Catechol-O-Methyltransferase Loss Drives Cell-Specific Nociceptive Signaling via the Enteric Catechol-O-Methyltransferase/microRNA-155/Tumor Necrosis Factor α Axis.

BACKGROUND & AIMS: The etiology of abdominal pain in postinfectious, diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown, and few treatment options exist. Catechol-O-methyltransferase (COMT), an enzyme that inactivates and degrades biologically active catecholamines, plays an important role in numerous physiologic processes, including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS: Colon neurons, epithelial cells, and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT, microRNA-155 (miR-155), and tumor necrosis factor (TNF) α expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-, colon-specific COMT-/-, and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS: Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-α were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-α in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-α) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS: Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-α axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections.

Macrophages and Gut Barrier Function: Guardians of Gastrointestinal Health in Post-Inflammatory and Post-Infection Responses.

The gut barrier is essential for protection against pathogens and maintaining homeostasis. Macrophages are key players in the immune system, are indispensable for intestinal health, and contribute to immune defense and repair mechanisms. Understanding the multifaceted roles of macrophages can provide critical insights into maintaining and restoring gastrointestinal (GI) health. This review explores the essential role of macrophages in maintaining the gut barrier function and their contribution to post-inflammatory and post-infectious responses in the gut. Macrophages significantly contribute to gut barrier integrity through epithelial repair, immune modulation, and interactions with gut microbiota. They demonstrate active plasticity by switching phenotypes to resolve inflammation, facilitate tissue repair, and regulate microbial populations following an infection or inflammation. In addition, tissue-resident (M2) and infiltration (M1) macrophages convert to each other in gut problems such as IBS and IBD via major signaling pathways mediated by NF-κB, JAK/STAT, PI3K/AKT, MAPK, Toll-like receptors, and specific microRNAs such as miR-155, miR-29, miR-146a, and miR-199, which may be good targets for new therapeutic approaches. Future research should focus on elucidating the detailed molecular mechanisms and developing personalized therapeutic approaches to fully harness the potential of macrophages to maintain and restore intestinal permeability and gut health.

Excitable Axonal Domains Adapt to Sensory Deprivation in the Olfactory System.

The axon initial segment (AIS), nodes of Ranvier, and the oligodendrocyte-derived myelin sheath have significant influence on the firing patterns of neurons and the faithful, coordinated transmission of action potentials (APs) to downstream brain regions. In the olfactory bulb (OB), olfactory discrimination tasks lead to adaptive changes in cell firing patterns, and the output signals must reliably travel large distances to other brain regions along highly myelinated tracts. Whether myelinated axons adapt to facilitate olfactory sensory processing is unknown. Here, we investigate the morphology and physiology of mitral cell (MC) axons in the olfactory system of adult male and female mice and show that unilateral sensory deprivation causes system-wide adaptations in axonal morphology and myelin thickness. MC spiking patterns and APs also adapted to sensory deprivation. Strikingly, myelination and MC physiology were altered on both the deprived and nondeprived sides, indicating system level adaptations to reduced sensory input. Our work demonstrates a previously unstudied mechanism of plasticity in the olfactory system.SIGNIFICANCE STATEMENT Successful transmission of information from the olfactory bulb (OB) to piriform cortex through the lateral olfactory tract (LOT) relies on synchronized arrival of action potentials (APs). The coincident arrival of APs is dependent on reliable generation of APs in the axon initial segment (AIS) and fast conduction mediated by axon myelination. Here, we studied changes in mitral cell (MC) firing and AIS structure as well as changes in myelination of the LOT on unilateral olfactory deprivation in the adult mouse. Strikingly, myelination and MC physiology were altered on both the deprived and nondeprived sides, indicating system level adaptations to reduced sensory input. Our work demonstrates a previously unstudied mechanism of plasticity in the olfactory system.

Beyond Extracorporeal Cardiopulmonary Resuscitation: Systems of Care Supporting Cardiac Arrest Patients.

Introduction: Out-of-hospital cardiac arrest (OHCA) is a major cause of death and disability in the United States. Cardiac arrest centers (CAC) are necessary for the management of these critically ill and complex post arrest patients due to their specialized services and provider expertise. We report the case of a patient with OHCA and the systems of care involved in his resuscitation and recovery. Case Report: Emergency medical services attended a 39-year-old male with ongoing bystander cardiopulmonary resuscitation (CPR) after a witnessed collapse. Despite receiving appropriate advanced cardiac life support, including three defibrillations, he remained in refractory ventricular fibrillation. A prehospital physician identified him as an extracorporeal cardiopulmonary resuscitation (ECPR) candidate due to his age, witnessed arrest, refractory rhythm, and functional status. He was expedited to a CAC but no longer qualified for ECPR due to the time limit. He was resuscitated by the multiple teams activated prior to his arrival. He eventually had sustained return of circulation, was taken to the catheterization lab for emergent percutaneous coronary intervention, and recovered with a good neurologic outcome. Conclusion: Cardiac arrest centers may be capable of advanced interventions including ECPR. However, the systems of care offered by these centers is itself a lifesaving intervention. As this case highlights, despite not receiving the specified intervention (ECPR) the systems of care required to offer such a resource led to this favorable outcome.

The Use of B-Cell Polysome Profiling to Validate Novel RPL5 (uL18) and RPL26 (uL24) Variants in Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome usually caused by heterozygous variants in ribosomal proteins (RP) and which leads to severe anemia. Genetic studies in DBA rely primarily on multigene panels that often result in variants of unknown significance. Our objective was to optimize polysome profiling to functionally validate new large subunit RP variants. We determined the optimal experimental conditions for B-cell polysome profiles then performed this analysis on 2 children with DBA and novel missense RPL5 (uL18) and RPL26 (uL24) variants of unknown significance. Both patients had reduced 60S and 80S fractions when compared with an unaffected parent consistent with a large ribosomal subunit defect. Polysome profiling using primary B-cells is an adjunctive tool that can assist in validation of large subunit RP variants of uncertain significance. Further studies are necessary to validate this method in patients with known DBA mutations, small RP subunit variants, and silent carriers.

Predicting Failure of Intravenous Access in Adults: The Value of Prior Difficulty.

BACKGROUND: When intravenous access cannot be established using traditional methods of inspection/palpation, advanced methods are often required, leading to substantial delays in care. Knowing the likelihood of intravenous access failure can improve emergency department (ED) efficiency. OBJECTIVE: Our aim was to validate prior need for an advanced technique to establish intravenous access as a predictor of failure to achieve access via traditional methods and to estimate the risk difference associated with this finding. METHODS: We re-analyzed data collected for a clinical trial that randomized ED patients requiring intravenous access to one of two types of intravenous catheter; gauge size was selected by the inserter. The re-analysis pools data from both groups to examine predictors of failure to establish intravenous access by traditional methods, with failure defined as abandonment or use of an advanced technique (ultrasound guidance or external jugular vein catheterization). Confidence intervals for the difference between proportions were calculated using a normal binomial approximation. RESULTS: We obtained data from 600 patients, with a median age of 52 years (interquartile range 36-63 years). We noted failure of traditional methods in 28 (4.7%) patients, including 17 of 109 (16%) with prior need for advanced techniques. The risk difference for prior need for advanced techniques versus no prior difficulty was 14% (95% confidence interval 7-22). CONCLUSIONS: Patients with a prior need for advanced techniques were 14% more likely to have failure of intravenous access by traditional methods than those without prior difficulty.

Active Bax and Bak are functional holins.

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.

Combination cisplatin and sulforaphane treatment reduces proliferation, invasion, and tumor formation in epidermal squamous cell carcinoma.

Epidermal squamous cell carcinoma is an extremely common type of cancer. Early tumors can be successfully treated by surgery, but recurrent disease is aggressive and resistant to therapy. Cisplatin is often used as a treatment, but the outcome is rarely satisfactory. For this reason new strategies are required. Sulforaphane is a diet-derived cancer prevention agent that is effective in suppressing tumor growth in animal models of skin cancer. We monitored the efficacy of sulforaphane and cisplatin as a combined therapy for squamous cell carcinoma. Both agents suppress cell proliferation, growth of cancer stem cell spheroids, matrigel invasion and migration of SCC-13 and HaCaT cells, and combination treatment is more efficient. In addition, SCC-13 cell derived cancer stem cells are more responsive to these agents than non-stem cancer cells. Both agents suppress tumor formation, but enhanced suppression is observed with combined treatment. Moreover, both agents reduce the number of tumor-resident cancer stem cells. SFN treatment of cultured cells or tumors increases apoptosis and p21Cip1 level, and both agents increase tumor apoptosis. We suggest that combined therapy with sulforaphane and cisplatin is efficient in suppressing tumor formation and may be a treatment option for advanced epidermal squamous cell carcinoma.

Structural mechanisms of PriA-mediated DNA replication restart.

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.

Inhibition of autophagic turnover in ß-cells by fatty acids and glucose leads to apoptotic cell death.

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired ß-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect ß-cell function by modulating autophagy. We report that exposure of ß-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in ß-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in ß-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in ß-cells, contributing to ß-cell failure in the presence of metabolic stress.

The carboxyl-terminal tail of Noxa protein regulates the stability of Noxa and Mcl-1.

The BH3-only protein Noxa is a critical mediator of apoptosis and functions primarily by sequestering/inactivating the antiapoptotic Bcl-2 family protein Mcl-1. Although Noxa is a highly labile protein, recent studies suggested that it is degraded by the proteasome in a ubiquitylation-independent manner. In the present study, we investigated the mechanism of Noxa degradation and its ability to regulate the stability of Mcl-1. We found that the ubiquitylation-independent degradation of Noxa does not require a physical association with Mcl-1. A short stretch of amino acid residues in the C-terminal tail was found to mediate the proteasome-dependent degradation of Noxa. Ectopic placement of this degron was able to render other proteins unstable. Surprisingly, mutation of this sequence not only attenuated the rapid degradation of Noxa, but also stabilized endogenous Mcl-1 through the BH3-mediated direct interaction. Together, these results suggest that the C-terminal tail of Noxa regulates the stability of both Noxa and Mcl-1.

Structure of the SSB-DNA polymerase III interface and its role in DNA replication.

Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.

WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability.

Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes.

Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins.

We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

Onset of Irritable Bowel Syndrome, Dyspepsia, Diarrhea, Bloating, and Constipation in Deployed Gulf War Veterans.

An estimated 694,550 United States service members were actively deployed to the Persian Gulf from 1990-1991. Many veterans who were deployed developed Persian Gulf War Syndrome along with chronic gastrointestinal symptoms after returning from the Persian Gulf. Our objective in this study was to determine the phenotypic expression of gastrointestinal symptom complexes in previously healthy veterans who had been stationed in the Persian Gulf. One hundred and four consecutive veterans (88 males, 16 females) who had previously been deployed in 1990-91 were evaluated for their bowel habits and gastrointestinal symptoms. A workup was completed to find identifiable causes of their symptoms and all veterans were asked to do a modified version of the Bowel Disease Questionnaire symptom survey. None of the veterans reported gastrointestinal symptoms before deployment. During deployment to the Persian Gulf: 22 veterans (21%) developed irritable bowel syndrome; 17 (16%) developed dyspepsia; 50 (48%) developed diarrhea; 11 (11%) developed bloating; and 4 (4%) developed constipation. The results of the current study suggest that the development of irritable bowel syndrome, dyspepsia, diarrhea, bloating, and constipation is frequently seen in deployed Gulf War Veterans and the gastrointestinal symptoms commonly persist upon returning home. These novel findings are very important for currently deployed veterans who are serving in the Middle East and are at a high risk of developing gastrointestinal disorders.

Structure and cellular dynamics of Deinococcus radiodurans single-stranded DNA (ssDNA)-binding protein (SSB)-DNA complexes.

The single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair.

Mechanism of Exonuclease I stimulation by the single-stranded DNA-binding protein.

Bacterial single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during cellular DNA replication, recombination and repair reactions. SSBs also form complexes with an array of genome maintenance enzymes via their conserved C-terminal tail (SSB-Ct) elements. In many cases, complex formation with SSB stimulates the biochemical activities of its protein partners. Here, we investigate the mechanism by which Escherichia coli SSB stimulates hydrolysis of ssDNA by Exonuclease I (ExoI). Steady-state kinetic experiments show that SSB stimulates ExoI activity through effects on both apparent k(cat) and K(m). SSB variant proteins with altered SSB-Ct sequences either stimulate more modestly or inhibit ExoI hydrolysis of ssDNA due to increases in the apparent Michaelis constant, highlighting a role for protein complex formation in ExoI substrate binding. Consistent with a model in which SSB stabilizes ExoI substrate binding and melts secondary structures that could impede ExoI processivity, the specific activity of a fusion protein in which ExoI is tethered to SSB is nearly equivalent to that of SSB-stimulated ExoI. Taken together, these studies delineate stimulatory roles for SSB in which protein interactions and ssDNA binding are both important for maximal activity of its protein partners.

Antioxidant activities, phenolic compounds, and mineral composition of seed from Acacia retinodes, A. Provincialis and A. Tenuissima.

Seeds of the species Acacia retinodes, A. provincialis, and A. tenuissima) from different growing locations were analysed for their mineral composition, free and bound polyphenols, and flavonoids. Previous research has studied these compounds in only a limited number of Acacia species, and only one study reports significant differences between three species. All species were rich in potassium (353 - 427 mg/100 g), sodium (14 - 240 mg/100 g) and iron (7 - 8 mg/100 g). The free polyphenol extracts of all species had higher total phenolic content, total flavonoid content and antioxidant activities than their bound counterparts, indicating the possibility of higher bioavailability than the bound polyphenol extracts. The predominant phenolic compounds found in the Acacia polyphenol seed extracts were 6-Hydroxy-2-methylindole and 2,2'-Methylenebis(6-tert-butyl-methylphenol), though no phenolic compounds were identified in the bound extracts of A. retinodes Grampians and A. provincialis Tarrington. Other compounds identified in the seed extracts include sucrose, d-fructofuranose and d-pinitol.