Bacterial translocation markers in type 2 diabetes mellitus: their association with glycemic control and diabetic kidney disease in Egyptian patients

Background. The involvement of bacterial translocation in the pathogenesis of type 2 diabetes mellitus (T2DM) has been highlighted in recent years. The objective of the current study was to evaluate the potential impact of lipopolysaccaride-binding protein (LBP) and DNA translocation on glycemic control and progression to diabetic kidney disease in T2DM patients. Material and methods. A total of 30 T2DM patients as well as 30 controls were included in a cross-sectional observational study. Plasma LBP levels were deter­mined using an enzyme linked immunoassay. DNA translocation was assessed using polymerase chain reaction targeting 16SrNA gene. Results. Plasma levels of LBP were significantly elevated in T2DM patients than in controls (p = 0.02). LBP level was significantly and positively correlated with fasting glucose level, glycated hemoglobin, C-reactive protein, albumin-creatinine ratio and negatively correlated with glomerular filtration rate. Receiver operating curve revealed that LBP with a cut off of 15.17 μg/mL succeeded to predict both glycemic control and dia­betic kidney disease in T2DM patients. The bacterial 16SrRNA was detected in almost all blood samples of T2DM patients (28/30) and in about half (16/30) of the control group (p < 0.001). Conclusion. Translocation products could trigger diabe­tes related complications. Future interventional work should target these products to reverse their effects

Charakterisierung von Emerin LEM-Domäne Fehlsinn-Mutationen bei Patienten mit exklusiven Herzdefekten

Emery-Dreifuss Muscular Dystrophy (EDMD) is among the most widely common human genetic muscular dystrophies. The cardiac involvement in the disease is the most life-threatening symptom and the major cause of mortality. The majority of cases of its X-linked type are due to mutations in a gene encoding for the nuclear envelope protein, emerin. Despite the considerable advances that have been achieved in terms of the characterization of emerin structure, various binding partners, and functions in the human body, the picture is still rather incomplete. Fifty years now after EDMD had been first documented, researchers still fall short of understanding the pathophysiology of the disease. Thereby, it comes as no surprise that, to date, there is no described treatment of EDMD. Accordingly, this thesis is an initial attempt to characterize three emerin LEM-domain missense mutations (P22L, DK37, and T43I) present in patients with exclusive cardiac defects. The main objective of this thesis is to investigate the effect of the three mutations on emerin structure, its self-assembly, and interactions with some of its well-described binding partners. The presented work highlights that albeit the localization of the three mutations in the only folded region of emerin, the variants show no global defect in their structure, except for the destabilization of the LEM-domain of the variant DK37. Importantly, the mutants are able to self-assemble, yet with astonishing fast polymerization kinetics. In addition, the investigations have illustrated that the three variants, despite the presence of the mutations in the BAF-binding domain, are surprisingly capable of binding BAF. The analysis did not reveal any differences in the mutants binding to Ig-fold domain of lamin A/C. Further, there is no defect in DK37 phosphorylation by Src kinase. Also, preliminary characterization of the DK37 mutation in immortalized human fibroblasts has featured no overt defects in mechanobiology, and in the expression of nuclear envelope or cytoskeletal proteins. Taken all together, the presented work outlines that the three emerin missense mutations display no defects in several prominent emerin properties, which are questioned in this thesis. On the basis of the results of the conducted research, considerable insight has been gained with regard to the consequences of the mutations of interest. In other words, the presented work lends support to following investigations in order to explore other unquestioned properties or functions of emerin that might be associated with the pathophysiology of EDMD

Caractérisation des mutations du domaine LEM de l'émérine présentes chez les patients avec uniquement des anomalies cardiaques auriculaires

Emery-Dreifuss Muscular Dystrophy (EDMD) is among the most widely common human genetic muscular dystrophies. The cardiac involvement in the disease is the most life-threatening symptom and the major cause of mortality. The majority of cases of its X-linked type are due to mutations in a gene encoding for the nuclear envelope protein, emerin. Despite the considerable advances that have been achieved in terms of the characterization of emerin structure, various binding partners, and functions in the human body, the picture is still rather incomplete. Fifty years now after EDMD had been first documented, researchers still fall short of understanding the pathophysiology of the disease. Thereby, it comes as no surprise that, to date, there is no described treatment of EDMD. Accordingly, this thesis is an initial attempt to characterize three emerin LEM-domain missense mutations (P22L, ΔK37, and T43I) present in patients with exclusive cardiac defects. The main objective of this thesis is to investigate the effect of the three mutations on: emerin structure, its self-assembly, and interactions with some of its well-described binding partners. The presented work highlights that albeit the localization of the three mutations in the only folded region of emerin, the variants show no global defect in their structure, except for the destabilization of the LEM-domain of the variant ΔK37. Importantly, the mutants are able to self-assemble, yet with astonishing fast polymerization kinetics. In addition, the investigations have illustrated that the three variants, despite the presence of the mutations in the BAF-binding domain, are surprisingly capable of binding BAF. The analysis did not reveal any differences in the mutants binding to Ig-fold domain of lamin A/C. Further, there is no defect in ΔK37 phosphorylation by Src kinase. Also, preliminary characterization of the ΔK37 mutation in immortalized human fibroblasts has featured no overt defects in mechanobiology, and in the expression of nuclear envelope or cytoskeletal proteins. Taken all together, the presented work outlines that the three emerin missense mutations display no defects in several prominent emerin properties, which are questioned in this thesis. On the basis of the results of the conducted research, considerable insight has been gained with regard to the consequences of the mutations of interest. In other words, the presented work lends support to following investigations in order to explore other unquestioned properties or functions of emerin that might be associated with the pathophysiology of EDMD.La dystrophie musculaire d’Emery-Dreifuss (DMED) est l'une des dystrophies musculaires génétiques humaines les plus répandues. L'implication cardiaque dans la maladie est le symptôme qui met le plus la vie en danger et la principale cause de mortalité. La majorité des cas de son type liée à l’X sont dus à des mutations dans un gène codant pour une protéine de l'enveloppe nucléaire, l'émérine. Malgré les progrès considérables qui ont été réalisés en termes de caractérisation de la structure de l'émerine, ses différents partenaires de liaison, et ses fonctions dans le corps humain, le tableau est encore assez incomplet. Cinquante ans après que la DMED ait été documentée pour la première fois, les chercheurs n'ont toujours pas compris la pathophysiologie de la maladie. Il n'est donc pas surprenant qu'à ce jour, il n'existe pas de traitement décrit pour la DMED. Cette thèse est une première tentative pour caractériser trois mutations faux-sens du domaine LEM de l'émerine (P22L, ΔK37, et T43I) présentes chez des patients présentant uniquement des symptômes cardiaques. L'objectif principal de cette thèse est d'étudier l'effet des trois mutations sur la structure de l'émerine, son auto-assemblage, et les interactions avec certains de ses partenaires de liaison bien décrits. Le travail présenté souligne que malgré la présence des trois mutations dans la seule région repliée de l'émerine, les variants ne présentent aucun défaut global dans leur structure, à l'exception de la déstabilisation du domaine LEM du variant ΔK37. Il est important de noter que les mutants sont capables de s'auto-assembler, mais avec une cinétique de polymérisation rapide. En outre, l’étude a montré que les trois variants, bien que mutés dans le domaine de liaison à BAF, sont étonnamment capables de se lier à la protéine BAF. De plus, l'analyse ne révèle aucune différence dans les interactions des variants avec l’Ig-fold de la lamine A/C. De plus, il n'y a pas de défaut dans la phosphorylation du variant ΔK37 par la kinase Src. La caractérisation préliminaire de la mutation ΔK37 dans des fibroblastes humains immortalisés n'a pas montré de défauts manifestes en mécanobiologie et dans l'expression des protéines de l'enveloppe nucléaire ou du cytosquelette. Pris dans son ensemble, le travail présenté souligne que les trois mutations faux-sens de l’émerine ne provoquent aucun défaut dans plusieurs propriétés importantes de l'émerine, qui sont testées dans cette thèse. Sur la base des résultats de la recherche menée, nous avons acquis des connaissances considérables en ce qui concerne les conséquences des mutations d'intérêt. En d'autres termes, le travail présenté montre qu’il faut continuer les recherches sur l’émerine, afin d'explorer d'autres propriétés ou fonctions de cette protéine qui pourraient être associées à la physiopathologie de la DMED

L-methionine protects against nephrotoxicity induced by methotrexate through modulation of redox status and inflammation

ABSTRACTObjective: Methotrexate (MTX) is a drug used in the treatment of cancer and autoimmune disorders; however, its clinical use is limited because of serious side effects including renal toxicity. This study aimed to investigate the protective effect of Lmethionine (L-Met) on MTX toxicity in the kidneys of rats.Methods: Thirty male rats were divided equally into five groups: control (saline), Met400 (400 mg/kg L-Met), MTX (20 mg/kg MTX), MTX-Met300 (300 mg/kg L-Met and 20 mg/kg MTX), and MTX-Met400 (400 mg/kg L-Met and 20 mg/kg MTX). Rats were euthanized one day after the last dose administration (day 16) and serum and renal tissue samples were collected. Renal function and injury indices, oxidative stress/antioxidant indices and proinflammatory cytokines were evaluated.Results: The results showed that L-Met could effectively counteract the nephrotoxic effects of MTX, in a dose-related manner, by improving most of the tested parameters. Furthermore, the higher dose of L-Met was able to restore several parameters to normal levels. In addition, investigation of MTX-induced hematological changes revealed a corrective potential of L-Met.Conclusion: L-Met can be an effective adjuvant therapy to modulate renal toxicity associated with MTX because of its antioxidant and antiinflammatory effects

Wskaźniki translokacji bakterii w cukrzycy typu 2 oraz ich związek z kontrolą glikemii i cukrzycową chorobą nerek w populacji egipskiej

Wstęp. W ostatnich latach podkreśla się udział trans­lokacji bakterii w patogenezie cukrzycy typu 2 (T2DM). Niniejsze badanie przeprowadzono w celu oceny potencjalnego wpływu stężenia białka wiążącego lipopolisacharydy (LBP) i translokacji DNA na kontrolę glikemii i progresję cukrzycowej choroby nerek u cho­rych na T2DM. Metody. Do przekrojowego badania obserwacyjnego włączono 30 chorych na T2DM oraz 30 osób tworzą­cych grupę kontrolną. Stężenia LBP w osoczu mierzono za pomocą metody immunoenzymatycznej. Do oceny translokacji DNA stosowano reakcję łańcuchową poli­merazy ukierunkowaną na gen 16S rRNA. Wyniki. Stężenia LBP w osoczu były istotnie wyższe u chorych na T2DM niż u osób z grupy kontrolnej (p = 0,02). Stwierdzono istotne dodatnie korelacje między stężeniem LBP a glikemią na czczo, odsetkiem hemoglobiny glikowanej, stężeniem białka C-reaktyw­nego (CRP), współczynnikiem albumina/kreatynina oraz ujemną korelację ze wskaźnikiem filtracji kłębuszko­wej. Na podstawie analizy krzywej ROC stężenia LBP wykazano, że parameter ten jest użytecznym czyn­nikiem predykcyjnym zarówno kontroli glikemii, jak i cukrzycowej choroby nerek u chorych na T2DM, a punkt odcięcia wynosi 15,17 μg/ml. Bakteryjne 16S rRNA wykryto w prawie wszystkich próbkach krwi chorych na T2DM (28/30) i w mniej więcej połowie próbek osób z grupy kontrolnej (16/30) (p < 0,001). Wnioski. Produkty translokacji mogą indukować roz­wój powikłań związanych z cukrzycą. Przyszłe bada­nia dotyczące protokołów interwencyjnych powinny być ukierunkowane na te produkty i poszukiwanie możliwości odwrócenia efektów ich oddziaływania

Wskaźniki translokacji bakterii w cukrzycy typu 2 oraz ich związek z kontrolą glikemii i cukrzycową chorobą nerek w populacji egipskiej

Wstęp. W ostatnich latach podkreśla się udział translokacji bakterii w patogenezie cukrzycy typu 2 (T2DM). Niniejsze badanie przeprowadzono w celu oceny potencjalnego wpływu stężenia białka wiążącego lipopolisacharydy (LBP) i translokacji DNA na kontrolę glikemii i progresję cukrzycowej choroby nerek u chorych na T2DM.Metody. Do przekrojowego badania obserwacyjnego włączono 30 chorych na T2DM oraz 30 osób tworzących grupę kontrolną. Stężenia LBP w osoczu mierzono za pomocą metody immunoenzymatycznej. Do oceny translokacji DNA stosowano reakcję łańcuchową polimerazyukierunkowaną na gen 16S rRNA.Wyniki. Stężenia LBP w osoczu były istotnie wyższe u chorych na T2DM niż u osób z grupy kontrolnej (p = 0,02). Stwierdzono istotne dodatnie korelacje między stężeniem LBP a glikemią na czczo, odsetkiem hemoglobiny glikowanej, stężeniem białka C-reaktywnego (CRP), współczynnikiem albumina/kreatynina oraz ujemną korelację ze wskaźnikiem filtracji kłębuszkowej. Na podstawie analizy krzywej ROC stężenia LBP wykazano, że parameter ten jest użytecznym czynnikiem predykcyjnym zarówno kontroli glikemii, jak i cukrzycowej choroby nerek u chorych na T2DM, a punkt odcięcia wynosi 15,17 μg/ml. Bakteryjne 16S rRNA wykryto w prawie wszystkich próbkach krwi chorych na T2DM (28/30) i w mniej więcej połowie próbek osób z grupy kontrolnej (16/30) (p < 0,001).Wnioski. Produkty translokacji mogą indukować rozwój powikłań związanych z cukrzycą. Przyszłe badania dotyczące protokołów interwencyjnych powinny być ukierunkowane na te produkty i poszukiwanie możliwości odwrócenia efektów ich oddziaływania

Relation between electrical compound action potential measures and speech perception in cochlear implanted children: audiological and phonological outcomes

Abstract Purpose To study ECAP measures (threshold and amplitude growth function 'AGF') in children CI users and find the relation between these ECAP measures and speech outcomes using audiological and phonological assessment. Subjects and method Twenty-one children were unilaterally implanted with Medel CI, and all subjects were submitted to phonological assessment, basic audiological assessment, speech recognition tests (WRS and BKB-SIN) and Medel maestro software measures (IFT, AutoART and AGF measures "thresholds and slopes" across apical, middle and basal electrodes). Results This study demonstrated no statistically significant difference between AGF thresholds at apical, middle and basal electrodes and a statistically significant difference between AGF slopes at apical and both middle and basal electrodes. There was no statistically significant correlation between the ECAP threshold and speech perception tests. In contrast, a positive statistically significant correlation was found between the AGF slope of the apical electrode and word recognition score, and a negative statistically significant correlation between AGF slopes at apical, middle and basal electrodes and SNR loss of BKB-SIN. High sensitivity and specificity of AGF slope at apical electrode were found to differentiate between good and poor performers as regards SNR loss of BKB-SIN and language test. Conclusions The AGF slope reflects neural survival better than the ECAP threshold. AGF slope at apical electrodes correlated with better CI performance in both phoniatric and audiological measures of speech perception and can be used as an objective tool to predict CI outcome

Rapid detection of urinary long non-coding RNA urothelial carcinoma associated one using a PCR-free nanoparticle-based assay

<div><p></p><p>We developed a specific hybridization assay for direct detection of long non-coding RNA urothelial carcinoma associated-1 (<i>lncRNA-UCA1</i>). Total RNA was extracted from urine pellet samples (bladder carcinoma patients and controls). Then, we compared the developed nanoassay with quantitative real time polymerase chain reaction (qRT-PCR) results in detection of urine <i>UCA1</i> in bladder cancer and control samples. The sensitivity and the specificity of <i>UCA1</i> nanoassay were 92.1% and 93.3%, respectively. The concordance of the two methods was 98%. Interestingly, all bilharzial benign cases showed negative <i>lncRNA-UCA1</i> using both methods. <i>UCA1</i>-nanoassay is a valid test for direct detection of urine <i>UCA1</i> for bladder cancer detection.</p></div

An Emerin LEM-Domain Mutation Impairs Cell Response to Mechanical Stress

International audienceEmerin is a nuclear envelope protein that contributes to genome organization and cell mechanics. Through its N-terminal LAP2-emerin-MAN1 (LEM)-domain, emerin interacts with the DNA-binding protein barrier-to-autointegration (BAF). Emerin also binds to members of the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Mutations in the gene encoding emerin are responsible for the majority of cases of X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). Most of these mutations lead to an absence of emerin. A few missense and short deletion mutations in the disordered region of emerin are also associated with X-EDMD. More recently, missense and short deletion mutations P22L, ∆K37 and T43I were discovered in emerin LEM-domain, associated with isolated atrial cardiac defects (ACD). Here we reveal which defects, at both the molecular and cellular levels, are elicited by these LEM-domain mutations. Whereas ∆K37 mutation impaired the correct folding of the LEM-domain, P22L and T43I had no impact on the 3D structure of emerin. Surprisingly, all three mutants bound to BAF, albeit with a weaker affinity in the case of ∆K37. In human myofibroblasts derived from a patient's fibroblasts, emerin ∆K37 was correctly localized at the inner nuclear membrane, but was present at a significantly lower level, indicating that this mutant is abnormally degraded. Moreover, SUN2 was reduced, and these cells were defective in producing actin stress fibers when grown on a stiff substrate and after cyclic stretches. Altogether, our data suggest that the main effect of mutation ∆K37 is to perturb emerin function within the LINC complex in response to mechanical stress

Emerin self‐assembly mechanism: role of the LEM domain

International audienceAt the nuclear envelope, the inner nuclear membrane protein emerin contributes to the interface between the nucleoskeleton and the chromatin. Emerin is an essential actor of the nuclear response to a mechanical signal. Genetic defects in emerin cause Emery-Dreifuss muscular dystrophy. It was proposed that emerin oligomerization regulates nucleoskeleton binding , and impaired oligomerization contributes to the loss of function of emerin disease-causing mutants. We here report the first structural characterization of emerin oligomers. We identified an N-terminal emerin region from amino acid 1 to amino acid 132 that is necessary and sufficient for formation of long curvilinear filaments. In emerin monomer, this region contains a globular LEM domain and a fragment that is intrinsically disordered. Solid-state nuclear magnetic resonance analysis identifies the LEM b-fragment as part of the oligomeric structural core. However, the LEM domain alone does not self-assemble into filaments. Additional residues forming a b-structure are observed within the filaments that could correspond to the unstructured region in emerin monomer. We show that the delK37 mutation causing muscular dystrophy triggers LEM domain unfolding and increases emerin self-assembly rate. Similarly, inserting a disulfide bridge that stabilizes the LEM folded state impairs emerin N-terminal region self-assembly, whereas reducing this disulfide bridge triggers self-assembly. We conclude that the LEM domain, responsible for binding to the chromatin protein BAF, undergoes a conformational change during self-assembly of emerin N-terminal region. The consequences of these structural rearrangement and self-assembly events on emerin binding properties are discussed. Abbreviations BAF, barrier-to-autointegration factor; DTT, dithiothreitol; EDMD, Emery-Dreifuss muscular dystrophy; LEM, Lap2-emerin-Man1; LINC, linker of nucleoskeleton and cytoskeleton; NMR, nuclear magnetic resonance; SDS/PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ThT, thioflavin T