Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Enhancing the tropism of bacteria via genetically programmed biosensors.

Engineered bacteria for therapeutic applications would benefit from control mechanisms that confine the growth of the bacteria within specific tissues or regions in the body. Here we show that the tropism of engineered bacteria can be enhanced by coupling bacterial growth with genetic circuits that sense oxygen, pH or lactate through the control of the expression of essential genes. Bacteria that were engineered with pH or oxygen sensors showed preferential growth in physiologically relevant acidic or oxygen conditions, and reduced growth outside the permissive environments when orally delivered to mice. In syngeneic mice bearing subcutaneous tumours, bacteria engineered with both hypoxia and lactate biosensors coupled through an AND gate showed increased tumour specificity. The multiplexing of genetic circuits may be more broadly applicable for enhancing the localization of bacteria to specified niches.

Engineered Bacterial Production of Volatile Methyl Salicylate.

The engineering of microbial metabolic pathways over the last two decades has led to numerous examples of cell factories used for the production of small molecules. These molecules have an array of utility in commercial industries and as in situ expressed biomarkers or therapeutics in microbial applications. While most efforts have focused on the production of molecules in the liquid phase, there has been increasing interest in harnessing microbes' inherent ability to generate volatile compounds. Here, we optimized and characterized the production of methyl salicylate, an aromatic compound found mainly in plants, using a common lab strain of E. coli. We utilized genetic components from both microbes and plants to construct the volatile metabolite circuit cassette. In order to maximize production, we explored expression of methyl salicylate precursors, upregulation of expression by increasing ribosomal binding strength and codon optimization of the methyl transferase gene obtained from plant Petunia x hybrida. Last, we validated and quantified the production of methyl salicylate with liquid chromatography or gas chromatography mass spectrometry (LC-MS or GC-MS) and found that the codon optimized strain with precursor supplementation yielded the highest production compared to the other strains. This work characterizes an optimized metabolite producing genetic circuit and sets the stage for creation of an engineered bacteria diagnostic to be used in volatile assays.

Can an interactive case-based activity help bridge the theory-practice gap in operative dentistry?

INTRODUCTION: A theory-practice gap in pre-doctoral dental education is a common source of stress for dental students. An interactive, small-group, case-based activity was designed to bridge the gap between pre-clinical and clinical experiences. The aim of our study was to assess the effectiveness of the case-based activity by evaluating students' comfort level in operative procedures. MATERIALS AND METHODS: Over 5 years, a total of 172 second-year students from the classes of 2017 through 2021 participated in the case-based activity delivered after the completion of the core operative dentistry course. The exercise included a pre-activity online quiz, an in-class case-based session and a laboratory exercise. Students' self-reported comfort levels in performing operative procedures were collected by surveys at three different times. They included the post-course survey distributed after the completion of the core operative dentistry course, the post-activity survey distributed after the completion of the case-based activity, and the follow-up survey distributed after students completed their first operative procedures in clinic. RESULTS: There was a 93% response rate. The average rating of all eight statements revealed statistically significant increase in students' comfort level after completing the case-based activity and after performing their first operative procedures in the teaching practice. CONCLUSION: This observation suggests that the case-based activity was effective in raising students' comfort levels. The activity may serve as an important tool in bridging the theory-practice gap between pre-clinical and clinical operative experiences.

A Systematic Network of Autism Primary Care Services (SYNAPSE): A Model of Coproduction for the Management of Autism Spectrum Disorder.

The prevalence of Autism Spectrum Disorder (ASD) is growing rapidly, affecting 1 in 59 children in the United States in 2018. Individuals with ASD currently receive fragmented care that threatens their health and well-being. Challenges of autism care include disconnections between the medical system and school supports, poor care coordination between primary care and specialists, and saturation of neuropsychiatry-based centers' capacity to care for the ASD population. ASD treatment also lacks of a coordinated system of care for patients' multi-system comorbidities. Families are calling for an ASD care delivery system to meet their needs and the needs of their children. To serve people with ASD and their medical and other providers, we propose a coordinated approach to care grounded in primary care. We call the model the "Systematic Network of Autism Primary Care Services (SYNAPSE)." We develop the model by applying the frameworks of "coproduction" of care and chronic disease management. In this Commentary we discuss the model's rationale, underpinnings, and the implications for clinical practice. We advance these ideas to align with policy makers' recognition of the importance of primary care for ASD, as reflected by the most recent Interagency Autism Coordinating Committee (IACC) meeting at the National Institute of Mental Health.

Advances in bacterial cancer therapies using synthetic biology.

Synthetic biology aims to apply engineering principles to biology by modulating the behavior of living organisms. An emerging application of this field is the engineering of bacteria as a cancer therapy by the programming of therapeutic, safety, and specificity features through genetic modification. Here, we review progress in this engineering including the targeting of bacteria to tumors, specific sensing and response to tumor microenvironments, remote induction methods, and controllable release of therapeutics. We discuss the most prominent bacteria strains used and their specific properties and the types of therapeutics tested thus far. Finally, we note current challenges, such as genetic stability, that researchers must address for successful clinical implementation of this novel therapy in humans.

Recovery of a human natural antibody against the noncollagenous-1 domain of type IV collagen using humanized models.

BACKGROUND: Anti-glomerular basement membrane nephritis and Goodpasture syndrome result from autoantibody (Ab)-mediated destruction of kidney and lung. Ab target the noncollagenous 1 (NC1) domain of alpha3(IV) collagen, but little is known about Ab origins or structure. This ignorance is due in part to the inability to recover monoclonal Ab by transformation of patients' blood cells. The aim of this study was to assess the suitability of two humanized models for this purpose. METHODS: NOD-scid-gamma immunodeficient mice were engrafted either with human CD34+ hematopoietic stem cells (HSC) (Hu-HSC mice) and immunized with alpha3(IV)NC1 collagen containing the Goodpasture epitopes or with nephritis patients' peripheral blood leukocytes (PBL) (Hu-PBL mice). After in vivo immune cell development and/or expansion, recovered human B cells were Epstein Barr virus (EBV)-transformed, screened for antigen (Ag) binding, electrofused with a mouse-human heterohybridoma, subcloned, and human Ab RNA sequenced by PCR after reverse transcription to cDNA. Flow cytometry was used to assess human B cell markers and differentiation in Hu-PBL mice. RESULTS: Sequence analysis of a human Ab derived from an immunized Hu-HSC mouse and reactive with alpha3(IV)NC1 collagen reveals that it is encoded by unmutated heavy and light chain genes. The heavy chain complementarity determining region 3, a major determinant of Ag binding, contains uncommon motifs, including an N-region somatically-introduced highly hydrophobic tetrapeptide and dual cysteines encoded by a uniquely human IGHD2-2 Ab gene segment that lacks a murine counterpart. Comparison of human and mouse autoantibodies suggests that structurally similar murine Ab may arise by convergent selection. In contrast to the Hu-HSC model, transformed human B cells are rarely recovered from Hu-PBL mice, in which human B cells terminally differentiate and lose expression of EBV receptor CD21, thus precluding their transformation and recovery. CONCLUSIONS: Hu-HSC mice reveal that potentially pathogenic B cells bearing unmutated Ig receptors reactive with the NC1 domain on alpha3(IV) collagen can be generated in, and not purged from, the human preimmune repertoire. Uniquely human gene elements are recruited to generate the antigen binding site in at least a subset of these autoantibodies, indicating that humanized models may provide insights inaccessible using conventional mouse models.