Transitional Care In Patients With Hirschsprung Disease: Those Left Behind.

BACKGROUND: The long term effects of Hirschsprung disease are clinically variable. Improved understanding of challenges patients may face as adults can help inform transitional care management. OBJECTIVE: To explore the outcomes and transitional care experiences in adult patients with Hirschsprung. DESIGN: Cohort study. SETTING: Single center. PATIENTS: All patients treated for Hirschsprung 1977-2001 (aged >18 at time of survey July 2018-2019). Eligible patients were sent validated multi-domain surveys as well as qualitative questions regarding their transitional care. MAIN OUTCOME MEASURES: Status of transitional care, bowel function and quality of life assessment. Qualitative analysis of transitional care experience. RESULTS: Of 139 patients, 20 had received transition care (10 had at least 1 visit but had been discharged and 10 were receiving ongoing follow-up). These patients had inferior bowel function and quality of life scores at follow-up. Twenty-three (17%) patients had issues with soiling at time of discharge, 7 received transitional care. Of these, 9/23 (39%) had a normal bowel function score (≥17), 5/23 (22%) had a poor score (<12) and one patient had since had a stoma formation. Eighteen (13%) patients had active moderate-severe issues related to bowel function, only 5 had been transitioned, and just 2 remained under ongoing care. Importantly, when these patients were discharged from our pediatric center, at a median age of 14 (IQR 12-16) years, 10/17 had no perceptible bowel issues, suggesting a worsening of function after discharge. LIMITATIONS: The retrospective design and reliance on clinical notes to gather information of discharge status as well as patient recall of events. CONCLUSION: There remains a small but significant proportion of Hirschsprung patients for whom bowel function either remains or becomes a major burden. These results support a need to better stratify patients requiring transitional care, and ensure a clear route to care if their status changes after discharge. See Video Abstract.

The electron transport chain of Shewanella oneidensis MR-1 can operate bidirectionally to enable microbial electrosynthesis.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.

Flux-Balance Analysis and Mobile CRISPRi-Guided Deletion of a Conditionally Essential Gene in Shewanella oneidensis MR-1.

Carbon-neutral production of valuable bioproducts is critical to sustainable development but remains limited by the slow engineering of photosynthetic organisms. Improving existing synthetic biology tools to engineer model organisms to fix carbon dioxide is one route to overcoming the limitations of photosynthetic organisms. In this work, we describe a pipeline that enabled the deletion of a conditionally essential gene from the Shewanella oneidensis MR-1 genome. S. oneidensis is a simple bacterial host that could be used for electricity-driven conversion of carbon dioxide in the future with further genetic engineering. We used Flux Balance Analysis (FBA) to model carbon and energy flows in central metabolism and assess the effects of single and double gene deletions. We modeled the growth of deletion strains under several alternative conditions to identify substrates that restore viability to an otherwise lethal gene knockout. These predictions were tested in vivo using a Mobile-CRISPRi gene knockdown system. The information learned from FBA and knockdown experiments informed our strategy for gene deletion, allowing us to successfully delete an "expected essential" gene, gpmA. FBA predicted, knockdown experiments supported, and deletion confirmed that the "essential" gene gpmA is not needed for survival, dependent on the medium used. Removal of gpmA is a first step toward driving electrode-powered CO2 fixation via RuBisCO. This work demonstrates the potential for broadening the scope of genetic engineering in S. oneidensis as a synthetic biology chassis. By combining computational analysis with a CRISPRi knockdown system in this way, one can systematically assess the impact of conditionally essential genes and use this knowledge to generate mutations previously thought unachievable.