The literature documenting the value of drug-like molecules found in natural products is vast. Although many dietary and herbal remedies have been found to be effective for treating intestinal inflammation, the identification of their active components has lagged behind. In this study, we find that a major ginger component, furanodienone (FDN), is a selective pregnane X receptor (PXR) ligand with agonistic transcriptional outcomes. We show that FDN binds within a sub-pocket of the PXR ligand binding domain (LBD), with subsequent alterations in LBD structure. Using male mice, we show that orally provided FDN has potent PXR-dependant anti-inflammatory outcomes that are colon-specific. Increased affinity and target gene activation in the presence of synergistically acting agonists indicates further opportunities for augmenting FDN activity, efficacy and safety. Collectively, these results support the translational potential of FDN as a therapeutic agent for the treatment and prevention of colonic diseases. Here the authors discover that furanodienone (FDN), a small molecule derived from ginger, exhibits PXR agonist activity selective for the intestine and show that when FDN is administered orally in pharmacological doses ameliorates induced colitis in male mice.

Uric acid disequilibrium is implicated in chronic hyperuricemia-related diseases. Long-term monitoring and lowering of serum uric acid levels may be crucial for diagnosis and effective management of these conditions. However, current strategies are not sufficient for accurate diagnosis and successful long-term management of hyperuricemia. Moreover, drug-based therapeutics can cause side effects in patients. The intestinal tract plays an important role in maintaining healthy serum acid levels. Hence, we investigated the engineered human commensal Escherichia coli as a novel method for diagnosis and long-term management of hyperuricemia. To monitor changes in uric acid concentration in the intestinal lumen, we developed a bioreporter using the uric acid responsive synthetic promoter, pucpro, and uric acid binding Bacillus subtilis PucR protein. Results demonstrated that the bioreporter module in commensal E. coli can detect changes in uric acid concentration in a dose-dependent manner. To eliminate the excess uric acid, we designed a uric acid degradation module, which overexpresses an E. coli uric acid transporter and a B. subtilis urate oxidase. Strains engineered with this module degraded all the uric acid (250 µM) found in the environment within 24 h, which is significantly lower (p < 0.001) compared to wild type E. coli. Finally, we designed an in vitro model using human intestinal cell line, Caco-2, which provided a versatile tool to study the uric acid transport and degradation in an environment mimicking the human intestinal tract. Results showed that engineered commensal E. coli reduced (p < 0.01) the apical uric acid concentration by 40.35% compared to wild type E. coli. This study shows that reprogramming E. coli holds promise as a valid alternative synthetic biology therapy to monitor and maintain healthy serum uric acid levels.

Clostridioides difficile infection (CDI) results in significant morbidity and mortality in hospitalised patients. The pathogenesis of CDI is intrinsically related to the ability of C. difficile to shuffle between active vegetative cells and dormant endospores through the processes of germination and sporulation. Here, we hypothesise that dysregulation of microbiome-mediated bile salt metabolism contributes to CDI and that its alleviation can limit the pathogenesis of CDI. We engineer a genetic circuit harbouring a genetically encoded sensor, amplifier and actuator in probiotics to restore intestinal bile salt metabolism in response to antibiotic-induced microbiome dysbiosis. We demonstrate that the engineered probiotics limited the germination of endospores and the growth of vegetative cells of C. difficile in vitro and further significantly reduced CDI in model mice, as evidenced by a 100% survival rate and improved clinical outcomes. Our work presents an antimicrobial strategy that harnesses the host-pathogen microenvironment as the intervention target to limit the pathogenesis of infection.

There is a global increase in cases of hyperuricemia over the last 10 years. A critical component of serum uric acid control is the transport of uric acid to the intestinal lumen, which accounts for 30% of the uric acid eliminated from the serum. This mini review looks at two important aspects of elevated uric acid: the dynamics of intestinal uric acid transport and hyperuricemia co-morbidities. Elevated serum uric acid can lead to gout and it can also impact other diseases such as diabetes, cardiovascular diseases and nervous system diseases. The level of uric acid in the intestine could be related to the potential for uric acid to impact other morbidities. We review the evidence for this and what it would mean for persons with elevated serum uric acid.

Reprogrammed glucose-responsive, insulin + cells (“β-like”) exhibit the potential to bypass the hurdles of exogenous insulin delivery in treating diabetes mellitus. Current cell-based therapies-transcription factor regulation, biomolecule-mediated enteric signaling, and transgenics - have demonstrated the promise of reprogramming either mature or progenitor gut cells into surrogate “β-like” cells. However, there are predominant challenges impeding the use of gut “β-like” cells as clinical replacements for insulin therapy. Reprogrammed “β-like” gut cells, even those of enteroendocrine origin, mostly do not exhibit glucose – potentiated insulin secretion. Despite the exceptionally low conversion rate of gut cells into surrogate “β-like” cells, the therapeutic quantity of gut “β-like” cells needed for normoglycemia has not even been established. There is also a lingering uncertainty regarding the functionality and bioavailability of gut derived insulin. Herein, we review the strategies, challenges, and opportunities in the generation of functional, reprogrammed “β-like” cells.

In rats, the effects of Piracetam (P), the prototype of nootropic drugs, were studied on a very widely used model of behavioral disturbance: the learned helplessness (LH) phenomenon. In this model, exposure to uncontrollable and unsignalled shocks impairs subsequent escape-avoidance learning. In a first experiment, this deficit was abolished by 200 mg/kg of P, and to a lesser extent, by a 100 mg/kg dose, administered before the training session. In non-stressed animals, no dose of P was able to have a facilitatory effect on escape-avoidance. In a second experiment, the administration of P, not before the training session as in Experiment I, but before the stress, had no effect on the LH phenomenon regardless of the dose.

The etiology of inflammatory bowel diseases (IBDs) frequently results in the uncontrolled inflammation of intestinal epithelial linings and the local environment. Here, we hypothesized that interferon-driven immunomodulation could promote anti-inflammatory effects. To test this hypothesis, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce and secrete a type III interferon, interferon lambda 1 (IFNL1), in response to nitric oxide (NO), a well-known colorectal inflammation marker. We then validated the anti-inflammatory effects of the engineered EcN strains in two in vitro models: a Caco-2/Jurkat T cell coculture model and a scaffold-based 3D coculture IBD model that comprises intestinal epithelial cells, myofibroblasts, and T cells. The IFNL1-expressing EcN strains upregulated Foxp3 expression in T cells and thereafter reduced the production of pro-inflammatory cytokines such as IL-13 and -33, significantly ameliorating inflammation. The engineered strains also rescued the integrity of the inflamed epithelial cell monolayer, protecting epithelial barrier integrity even under inflammation. In the 3D coculture model, IFNL1-expressing EcN treatment enhanced the population of regulatory T cells and increased anti-inflammatory cytokine IL-10. Taken together, our study showed the anti-inflammatory effects of IFNL1-expressing probiotics in two in vitro IBD models, demonstrating their potential as live biotherapeutics for IBD immunotherapy.

John C. March Matthew Wook Chang There has been considerable hype surrounding synthetic biology since its inception. Researchers laid claim to the potential of finally realizing our biological future through genetic manipulation. Unfortunately, early work indicated just how challenging harnessing biology to work for us in the way that electricity and chemistry already had. However, over the past 15 to 20 years the field has learned a great deal about the possibilities and pitfalls of forging microbes for specific applications. This issue presents some of the successes that have finally begun to pay off for this still-nascent field. We have gathered six articles from some of the most promising young minds in the field of synthetic biology: looking for their take on where the field is going and to share with the community their latest developments. You will find herein a meticulous review by Rottinghaus, Amrofell, and Moon, describing the very latest in microbial engineering for medical applications (https://doi.org/10.1002/biot.201900319). It is in this area that the field has made the perhaps most progress and this review captures some of the most promising efforts in diagnosing, treating, and preventing disease. The authors also discuss one of the biggest bottlenecks in the development of new strains to treat novel diseases: that of an experimental system akin to the high throughput systems that are frequently used to build strains. The limits of the animal model system are also discussed with ideas about how to replace it. An article by Tham and co-workers digs deeper into one specific medical presentation, that of atopic dermatitis (https://doi.org/10.1002/biot.201900322). Here, the concept of the biome is stretched to include skin microbes. Such an approach holds promise for many epidermal maladies. The authors discuss the ramifications of altering the skin microbiome to have less of Staphylococcus aureus and more “beneficial” microbial communities. Such tinkering can lead to unintended dysbiosis and the authors discuss how this can be managed. One of the ways that microbial pathogens evade detection and removal from the body is to gather in biofilms. The paper by Liu and co-workers from Chun Loong Ho's lab examines the role of the biofilm in protecting harmful bacteria and the biochemical processes that maintain it (https://doi.org/10.1002/biot.201900320). There are three classes of attack used against biofilms: physical, chemical, and biological. This review presents these with a perspective on what works and what is unlikely to do so. Over the last ten years, the relationship between gut microbiomes and obesity has been increasingly elucidated. Lim, Lee, and Ooi have provided some clues as to what causative mechanisms in that relationship are (https://doi.org/10.1002/biot.202000013). The explosion of obesity as a global pandemic has been widely reported. What is less well-known is how obesity and factors such as host genetics, mode of childbirth delivery, diet, lifestyle habits, and use of antibiotics can all play deciding roles in gut microbial health as well as in obesity. Connecting the temporal events is challenging and this review gives the reader a great place to start putting together the pieces of this complex relationship. It has been known for some time that the biochemical landscape of the human intestine changes constantly with changes in our health and diet. What has been much less known is how this plays out in controlling metabolism. The interaction between microbial populations in the gut and their host is mediated through numerous chemical transformations and exchanges. One such exchange has been linked to several metabolic disorders, including diabetes. Sasaki, Sasaki, and Kondo present their original research on how molecular structure plays a role in the balance of organic acids in the large bowel (https://doi.org/10.1002/biot.201900523). The same acids that facilitate interspecies communication in the gut play a role in directing the body's response to nutrients. Changes in how gut bacteria parse propionate, acetate, and butyrate can have profound impacts on how we metabolize and respond to nutrients. This fascinating paper dives into the conversion chemistry of different metabolites that keep metabolism tightly regulated. Finally, the last article from Lei Dai's group revisits the concept of “live biotherapeutics” and how they can be improved upon to be more effective with fewer side-effects (https://doi.org/10.1002/biot.202000155). This article deftly moves between many of the success stories now happening in synthetic biology. From phage therapy for controlling microbial population densities in a consortium to deploying engineered bacteria that can recognize a tumor, swim to it, and dispatch it from the intestine, the authors do a great job of presenting material that demonstrates that success has started to infiltrate the synthetic biological ranks. These are just a handful of bright young investigators, building more sustainable and compatible technologies for healing. It is expected that a future issue of this magazine might include work on sustainable technologies, such as in chemical production or waste conversion to value-added products or electricity from batteries comprised of electrochemical microbes. We are proud to present these innovators of the biological realm and hope you will check back regularly to see what they and yet to be named others are working on next. Thank you and enjoy!

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

The goal of this study was to develop a scaffold for the generation of an artificial intestine that specifically mimics the architecture and biomechanical properties of the native small intestine, and to evaluate the scaffold in vitro and in vivo. Scaffolds mimicking the microarchitecture of native intestine were fabricated from poly(glycerol sebacate) (PGS) with a thickness of 647 mu m (+/- 241 mu m) and villus height of 340 mu m (+/- 29.5 mu m). The scaffolds showed excellent biological properties, as 71.4% (+/- 7.2%) and 58.7% (+/- 12.7%) mass remained after 5 weeks of in vitro exposure to control and digestive media, respectively. Tensile properties of the scaffolds approached those of native porcine intestine and scaffolds maintained their mechanical properties over 6 weeks based on rheometer measurements. Scaffolds accommodated intestinal epithelial stem cells and demonstrated maintenance of size and microarchitecture after 12 weeks of omental implantation in mice. There was an expected amount of inflammation, but less tissue infiltration and tissue formation than anticipated. In conclusion, we developed novel scaffolds using PGS that mimic the microarchitecture and mechanical properties of native intestine with promise for use in artificial intestine for individuals with short bowel syndrome. Graphical abstract Impact Statement This study is significant because it demonstrates an attempt to design a scaffold specifically for small intestine using a novel fabrication method, resulting in an architecture that resembles intestinal villi. In addition, we use the versatile polymer poly(glycerol sebacate) (PGS) for artificial intestine, which has tunable mechanical and degradation properties that can be harnessed for further fine-tuning of scaffold design. Moreover, the utilization of PGS allows for future development of growth factor and drug delivery from the scaffolds to promote artificial intestine formation.

Background: Obsessive-compulsive disorder (OCD) is often a life-long disorder with high psychosocial impairment. Serotonin reuptake inhibitors (SRIs) are the only FDA approved drugs, and approximately 50% of patients are non-responders when using a criterion of 25% to 35% improvement with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS). About 30% are non-responders to combined first-line therapies (SRIs and exposure and response prevention). Previous research (one open, one randomized clinical trial) has demonstrated that Kundalini Yoga (KY) meditation can lead to an improvement in symptoms of obsessive-compulsive severity. We expand here with a larger trial. Design: This trial compared two parallel run groups [KY vs. Relaxation Response meditation (RR)]. Patients were randomly allocated based on gender and Y-BOCS scores. They were told two different (unnamed) types of meditation would be compared, and informed if one showed greater benefits, the groups would merge for 12 months using the more effective intervention. Raters were blind in Phase One (0-4.5 months) to patient assignments, but not in Phase Two. Main Outcome Measures: Primary outcome variable, clinician-administered Y-BOCS. Secondary scales: Dimensional Yale-Brown Obsessive Compulsive Scale (clinician-administered), Profile of Mood Scales, Beck Anxiety Inventory, Beck Depression Inventory, Clinical Global Impression, Short Form 36 Health Survey. Results: Phase One: Baseline Y-BOCS scores: KY mean = 26.46 (SD 5.124; N = 24), RR mean = 26.79 (SD = 4.578; N = 24). An intent-to-treat analysis with the last observation carried forward for dropouts showed statistically greater improvement with KY compared to RR on the Y-BOCS, and statistically greater improvement on five of six secondary measures. For completers, the Y-BOCS showed 40.4% improvement for KY (N = 16), 17.9% for RR (N = 11); 31.3% in KY were judged to be in remission compared to 9.1% in RR. KY completers showed greater improvement on five of six secondary measures. At the end of Phase Two (12 months), patients, drawn from the initial groups, who elected to receive KY continued to show improvement in their Y-BOCS scores. Conclusion: KY shows promise as an add-on option for OCD patients unresponsive to first line therapies. Future studies will establish KY's relative efficacy compared to Exposure and Response Prevention and/or medications, and the most effective treatment schedule.

Short bowel syndrome (SBS) is a major cause of morbidity and mortality in the pediatric population, for which treatment options are limited. To develop novel approaches for the treatment of SBS, we now focus on the development of a tissue-engineered intestine (also known as an "artificial intestine"), in which intestinal stem cells are cultured onto an absorbable bioscaffold, followed by implantation into the host. To enhance the translational potential of these preclinical studies, we have developed three clinically relevant models in neonatal piglets, which approximate the size of the human infant and were evaluated after implantation and establishment of intestinal continuity over the long term. The models included (1) a staged, multioperation approach; (2) a single operation with a de-functionalized loop of small intestine; and (3) a single operation with an intestinal bypass. The first model had complications related to multiple operations in a short time period, including surgical site infections and wound hernias. The second model avoided wound complications, but was associated with high ostomy output, local skin breakdown, and systemic dehydration with associated electrolyte imbalances. The third model was the most effective, although resulted in stoma prolapse. In summary, we have now developed and evaluated three operative methods for the long-term evaluation of the artificial intestine in the piglet, and conclude that a single operation with a de-functionalized loop of small intestine may be an optimal approach for evaluation over the long term.

Nontyphoidal serovars of Salmonella enterica are pathogenic bacteria that are common causes of food poisoning. Whereas Salmonella mechanisms of host cell invasion, inflammation, and pathogenesis are mostly well established, a new possible mechanism of immune evasion is being uncovered. Programmed death ligand 1 (PD-L1) is an immunosuppressive membrane protein that binds to activated T cells via their PD-1 receptor and thereby halts their activation. PD-L1 expression plays an essential role in the immunological tolerance of self-antigens but is also exploited for immune evasion by pathogen-infected cells and cancer cells. Here, we show for the first time that Salmonella infection of intestinal epithelial cells causes the induction of PD-L1. The increased expression of PD-L1 through Salmonella infection was seen in both human and rat intestinal epithelial cell lines. We determined that cellular invasion by the bacteria is necessary for PD-L1 induction, potentially indicating that Salmonella strains are delivering mediators from inside the host cell that trigger the increased PD-L1 expression. Using knockout mutants, we determined that this effect largely originates from the Salmonella pathogenicity island 2. We also show for the first time in any cell type that Salmonella combined with gamma interferon (IFN-γ) causes a synergistic induction of PD-L1. Finally, we show that Salmonella plus IFN-γ induction of PD-L1 decreased the cytokine production of activated T cells. Understanding Salmonella immune evasion strategies could generate new therapeutic targets and help to manipulate PD-L1 expression in other diseases.

For therapies targeting diseases of the gastrointestinal tract, we and others envision probiotic bacteria that synthesize and excrete biotherapeutics at disease sites. Toward this goal, we have engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide (NO), an intestinal biomarker for Crohn's disease (CD). This is accomplished by co-expressing the pore forming protein TolAIII with the biologic, granulocyte macrophage-colony stimulating factor (GM-CSF). We have additionally engineered these bacteria to accumulate at sites of elevated NO by engineering their motility circuits and controlling pseudotaxis. Importantly, because we have focused on in vitro test beds, motility and biotherapeutics production are spatiotemporally characterized. Together, the targeted recognition, synthesis, and biomolecule delivery comprises a "smart" probiotics platform that may have utility in the treatment of CD. Further, this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively.

BACKGROUND:Human hookworm larvae arrest development until they enter an appropriate host. This makes it difficult to access the larvae for studying larval development or host-parasite interactions. While there are in vivo and in vitro animal models of human hookworm infection, there is currently no human, in vitro model. While animal models have provided much insight into hookworm biology, there are limitations to how closely this can replicate human infection. Therefore, we have developed a human, in vitro model of the initial phase of hookworm infection using intestinal epithelial cell culture. RESULTS:Co-culture of the human hookworm Ancylostoma ceylanicum with the mucus-secreting, human intestinal epithelial cell line HT-29-MTX resulted in activation of infective third-stage larvae, as measured by resumption of feeding. Larvae were maximally activated by direct contact with fully differentiated HT-29-MTX intestinal epithelial cells. HT-29-MTX cells treated with A. ceylanicum larvae showed differential gene expression of several immunity-related genes. CONCLUSIONS:Co-culture with HT-29-MTX can be used to activate A. ceylanicum larvae. This provides an opportunity to study the interaction of activated larvae with the human intestinal epithelium.

The development of in vitro artificial small intestines that realistically mimic in vivo systems will enable vast improvement of our understanding of the human gut and its impact on human health. Synthetic in vitro models can control specific parameters, including (but not limited to) cell types, fluid flow, nutrient profiles and gaseous exchange. They are also "open" systems, enabling access to chemical and physiological information. In this work, we demonstrate the importance of gut surface topography and fluid flow dynamics which are shown to impact epithelial cell growth, proliferation and intestinal cell function. We have constructed a small intestinal bioreactor using 3-D printing and polymeric scaffolds that mimic the 3-D topography of the intestine and its fluid flow. Our results indicate that TEER measurements, which are typically high in static 2-D Transwell apparatuses, is lower in the presence of liquid sheer and 3-D topography compared to a flat scaffold and static conditions. There was also increased cell proliferation and discovered localized regions of elevated apoptosis, specifically at the tips of the villi, where there is highest sheer. Similarly, glucose was actively transported (as opposed to passive) and at higher rates under flow.

This chapter begins by stating the importance of considering the concept of anxiety and its heterogeneity. It defines anxiety to set the scene for the rest of the discussion in this chapter. The chapter then makes a distinction between anxiety and anxiety disorders. The chapter looks at research in the areas of anxiety and anxiety disorders. Recent research has expanded our understanding of the phenomena linked to the concepts of anxiety and anxiety disorder. The chapter states a hope that major advances in research will now occur thanks to a better understanding of the conditions, which hopefully will lead to a more fruitful set of psychiatric classifications.

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Short bowel syndrome is a major cause of morbidity and mortality in children. Despite decades of experience in the management of short bowel syndrome, current therapy is primarily supportive. Definitive treatment often requires intestinal transplantation, which is associated with significant morbidity and mortality. In order to develop novel approaches to the treatment of short bowel syndrome, we and others have focused on the development of an artificial intestine, by placing intestinal stem cells on a bioscaffold that has an absorptive surface resembling native intestine, and taking advantage of neovascularization to develop a blood supply. This review will explore recent advances in biomaterials, vascularization, and progress toward development of a functional epithelium and mesenchymal niche, highlighting both success and ongoing challenges in the field.