The protective role of commensal gut microbes and their metabolites against bacterial pathogens.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Pushing Backwards: Evaluating Effectiveness of Conservative Treatment of Intestinal Intussusception.

OBJECTIVE: Intestinal intussusception (II) is a common cause for acute abdomen in children, occurring in 0.33 to 0.71 per 1000 children per year. Early diagnosis and treatment are fundamental for prevention of irreversible intestinal damage. The first line of treatment is conservative, with saline reduction enema or air reduction enema. Our goal is to evaluate results with conservative treatment of II in children. METHODS: A retrospective single-center review of all patients with diagnosis of II from January 2014 to December 2019 was performed. Demographics, clinical data, treatment option, and results were assessed. RESULTS: Thirty-eight cases were identified. The mean age was 26 months, and 68% were males. Most presented with abdominal pain (95%) and vomiting (66%), after an average of 30 hours. Rectal bleeding was present in 32% of patients. Abdominal ultrasound was performed in all patients for diagnosis. Conservative treatment was first option in 95% of patients, with a global effectiveness of 83% after 1 attempt. Saline reduction enema was more effective than air reduction enema (88% vs 70%), and patients with successful reduction were younger (24 vs 33 months), but neither reached statistical significance. Two patients had a subsequent II episode within 1 week after hospital discharge. Neither age, sex, symptoms and respective duration, rotavirus inoculation, intussuscepted bowel length, nor technique used was predictive of treatment failure or II relapse. CONCLUSIONS: Conservative treatment in II is a safe and effective option, preventing invasive surgical procedures. Effectiveness of such treatments may be as high as 88% after 1 attempt, with rapid diet reintroduction. Same-day discharge after oral feeding toleration is safe.

Drug-Related Adverse Reactions in Pediatric Inflammatory Bowel Disease.

The therapeutic approach to inflammatory bowel disease (IBD) is complex, often involving multiple pharmacologic classes. We aimed to evaluate the prevalence of drug-related adverse reactions (ARs) associated with therapies used in pediatric IBD. We conducted a retrospective study of pediatric patients with IBD followed in a tertiary hospital from 2010 to 2022. Ninety-nine patients were included (62.6% were male), with a median age at diagnosis of 13 years (interquartile range [IQR] 11-15 years). The majority had Crohn's disease (69.7%), followed by ulcerative colitis (21.2%) and unclassified IBD (9.1%). The most prescribed therapies were: immunomodulators (n = 75, 75.8%), exclusive enteral nutrition (n = 61, 61.6%), and biologics (n = 58, 58.6%). During a median follow-up time of 31 months (IQR 11-51 months), the incidence of ARs was 16.2% (16 ARs occurred in 14 patients). The main drug involved was azathioprine (12/16) and the most frequent AR was hepatitis (5/16). Drug discontinuation was necessary in all but 1 case. Of the ARs recorded, 75% were mild to moderate and 81.3% did not require specific treatment; all patients had clinical and/or analytical normalization. There was a positive association between the cumulative number of prescribed drugs and the occurrence of ARs (P = .044). The incidence of ARs was similar to the rates reported in the few existing previous studies. The majority of ARs were mild, but implied the discontinuation of therapy or dose reduction, with a possible impact on disease control.

Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.

The human body has evolved to remove xenobiotics through a multistep clearance process. Non-endogenous metabolites are converted through a series of phase I and different phase II enzymes into compounds with higher hydrophilicity. These compounds are important for diverse research fields such as toxicology, nutrition, biomarker discovery, doping control, and microbiome metabolism. One of the challenges in these research fields has been the investigation of the two major phase II modifications, sulfation and glucuronidation, and the corresponding unconjugated aglycon independently. We have now developed a new methodology utilizing an immobilized arylsulfatase and an immobilized ß-glucuronidase to magnetic beads for treatment of human urine samples. The enzyme activities remained the same compared to the enzyme in solution. The separate mass spectrometric investigation of each metabolite class in a single sample was successfully applied to obtain the dietary glucuronidation and sulfation profile of 116 compounds. Our new chemical biology strategy provides a new tool for the investigation of metabolites in biological samples with the potential for broad-scale application in metabolomics, nutrition, and microbiome studies.

Quality of life after otoplasty for prominent ears in children

Prominent ears are a common congenital malformation and are associated with low self-esteem, social isolation and diminished school performance. Our goal was to evaluate the influence of otoplasty on children’s quality of life (QoL).Material and methodsPatients submitted to otoplasty from 2016 to 2018 were summoned for a reevaluation. Seventy patients and respective caregivers agreed to participate. Surgical, demographic and clinical data were reviewed from electronic registries.Two sets of inquiries were performedPediatric Quality of Life Inventory 4.0 (for parent and child) and an adaptation of the Glasgow Children Benefit Inventory (GCBI-b). Fifteen patients were excluded for incomplete inquiries.ResultsFifty-five patients were included, 70.9% were males. Median age at surgery was 7.7 ± 3.3 years. Aesthetic dissatisfaction was the main previous negative experience. Median self-report quality of life was 85.6% and parent-report was 86.9%. Median GCBI-b was +20.5, indicating an improvement in patients’ QoL. Bullying and high parental expectations for life change post-surgery were predictive of higher GCBI-b scores (p < 0.05). Ninety-six percent of parents would recommend surgery to other children.ConclusionsOtoplasty is a valid treatment option for prominent ears in children,improving not only aesthetics but also health-related QoL. (AU)

Mapping peripheral and abdominal sarcopenia acquired in the acute phase of COVID-19 during 7 days of mechanical ventilation.

Our aim was to map acquired peripheral and abdominal sarcopenia in mechanically ventilated adults with COVID-19 through ultrasound measurements. On Days 1, 3, 5 and 7 after admission to critical care, the muscle thickness and cross-sectional area of the quadriceps, rectus femoris, vastus intermedius, tibialis anterior, medial and lateral gastrocnemius, deltoid, biceps brachii, rectus abdominis, internal and external oblique, and transversus abdominis were measured using bedside ultrasound. A total of 5460 ultrasound images were analyzed from 30 patients (age: 59.8 ± 15.6 years; 70% men). Muscle thickness loss was found in the bilateral anterior tibial and medial gastrocnemius muscles (range 11.5-14.6%) between Days 1 and 3; in the bilateral quadriceps, rectus femoris, lateral gastrocnemius, deltoid, and biceps brachii (range 16.3-39.1%) between Days 1 and 5; in the internal oblique abdominal (25.9%) between Days 1 and 5; and in the rectus and transversus abdominis (29%) between Days 1 and 7. The cross-sectional area was reduced in the bilateral tibialis anterior and left biceps brachii (range 24.6-25.6%) between Days 1 and 5 and in the bilateral rectus femoris and right biceps brachii (range 22.9-27.7%) between Days 1 and 7. These findings indicate that the peripheral and abdominal muscle loss is progressive during the first week of mechanical ventilation and is significantly higher in the lower limbs, left quadriceps and right rectus femoris muscles in critically ill patients with COVID-19.

Management of infantile hemangiomas-experience of a tertiary hospital.

The purpose of the study is to describe the experience of a multidisciplinary team in a tertiary hospital regarding the management of Infantile Hemangiomas (IH). The method employed is a retrospective analysis of patients with IH followed in a tertiary pediatric hospital between January 2010 and May 2022. A total of 393 IH were diagnosed (56.7% female), with a median age of 5 months (interquartile range (IQR), 3-10). Imaging investigation was necessary for diagnosis and for exclusion of other IH in 9.2% and 14.3%, respectively. Focal (74.0%) and superficial (59.7%) lesions were more frequent as was facial location (35.9%). Pre-treatment ulceration or hemorrhage occurred in 6.6%. At follow-up, 87.4% regressed partially and 12.6% completely; 2.7% relapsed. Propranolol was started in 30.0% of cases for a median period of 9 months (IQR, 6-12), mainly due to esthetic concerns (41.9%). Side effects occurred in 8.3% (sleep disturbance in 5.1%). Only 1.7% were refractory and 5.9% had a rebound effect. Eleven patients were treated with topical timolol and 41 underwent surgery. Patients that were treated with propranolol had more risk factors (p = 0.016) and presented deeper lesions (p < 0.001) with a larger diameter (p < 0.001); total IH regression was less frequent (p < 0.001). Since 2020, twice-daily dosage was more frequently prescribed than three times daily (p = 0.007) and inpatient initiation of propranolol decreased (p = 0.750), without significant difference in the incidence of adverse reactions, duration of treatment, and lesion evolution.  Conclusions: Our protocol proved to be safe and feasible in an outpatient setting and twice daily administration of propranolol was effective. The majority of IH showed at least partial regression. Early detection of high-risk IH is paramount and a multidisciplinary assessment by a specialized team is essential for adequate management. What is Known: • IH are the most common vascular tumors in childhood. Although the majority evolves favorably, treatment may be warranted in selected cases. • Early detection of high-risk IH is paramount, and a multidisciplinary assessment by a specialized team is essential for adequate management. What is New: • One-third of our sample was treated with propranolol. These patients had more risk factors and presented deeper lesions with a larger diameter, and tumor total regression was less frequent. • Our results reinforce safety and feasibility of propranolol initiation in an outpatient setting, including twice daily dosage.

Quality of life after otoplasty for prominent ears in children.

AIM: Prominent ears are a common congenital malformation and are associated with low self-esteem, social isolation and diminished school performance. Our goal was to evaluate the influence of otoplasty on children's quality of life (QoL). MATERIAL AND METHODS: Patients submitted to otoplasty from 2016 to 2018 were summoned for a reevaluation. Seventy patients and respective caregivers agreed to participate. Surgical, demographic and clinical data were reviewed from electronic registries. TWO SETS OF INQUIRIES WERE PERFORMED: Pediatric Quality of Life Inventory 4.0 (for parent and child) and an adaptation of the Glasgow Children Benefit Inventory (GCBI-b). Fifteen patients were excluded for incomplete inquiries. RESULTS: Fifty-five patients were included, 70.9% were males. Median age at surgery was 7.7 ±â€¯3.3 years. Aesthetic dissatisfaction was the main previous negative experience. Median self-report quality of life was 85.6% and parent-report was 86.9%. Median GCBI-b was +20.5, indicating an improvement in patients' QoL. Bullying and high parental expectations for life change post-surgery were predictive of higher GCBI-b scores (p < 0.05). Ninety-six percent of parents would recommend surgery to other children. CONCLUSIONS: Otoplasty is a valid treatment option for prominent ears in children,improving not only aesthetics but also health-related QoL.

Mucosal and Plasma Metabolomes in New-onset Paediatric Inflammatory Bowel Disease: Correlations with Disease Characteristics and Plasma Inflammation Protein Markers.

BACKGROUND AND AIMS: To advance the understanding of inflammatory bowel disease [IBD] pathophysiology, we compared the mucosal and plasma metabolomes between new-onset paediatric IBD patients and symptomatic non-IBD controls, and correlated plasma inflammation markers and disease characteristics with the altered metabolites. METHODS: Paired colonic and ileal biopsies and plasma from 67 treatment-naïve children with incident Crohn's disease [CD; n = 47], ulcerative colitis [UC; n = 9], and non-IBD controls [n = 11] were analysed using ultra-performance liquid chromatography-mass spectrometry [UPLC-MS/MS]. Inflammatory plasma proteins [n = 92] were assessed. RESULTS: The metabolomes in inflamed mucosal biopsies differed between IBD patients and controls. In CD, mucosal levels of several lysophospholipids [lysophosphatidylcholines, lysophosphatidyletanolamines, lysophosphatidylinositols, and lysophosphatidylserines] were decreased, correlating with various plasma metabolites including amino acid analogues and N-acetylated compounds. In both CD and UC, mucosal sphingolipids, including ceramide [d18:2/24:1, d18:1/24:2], lactosyl-N-palmitoyl-sphingosine [d18:1/16:0], behenoyl sphingomyelin [d18:1/22:0], lignoceroyl sphingomyelin [d18:1/24:0], and/or sphingomyelin [d18:1/24:1, d18:2/24:0] were increased, correlating with sphingolipids, bile acids, and/or N-acetylated metabolites in plasma. Among proteins associated with CD, interleukin-24 correlated with plasma metabolites, including lactosyl-N-palmitoyl sphingosine [d18:1/16:0] and phosphatidyletanolamine [18:1/18:1], haemoglobin, and faecal calprotectin. In UC, interleukin-24, interleukin-17A, and C-C motif chemokine 11 correlated with several plasma metabolites, including N-acetyltryptophan, tryptophan, glycerate, and threonate, and with the Paediatric Ulcerative Colitis Activity Index, C-reactive protein, and faecal calprotectin. CONCLUSIONS: Mucosal perturbations of lysophospholipids and sphingolipids characterised the metabolome in new-onset paediatric IBD and correlated with plasma metabolites. By integrating plasma metabolomics data with inflammatory proteins and clinical data, we identified clinical and inflammatory markers associated with metabolomic signatures for IBD.

Mediastinal Teratoma In Children - Case Report.

Mediastinal teratomas presenting in the pediatric age are extremely rare. We report three cases of mediastinal teratomas in children aged 15 months to 9 years. Patients were submitted to complete tumor resection, with an uneventful postoperative course and follow-up. Our report emphasizes the importance of a detailed patient examination and careful interpretation of routinely performed image studies.

Enhanced cytotoxicity of a novel family of ATPase inhibitors in colorectal cancer cells with high NAT2 activity.

Loss of heterozygosity (LOH) is a hallmark feature of cancer genomes that reduces allelic variation, thereby creating tumor specific vulnerabilities which could be exploited for therapeutic purposes. We previously reported that loss of drug metabolic arylamine N-acetyltransferase 2 (NAT2) activity following LOH at 8p22 could be targeted for collateral lethality anticancer therapy in colorectal cancer (CRC). Here, we report a novel compound CBK034026C that exhibits specific toxicity towards CRC cells with high NAT2 activity. Connectivity Map analysis revealed that CBK034026C elicited a response pattern related to ATPase inhibitors. Similar to ouabain, a potent inhibitor of the Na+/K+-ATPase, CBK034026C activated the Nf-kB pathway. Further metabolomic profiling revealed downregulation of pathways associated with antioxidant defense and mitochondrial metabolism in CRC cells with high NAT2 activity, thereby weakening the protective response to oxidative stress induced by CBK034026C. The identification of a small molecule targeting metabolic vulnerabilities caused by NAT2 activity provides novel avenues for development of anticancer agents.

Analysis of Growth Phases of Enterotoxigenic Escherichia coli Reveals a Distinct Transition Phase before Entry into Early Stationary Phase with Shifts in Tryptophan, Fucose, and Putrescine Metabolism and Degradation of Neurotransmitter Precursors.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli. IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of certain amino acids. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described "indole peak," rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolome is short-lived and disappears when bacteria enter the early stationary phase. We suggest that these changes mainly prepare bacteria for ceased growth, but based on the pathways involved, we could suggest that this transient phase substantially influences survival and virulence.

Comparison of the CIPHER prognostic model with the existing scores in predicting severe maternal outcomes during intensive care unit admission.

OBJECTIVE: To compare the performance of the Collaborative Integrated Pregnancy High-Dependency Estimate of Risk (CIPHER) model in predicting maternal death and near-miss morbidity (Severe Maternal Outcome [SMO]) with the Sequential Organ Failure Assessment (SOFA), the Acute Physiology and Chronic Health Evaluation (APACHE) II, and the Simplified Acute Physiology Score (SAPS) III scores. METHODS: A retrospective and a prospective study was conducted at two centers in Brazil. For each score, area under curve (AUC) was used and score calibration was assessed using the Hosmer-Lemeshow statistic (H-L) test and the standardized mortality ratio (SMR). RESULTS: A cohort of 590 women was analyzed. A SMO was observed in 216 (36.6%) women. Of these, 13 (2.2%) were maternal deaths and 203 (34.4%) met one or more maternal near-miss criteria. The CIPHER model did not show significant diagnostic ability (AUC 0.52) and consequently its calibration was poor (H-L P < 0.05). The SAPS III had the best performance (AUC 0.77, H-L P > 0.05 and SMR 0.85). CONCLUSION: The performance of the CIPHER model was lower compared to the other scores. Since the CIPHER model is not ready for clinical use, the SAPS III score should be considered for the prediction of SMO.

Compensatory sweating after thoracoscopic sympathectomy for primary focal hyperhidrosis in children: Are there patient-related risk factors?

INTRODUCTION: Compensatory sweating (CS) is a common complication after thoracoscopic sympathectomy (TS) and is mainly associated with surgical technique. Our aim was to identify potential risk-factors for CS following TS for primary focal hyperhidrosis in children. METHODS: A retrospective, single-center review of all bilateral TS was performed between 2017 and 2019. Hyperhidrosis disease severity scale was used for pre-operative severity assessment. Post-operative evaluations were performed after three and six months. RESULTS: More than the 36-month period, 41 patients were submitted to T2-T4 TS, and 25 were females (60.9%). Median age at surgery was 15.5 years. CS was identified at the 3rd month in 17 (41%) children with most in the dorsolumbar region (56%). By the 6th month, there was a significant reduction in CS (41 to 32%, p = 0.02). The probability of resolution of CS by 6 months is about 50% in both the dorsolumbar and abdominal regions. Neither age, gender, body mass index, family history, or concomitant illnesses seemed to influence CS (p > 0.05). Axillary hyperhidrosis appears to be associated with the development of dorsolumbar CS (p = 0.037). CONCLUSION: Thoracoscopic sympathectomy for PFH is a safe and effective procedure, but compensatory sweating can be a common and debilitating side effect. Age, gender, and body mass index do not appear to influence CS. Axillary hyperhidrosis may be related to the development of dorsolumbar CS. LEVEL OF EVIDENCE: III.

Prognostic Value of an Estimate-of-Risk Model in Critically Ill Obstetric Patients in Brazil.

OBJECTIVE: To externally validate the CIPHER (Collaborative Integrated Pregnancy High-Dependency Estimate of Risk) prognostic model for pregnant and postpartum women admitted to the intensive care unit. METHODS: A retrospective and a prospective validation study were conducted at two reference centers in Brazil. A composite outcome was defined as maternal death or need for prolonged organ support (more than 7 days) or acute lifesaving intervention. To evaluate the performance of the CIPHER model, a receiver operating characteristic curve was used and score calibration was assessed by the Hosmer-Lemeshow test. We conducted a descriptive analysis comparing the results of the current study with the results of the model development study. RESULTS: A total of 590 women were included. The composite outcome was observed in 90 (15.2%) women. Of these, 13 (2.2%) were maternal deaths and 77 (13%) required one or more component of organ support or lifesaving intervention. The CIPHER model's area under the curve (AOC) did not show significant predictive ability (AOC 0.53, 95% CI 0.46-0.60), and consequently its calibration was poor (Hosmer-Lemeshow test P<.05). CONCLUSION: The CIPHER model for prediction of mortality and need for interventions in critically ill obstetric patients did not perform well in our Brazilian population. Different predictors of morbidity and mortality may need to be used for patients receiving care in public hospitals in low- and middle-income countries.

Differential regulation of oxidative stress, microbiota-derived, and energy metabolites in the mouse brain during sleep.

Sleep has evolved as a universal core function to allow for restorative biological processes. Detailed knowledge of metabolic changes necessary for the sleep state in the brain is missing. Herein, we have performed an in-depth metabolic analysis of four mouse brain regions and uncovered region-specific circadian variations. Metabolites linked to oxidative stress were altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. These findings provide molecular evidence of a significant metabolic shift of the brain energy metabolism. Specific alterations were observed for brain metabolites that have previously not been associated with a circadian function including the microbiome-derived metabolite ergothioneine that suggests a regulatory function. The pseudopeptide ß-citryl-glutamate has been linked to brain development and we have now discovered a previously unknown regioisomer. These metabolites altered by the circadian rhythm represent the foundation for hypothesis-driven studies of the underlying metabolic processes and their function.

Pediatric Burns With Cyanoacrylate Glue: An Inconspicuous Danger.

Burns are a common cause of pediatric injury and represent significant morbidity and mortality in this age group. In children, most burns are thermal, resulting from exposure to a hot surface, liquid, or fire. Cyanoacrylate is a liquid compound commonly found in households, mainly in "superglue" and nail glue. This compound solidifies through an exothermic reaction that is magnified when in contact with certain fibers that act as catalysts, such as cotton. In these circumstances, intense heat is produced in the contact area, potentially causing severe thermal burns. Despite its widespread availability, there is a paucity of safety information about cyanoacrylate and its role as a cause for burns. In the literature, only 18 cases of cyanoacrylate burns are reported in children. We present two cases of pediatric burns with cyanoacrylate and a review of the literature.

Investigation of the individual human sulfatome in plasma and urine samples reveals an age-dependency.

Metabolic microbiome interaction with the human host has been linked to human physiology and disease development. The elucidation of this interspecies metabolite exchange will lead to identification of beneficial metabolites and disease modulators. Their discovery and quantitative analysis requires the development of specific tools and analysis of specific compound classes. Sulfated metabolites are considered a readout for the co-metabolism of the microbiome and their host. This compound class is part of the human phase II clearance process of xenobiotics and is the main focus in drug or doping metabolism and also includes dietary components and microbiome-derived compounds. Here, we report the targeted analysis of sulfated metabolites in plasma and urine samples in the same individuals to identify the core sulfatome and similarities between these two sample types. This analysis of 27 individuals led to the identification of the core sulfatome of 41 metabolites in plasma and urine samples as well as an age effect for 15 metabolites in both sample types.

Comparison of two arylsulfatases for targeted mass spectrometric analysis of microbiota-derived metabolites.

Sulfation of metabolites is the second highest phase II modification in humans, which plays a critical role in the xenobiotics clearance process and gut microbiota-host co-metabolism. Besides the main function to remove xenobiotics from the body, sulfated metabolites have also been linked to inflammation, bacterial pathogenesis and metabolic disorders. A better understanding of how these metabolites impact the human body has turned into an important research area. Analytical methods for selective identification of this metabolite class are scarce. We have recently developed an assay utilizing the arylsulfatase from Helix pomatia due to a high substrate promiscuity combined with state-of-the-art metabolomics bioinformatic analysis for the selective identification of O-sulfated metabolites in human samples. This enzyme requires a multistep purification process as highest purity is needed for the developed mass spectrometric assay. In this study, we have utilized a new and recombinant overexpressed arylsulfatase (ASPC) for the selective identification of organic sulfate esters in human urine samples. We have compared the substrate conversion in urine samples and substrate specificity of this enzyme with purified arylsulfatase from Helix pomatia. Our analysis of urine samples revealed that both enzymes can be utilized for the selective analysis and discovery of sulfated metabolites with high promiscuity as demonstrated by equal hydrolysis of 108 substrates including sulfated conjugates of 27 metabolites of microbial origin. Importantly, we also identified 21 substrates in human urine samples that are exclusively hydrolyzed by ASPC and application of this enzyme increases the discovery of unknown sulfated metabolites with a higher scaffold diversity.

Regional Brain Analysis of Modified Amino Acids and Dipeptides during the Sleep/Wake Cycle.

Sleep is a state in which important restorative and anabolic processes occur. Understanding changes of these metabolic processes during the circadian rhythm in the brain is crucial to elucidate neurophysiological mechanisms important for sleep function. Investigation of amino acid modifications and dipeptides has recently emerged as a valuable approach in the metabolic profiling of the central nervous system. Nonetheless, very little is known about the effects of sleep on the brain levels of amino acid analogues. In the present study, we examined brain regional sleep-induced alterations selective for modified amino acids and dipeptides using Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) based metabolomics. Our approach enabled the detection and identification of numerous amino acid-containing metabolites in the cortex, the hippocampus, the midbrain, and the cerebellum. In particular, analogues of the aromatic amino acids phenylalanine, tyrosine and tryptophan were significantly altered during sleep in the investigated brain regions. Cortical levels of medium and long chain N-acyl glycines were higher during sleep. Regional specific changes were also detected, especially related to tyrosine analogues in the hippocampus and the cerebellum. Our findings demonstrate a strong correlation between circadian rhythms and amino acid metabolism specific for different brain regions that provide previously unknown insights in brain metabolism.