new molecules with activity against A. baumannii. Through this approach, we discovered abaucin, an antibacterial compound with narrow-spectrum activity against A. baumannii. Further investigations revealed that abaucin perturbs lipoprotein trafficking through a mechanism involving LolE. Moreover, abaucin could control an A. baumannii infection in a mouse wound model. This work highlights the utility of machine learning in antibiotic discovery and describes a promising lead with targeted activity against a challenging Gram-negative pathogen.

Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function.

Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.

Lipid Nanoparticles for Nucleic Acid Delivery to Endothelial Cells.

Endothelial cells play critical roles in circulatory homeostasis and are also the gateway to the major organs of the body. Dysfunction, injury, and gene expression profiles of these cells can cause, or are caused by, prevalent chronic diseases such as diabetes, cardiovascular disease, and cancer. Modulation of gene expression within endothelial cells could therefore be therapeutically strategic in treating longstanding disease challenges. Lipid nanoparticles (LNP) have emerged as potent, scalable, and tunable carrier systems for delivering nucleic acids, making them attractive vehicles for gene delivery to endothelial cells. Here, we discuss the functions of endothelial cells and highlight some receptors that are upregulated during health and disease. Examples and applications of DNA, mRNA, circRNA, saRNA, siRNA, shRNA, miRNA, and ASO delivery to endothelial cells and their targets are reviewed, as well as LNP composition and morphology, formulation strategies, target proteins, and biomechanical factors that modulate endothelial cell targeting. Finally, we discuss FDA-approved LNPs as well as LNPs that have been tested in clinical trials and their challenges, and provide some perspectives as to how to surmount those challenges.

Quality Assurance for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae in Canada, 2003 to 2012.

Data from the Canadian National Gonococcal Antimicrobial Susceptibility Comparison Program, including results from 25 proficiency panels distributed between 2003 and 2012, were analyzed. The average MIC agreement between the participating laboratories ranged from 85.6% to 98.8% over the 10-year period, with the interpretation agreement ranging from 85.7% to 98.1%.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection.

Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (∼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications.

Antimicrobial susceptibilities and distribution of sequence types of Neisseria gonorrhoeae isolates in Canada: 2010.

The monitoring of antimicrobial susceptibilities in Neisseria gonorrhoeae isolates and characterization of N. gonorrhoeae multiantigen sequence types (NG-MAST, ST) provide important surveillance data as resistance rates continue to rise. A total of 2970 N. gonorrhoeae isolates were collected by Canadian provincial public health laboratories in 2010, and 1233 were submitted to the National Microbiology Laboratory for testing. The NG-MAST and minimum inhibitory concentration (MIC) by agar dilution were determined for each isolate. Of the 2970 isolates, 25.1% were resistant to penicillin, 34.6% resistant to tetracycline, 31.5% resistant to erythromycin, 35.9% resistant to ciprofloxacin, and 1.2% resistant to azithromycin. Decreased susceptibility to cefixime (MIC ≥ 0.25 mg/L) and ceftriaxone (MIC ≥ 0.125 mg/L) was identified in 3.2% and 7.3% of the isolates, respectively. The most common STs found in Canada were ST1407 (13.3%), ST3150 (11.3%), and ST3158 (9.0%), with 249 different STs identified among the isolates. Within the ST1407 group, 19.5% and 43.3% isolates have decreased susceptibility to cefixime and ceftriaxone, respectively. ST1407, the most prevalent NG-MAST in Canada in 2010, has been associated with high-level ceftriaxone MICs and with cefixime treatment failure cases worldwide. Identification and monitoring of STs and corresponding antimicrobial resistance profiles may be useful in surveillance programs and be used to inform public health actions.

Applications of machine learning in microbial natural product drug discovery.

INTRODUCTION: Natural products (NPs) are a desirable source of new therapeutics due to their structural diversity and evolutionarily optimized bioactivities. NPs and their derivatives account for roughly 70% of approved pharmaceuticals. However, the rate at which novel NPs are discovered has decreased. To accelerate the microbial NP discovery process, machine learning (ML) is being applied to numerous areas of NP discovery and development. AREAS COVERED: This review explores the utility of ML at various phases of the microbial NP drug discovery pipeline, discussing concrete examples throughout each major phase: genome mining, dereplication, and biological target prediction. Moreover, the authors discuss how ML approaches can be applied to semi-synthetic approaches to drug discovery. EXPERT OPINION: Despite the important role that microbial NPs play in the development of novel drugs, their discovery has declined due to challenges associated with the conventional discovery process. ML is positioned to overcome these limitations given its ability to model complex datasets and generalize to novel chemical and sequence space. Unsurprisingly, ML comes with its own limitations that must be considered for its successful implementation. The authors stress the importance of continuing to build high quality and open access NP datasets to further increase the utility of ML in NP discovery.

A brief guide to machine learning for antibiotic discovery.

Rising antibiotic resistance and an alarmingly lean antibiotic pipeline require the adoption of novel approaches to rapidly discover new structural and functional classes of antibiotics. Excitingly, algorithmic approaches to antibiotic discovery are sufficiently advanced to meaningfully influence the antibiotic discovery process. Indeed, once trained on high-quality datasets, contemporary machine-learning and deep-learning models can be used to perform predictions for new antibiotics across vast chemical spaces, orders of magnitude more rapidly than compounds can be screened in the laboratory. This increases the probability of discovering new antibiotics with desirable properties. In this short review, we briefly describe the utility of contemporary machine-learning and deep-learning approaches to guide the discovery of new small-molecule antibiotics and unidentified natural products. We then propose a call to action for more open sharing of high-quality screening datasets to accelerate the rate at which forthcoming antibiotic-prediction models can be trained. Together, we aim to introduce antibiotic discoverers to a sample of recent applications of contemporary algorithmic methods to facilitate the wider adoption of these powerful computational approaches.

Provider perceptions of telehealth and in-person exposure and response prevention for obsessive-compulsive disorder.

Until recently, psychotherapies, including exposure and response prevention (ERP) for obsessive-compulsive disorder (OCD), have primarily been delivered in-person. The COVID-19 pandemic required OCD providers delivering ERP to quickly transition to telehealth services. While evidence supports telehealth ERP delivery, limited research has examined OCD provider perceptions about patient characteristics that are most appropriate for this modality, as well as provider abilities to identify and address factors interfering with effective telehealth ERP. In the present study, OCD therapists (N = 113) rated the feasibility of delivering telehealth ERP relative to in-person for different (1) patient age-groups, (2) levels of OCD severity, and (3) provider ability to identify and address factors interfering with ERP during in-person and telehealth ERP (e.g., cognitive avoidance, reassurance seeking, etc.). Providers reported significantly greater feasibility of delivering telehealth ERP to individuals ages 13-to-65-years relative to other age groups assessed. Greater perceived feasibility for telehealth relative to in-person ERP was reported for lower versus higher symptom severity levels. Lastly, providers felt better able to identify and address problematic factors in-person. These findings suggest that providers should practice appropriate caution when offering telehealth ERP for certain patients with OCD. Future research may examine how to address these potential limitations of telehealth ERP delivery.

Trends in antimicrobial resistance in Neisseria gonorrhoeae isolated in Canada: 2000-2009.

BACKGROUND: Canada conducts surveillance of penicillin, tetracycline, erythromycin, spectinomycin, ciprofloxacin, cefixime, and ceftriaxone susceptibilities in Neisseria gonorrhoeae isolates to support development of national treatment guidelines for sexually transmitted infections. METHODS: N. gonorrhoeae isolates were collected by Canadian provincial public health laboratories and included isolates from males and females ranging in age from 1 to 86 years. Minimum inhibitory concentrations (MICs) were determined by agar dilution at the National Microbiology Laboratory, Public Health Agency of Canada, and MIC interpretations were based on the criteria of the Clinical Laboratory Standards Institute. RESULTS: From 2000 to 2009, 40,875 isolates of N. gonorrhoeae were tested by provincial laboratories and 10,993 of these were characterized by the Public Health Agency of Canada. There was an increasing incidence of N. gonorrhoeae isolates that were chromosomally resistant to penicillin, tetracycline, and erythromycin while the plasmid-mediated resistance strains (penicillinase-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae, and PP/tetracycline-resistant N. gonorrhoeae strain all had a declining trend. The percentage of isolates resistant to ciprofloxacin significantly increased from 1.3% in 2000 to 25.5% in 2009. Only 0.17% of isolates tested were azithromycin resistant. Between 2000 and 2009, the modal MICs for ceftriaxone increased from 0.016 µg/mL to 0.063 µg/mL. CONCLUSIONS: Ciprofloxacin resistance in N. gonorrhoeae within Canada has increased to a level where quinolones are no longer the preferred drugs for the treatment of gonococcal infections and the modal MICs for the third-generation cephalosporins have increased over time. Close monitoring of antibiotic susceptibilities are required to inform treatment options.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neural correlates of cognitive behavioral therapy response in youth with negative valence disorders: A systematic review of the literature.

BACKGROUND: Cognitive-behavioral therapy (CBT) is the gold-standard psychotherapeutic treatment for pediatric negative valence disorders. However, some youths do not respond optimally to treatment, which may be due to variations in neural functioning. METHODS: We systematically reviewed functional magnetic resonance imaging studies in youths with negative valence disorders to identify pre- and post-treatment neural correlates of CBT response. RESULTS: A total of 21 studies were identified, of overall weak to moderate quality. The most consistent findings across negative valence disorders consisted of associations of treatment response with pre- and post-treatment task-based activation and/or functional connectivity within and between the prefrontal cortex, the medial temporal lobe, and other limbic regions. Associations of CBT response with baseline and/or post-treatment activity in the striatum, precentral and postcentral gyri, medial and posterior cingulate cortices, and parietal cortex, connectivity within and between the default-mode, cognitive control, salience, and frontoparietal networks, and metrics of large-scale brain network organization, were also reported, although less consistently. LIMITATIONS: The poor quality and limited number of studies and the important heterogeneity of study designs and results considerably limit the conclusions that can be drawn from this literature. CONCLUSIONS: Despite these limitations, these findings provide preliminary evidence suggesting youths presenting certain patterns of brain function may respond better to CBT, whereas others may benefit from alternative or augmented forms of treatment.

Olfactory bulb astrocytes mediate sensory circuit processing through Sox9 in the mouse brain.

The role of transcription factors during astrocyte development and their subsequent effects on neuronal development has been well studied. Less is known about astrocytes contributions towards circuits and behavior in the adult brain. Astrocytes play important roles in synaptic development and modulation, however their contributions towards neuronal sensory function and maintenance of neuronal circuit architecture remain unclear. Here, we show that loss of the transcription factor Sox9 results in both anatomical and functional changes in adult mouse olfactory bulb (OB) astrocytes, affecting sensory processing. Indeed, astrocyte-specific deletion of Sox9 in the OB results in decreased odor detection thresholds and discrimination and it is associated with aberrant neuronal sensory response maps. At functional level, loss of astrocytic Sox9 impairs the electrophysiological properties of mitral and tufted neurons. RNA-sequencing analysis reveals widespread changes in the gene expression profiles of OB astrocytes. In particular, we observe reduced GLT-1 expression and consequential alterations in glutamate transport. Our findings reveal that astrocytes are required for physiological sensory processing and we identify astrocytic Sox9 as an essential transcriptional regulator of mature astrocyte function in the mouse OB.

Nanoparticles exhibit greater accumulation in kidney glomeruli during experimental glomerular kidney disease.

Loss and dysfunction of glomerular podocytes result in increased macromolecule permeability through the glomerular filtration barrier and nephrotic syndrome. Current therapies can induce and maintain disease remission, but cause serious and chronic complications. Nanoparticle drug carriers could mitigate these side effects by delivering drugs to the kidneys more efficiently than free drug through tailoring of carrier properties. An important extrinsic factor of nanoparticle biodistribution is local pathophysiology, which may drive greater nanoparticle deposition in certain tissues. Here, we hypothesized that a "leakier" filtration barrier during glomerular kidney disease would increase nanoparticle distribution into the kidneys. We examined the effect of nanoparticle size and disease state on kidney accumulation in male BALB/c mice. The effect of size was tested using a panel of fluorescent polystyrene nanoparticles of size 20-200 nm, due to the relevance of this size range for drug delivery applications.Experimental focal segmental glomerulosclerosis was induced using an anti-podocyte antibody that causes abrupt podocyte depletion. Nanoparticles were modified with carboxymethyl-terminated poly(ethylene glycol) for stability and biocompatibility. After intravenous injection, fluorescence from nanoparticles of size 20 and 100 nm, but not 200 nm, was observed in kidney glomeruli and peritubular capillaries. During conditions of experimental focal segmental glomerulosclerosis, the number of fluorescent nanoparticle punctae in kidney glomeruli increased by 1.9-fold for 20 and 100 nm nanoparticles compared to normal conditions. These findings underscore the importance of understanding and leveraging kidney pathophysiology in engineering new, targeted drug carriers that accumulate more in diseased glomeruli to treat glomerular kidney disease.

Beta-Catenin Mutation with Complex Chromosomal Changes in Desmoid Tumor of the Scalp: A Case Report.

Gain-of-function mutations in the beta-catenin gene ( CTNNB1 ) drive genomic instability within different cancers. However, it is unclear whether alterations in beta-catenin signaling can still lead to chromosomal rearrangements in neoplasms without metastatic potential. Here, we report a unique case, whereby a desmoid tumor of the scalp contains a missense mutation in CTNNB1 . This mutation is located at the T41 phosphorylation site-previously reported to be necessary for proper beta-catenin degradation. Online database analysis then revealed that our mutation is likely causative of many different cancers and also absent in the healthy public. Karyotyping of the desmoid tumor cells then showed complex chromosomal changes in 16 out of 20 cells examined. To treat this patient, we surgically removed both the neoplasm and underlying calvarium and then successfully reconstructed the skull and scalp. Taken together, our data suggest that increased beta-catenin signaling can lead to genomic instability in the absence of metastatic potential.

Optimized nonviral gene delivery for primary urinary renal progenitor cells to enhance cell migration.

Progressive loss of glomerular podocytes during kidney disease leads to irreversible kidney failure, and is exacerbated by the fact that podocytes are terminally differentiated epithelial cells and unable to proliferate. Regeneration of lost podocytes must therefore derive from nonpodocyte sources. Human urine-derived renal progenitor cells (uRPCs) are attractive podocyte progenitors for cell therapy applications due to their availability from patient urine and ability to migrate to injured glomeruli and differentiate into de novo podocytes after intravenous administration. Because gene delivery has emerged as an important strategy to augment the functionality and survival of cell therapies prior to injection, in this work we optimized nonviral gene delivery conditions (cell density, DNA dose, % FBS, and transfection material composition) to primary uRPCs. Using the cationic polymer-peptide conjugate VIPER for gene delivery and the Sleeping Beauty transposon/transposase constructs for gene integration, we optimized transfection parameters to achieve efficient transgene expression (up to 55% transfected cells) and stable transgene expression (>65% integration efficiency) lasting up to 10 days. With these methods, we transfected uRPCs to overexpress CXCR4, an important chemokine receptor that mediates uRPC migration to the kidneys after intravenous injection, and demonstrate that CXCR4-uRPCs exhibit enhanced migration compared to mock-transfected cells.

Target specific functions of EPL interneurons in olfactory circuits.

Inhibitory interneurons are integral to sensory processing, yet revealing their cell type-specific roles in sensory circuits remains an ongoing focus. To Investigate the mouse olfactory system, we selectively remove GABAergic transmission from a subset of olfactory bulb interneurons, EPL interneurons (EPL-INs), and assay odor responses from their downstream synaptic partners - tufted cells and mitral cells. Using a combination of in vivo electrophysiological and imaging analyses, we find that inactivating this single node of inhibition leads to differential effects in magnitude, reliability, tuning width, and temporal dynamics between the two principal neurons. Furthermore, tufted and not mitral cell responses to odor mixtures become more linearly predictable without EPL-IN inhibition. Our data suggest that olfactory bulb interneurons, through exerting distinct inhibitory functions onto their different synaptic partners, play a significant role in the processing of odor information.