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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Glomerular disease augments kidney accumulation of synthetic anionic polymers.

Polymeric drug carriers can alter the pharmacokinetics of their drug cargoes, thereby improving drug therapeutic index and reducing side effects. Understanding and controlling polymer properties that drive tissue-specific accumulation is critical in engineering targeted drug delivery systems. For kidney disease applications, targeted drug delivery to renal cells that reside beyond the charge- and size-selective glomerular filtration barrier could have clinical potential. However, there are limited reports on polymer properties that might enhance kidney accumulation. Here, we studied the effects of molecular weight and charge on the in vivo kidney accumulation of polymers in health and disease. We synthesized a panel of well-defined polymers by atom transfer radical polymerization to answer several questions. First, the biodistribution of low molecular weight (23-27 kDa) polymers composed of various ratios of neutral:anionic monomers (1:0, 1:1, 1:4) in normal mice was determined. Then, highly anionic (1:4 monomer ratio) low molecular and high molecular weight (47 kDa) polymers were tested in both normal and experimental focal segmental glomerulosclerosis (FSGS) mice, a model that results in loss of glomerular filtration selectivity. Through these studies, we observed that kidney-specific polymer accumulation increases with anionic monomer content, but not molecular weight; experimental FSGS increases kidney accumulation of anionic polymers; and anionic polymers accumulate predominantly in proximal tubule cells, with some distribution in kidney glomeruli. These findings can be applied to the design of polymeric drug carriers to enhance or mitigate kidney accumulation.

Dynamic Cellular Integration Drives Functional Assembly of the Heart's Pacemaker Complex.

Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN's structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity.

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2-4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Boronic acid copolymers for direct loading and acid-triggered release of Bis-T-23 in cultured podocytes.

We report an acid-reversible linker for triggered release of Bis-T-23, an experimental small molecule drug for kidney disease treatment that restores podocyte morphology during disease. Bis-T-23 contains catechols, which form an acid-reversible, covalent boronate ester bond with boronic acids. We synthesized phenylboronic acid-containing polymers using reversible addition-fragmentation chain transfer polymerization that were able to directly load and solubilize Bis-T-23. Because of the reversibility of the boronic ester bond, drug was released in its native form in a pH-dependent manner. The polymers rapidly trafficked into acidic compartments and did not exhibit cytotoxicity, and polymer-drug conjugates successfully delivered Bis-T-23 into cultured podocytes.

Olfactory Cued Learning Paradigm.

Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an olfactory cued learning paradigm in which specific odor pairs are coupled to either a reward or punishment to study downstream circuit changes. The following protocol outlines the basic set up for our learning paradigm. Here, we describe the equipment setup, programming of software, and method of behavioral training.

Photonic Crystal Optical Tweezers with High Efficiency for Live Biological Samples and Viability Characterization.

We propose and demonstrate a new optical trapping method for single cells that utilizes modulated light fields to trap a wide array of cell types, including mammalian, yeast, and Escherichia coli cells, on the surface of a two-dimensional photonic crystal. This method is capable of reducing the required light intensity, and thus minimizing the photothermal damage to living cells, thereby extending cell viability in optical trapping and cell manipulation applications. To this end, a thorough characterization of cell viability in optical trapping environments was performed. This study also demonstrates the technique using spatial light modulation in patterned manipulation of live cell arrays over a broad area.