Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant

Amino acid sequence alignments of for various IRPs.

<p>The alignment of the predicted amino acid sequence of <i>E. andrei</i> IRP with <i>H. sapiens</i> IRP-1, <i>D. rerio</i> IRP-1, <i>A. thaliana</i> aconitase, <i>P. falciparum</i> IRP-like protein, <i>D. melanogaster</i> IRP-1A, <i>P. leniusculus</i> IRP-1-like protein and <i>C. elegans</i> aconitase-1 using the ClustalW multiple sequence alignment program. Putative conserved domains and binding sites were detected by NCBI-CDD. Two conserved domains of IRP are in gray: (Arg84-Val568: Aconitase catalytic domain, Asn672-Ile839: Aconitase swivel domain). The aconitase catalytic domain includes a ligand binding site that binds to the Fe-S cluster required for the activity. The aconitase swivel domain includes a substrate binding site with residues participating in the active site of the catalytic domain. Asterisks show homology in amino acids in all aligned proteins. Black arrows (▾) indicate three cysteine residues binding the Fe-S cluster. Further ligand binding sites are indicated by white arrows (Δ). Putative amino acid residues involved in RNA binding are underlined (based on NCBI-CDD prediction or from the comparison with human IRP1). The square (▪) shows the position of a serine that can be phosphorylated.</p

Comparison of the predicted secondary structure of the 5′-UTR sequence of the consensus ferritin IRE to the ferritins of <i>H. sapiens</i> and two possible structures of <i>E. Andrei.</i>

<p>The Mfold program was used for the design and comparison of the secondary structure of ferritin 5′-UTR. The EaIRE site of earthworm ferritin mRNA creates an unpaired uracil one nucleotide closer to the loop as an optimal secondary conformation instead of the conventional bulged cytosine of the typical IRE. The conventional structure with a bulged cytosine can be formed as well, but with very low probability (0.9% calculated by the Sfold server).</p

Coomassie staining of SDS-PAGE of <i>E. coli</i> lysate proteins transformed with pRSET B-<i>EaIRP</i> and purified rEaIRP.

<p>Lanes: M – MW markers, a - transformed <i>E. coli</i> BL21, b - transformed <i>E. coli</i> BL21 induced with 1 mM IPTG, c – purified rEaIRP, d- purified rEaIRP reduced with 1 mM 2-mercaptoethanol.</p

The expression of <i>Ea</i>IRP in different tissues.

<p>The expression of <i>EaIRP</i> in coelomocytes and different tissues was normalized to two different housekeeping genes: RPL13, RPL17. <i>EaIRP</i> was constitutively expressed in coelomocytes and in all tested tissues (EP – epidermis, CS – coelomocytes, SV – seminal vesicles, PH – pharynx, ES – esophagus, CR – crop, GZ – gizzard, INT – intestine). The expression was related to the expression in the epidermis, where the expression of <i>Ea</i>IRP was the lowest. The highest level of <i>EaIRP</i> expression was found in the part of the digestive tract that included the crop, gizzard and intestine. One-way ANOVA with Dunnett’s post-test was performed, using GraphPad Prism software to evaluate the significance of the data. Differences were considered significant when P<0.05, 0.001.</p

NMR- and MS-Based Untargeted Metabolomic Study of Stool and Serum Samples from Patients with Anorexia Nervosa

Anorexia nervosa (AN), a pathological restriction of food intake, leads to metabolic dysregulation. We conducted a metabolomics study to reveal changes caused by AN and the effect of hospital realimentation on metabolism. Both stool and serum from patients with AN and healthy controls were analyzed by NMR and MS. Statistical analysis revealed several altered biochemical and anthropometric parameters and 50 changed metabolites, including phospholipids, acylcarnitines, amino acids, derivatives of nicotinic acid, nucleotides, and energy metabolism intermediates. Biochemical and anthropometric parameters were correlated with metabolomic data. Metabolic changes in patients with AN described in our study imply serious system disruption defects, such as the development of inflammation and oxidative stress, changed free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels, a deficit of vitamins, muscle mass breakdown, and a decrease in ketone bodies as an important source of energy for the brain and heart. Furthermore, our data indicate only a very slight improvement after treatment. However, correlations of metabolomic results with body weight, interleukin 6, tumor necrosis factor α, fT4, and TSH might entail better prognoses and treatment effectiveness in patients with better system parameter status. Data sets are deposited in MassIVE: MSV000087713, DOI: 10.25345/C57R7X

NMR- and MS-Based Untargeted Metabolomic Study of Stool and Serum Samples from Patients with Anorexia Nervosa

Anorexia nervosa (AN), a pathological restriction of food intake, leads to metabolic dysregulation. We conducted a metabolomics study to reveal changes caused by AN and the effect of hospital realimentation on metabolism. Both stool and serum from patients with AN and healthy controls were analyzed by NMR and MS. Statistical analysis revealed several altered biochemical and anthropometric parameters and 50 changed metabolites, including phospholipids, acylcarnitines, amino acids, derivatives of nicotinic acid, nucleotides, and energy metabolism intermediates. Biochemical and anthropometric parameters were correlated with metabolomic data. Metabolic changes in patients with AN described in our study imply serious system disruption defects, such as the development of inflammation and oxidative stress, changed free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels, a deficit of vitamins, muscle mass breakdown, and a decrease in ketone bodies as an important source of energy for the brain and heart. Furthermore, our data indicate only a very slight improvement after treatment. However, correlations of metabolomic results with body weight, interleukin 6, tumor necrosis factor α, fT4, and TSH might entail better prognoses and treatment effectiveness in patients with better system parameter status. Data sets are deposited in MassIVE: MSV000087713, DOI: 10.25345/C57R7X

Microbiota, Microbial Metabolites, and Barrier Function in A Patient with Anorexia Nervosa after Fecal Microbiota Transplantation

The change in the gut microbiome and microbial metabolites in a patient suffering from severe and enduring anorexia nervosa (AN) and diagnosed with small intestinal bacterial overgrowth syndrome (SIBO) was investigated. Microbial gut dysbiosis is associated with both AN and SIBO, and therefore gut microbiome changes by serial fecal microbiota transplantation (FMT) is a possible therapeutic modality. This study assessed the effects of FMT on gut barrier function, microbiota composition, and the levels of bacterial metabolic products. The patient treatment with FMT led to the improvement of gut barrier function, which was altered prior to FMT. Very low bacterial alpha diversity, a lack of beneficial bacteria, together with a great abundance of fungal species were observed in the patient stool sample before FMT. After FMT, both bacterial species richness and gut microbiome evenness increased in the patient, while the fungal alpha diversity decreased. The total short-chain fatty acids (SCFAs) levels (molecules presenting an important source of energy for epithelial gut cells) gradually increased after FMT. Contrarily, one of the most abundant intestinal neurotransmitters, serotonin, tended to decrease throughout the observation period. Overall, gut microbial dysbiosis improvement after FMT was considered. However, there were no signs of patient clinical improvement. The need for an in-depth analysis of the donor's stool and correct selection pre-FMT is evident

Fetidin-lysenins genes alignment.

<div><p>The alignment of partial sequences of fetidin-lysenins genes.</p> <p>Universal primers (highlighted) were designed on the basis of presence of homologous regions.</p></div

Alignment of <i>E. andrei</i> and <i>E. fetida</i> COI sequences.

<p>Alignment of <i>E. andrei</i> and <i>E. fetida</i> COI sequences. Oligonucleotides used as discriminating primers for <i>E. andrei</i> or <i>E. fetida</i> COI are underlined or in bold (refer to the Table 1).</p