Shared molecular amino acid signature in the HLA-DR peptide binding pocket predisposes to both autoimmune diabetes and thyroiditis

There is strong genetic association between type 1A diabetes (T1D) and autoimmune thyroid disease (AITD). T1D and AITD frequently occur together in the same individual, a condition classified as a variant of the autoimmune polyglandular syndrome type 3 (APS3). Because T1D and AITD are individually strongly associated with different HLA class II sequences, we asked which HLA class II pocket sequence and structure confer joint susceptibility to both T1D and AITD in the same individual (APS3v). We sequenced the HLA-DR gene in 105 APS3v patients and 153 controls, and identified a pocket amino acid signature, DRβ-Tyr-26, DRβ-Leu-67, DRβ-Lys-71, and DRβ-Arg-74, that was strongly associated with APS3v (P = 5.4 × 10⁻¹⁴, odds ratio = 8.38). Logistic regression analysis demonstrated that DRβ-Leu-67 (P = 9.4 × 10⁻¹³) and DRβ-Arg-74 (P = 1.21 × 10⁻¹³) gave strong independent effects on disease susceptibility. Structural modeling studies demonstrated that pocket 4 was critical for the development of T1D+AITD; all disease-associated amino acids were linked to areas of the pocket that interact directly with the peptide and, therefore, influence peptide binding. The disease-susceptible HLA-DR pocket was more positively charged (Lys-71, Arg-74) compared with the protective pocket (Ala-71, Gln-74). We conclude that a specific pocket amino acid signature confers joint susceptibility to T1D+AITD in the same individual by causing significant structural changes in the MHC II peptide binding pocket and influencing peptide binding and presentation. Moreover, Arg-74 is a major amino acid position for the development of several autoimmune diseases. These findings suggest that blocking the critical Arg-74 pocket might offer a method for treating certain autoimmune conditions.     

The effect of music relaxation versus progressive muscular relaxation on insomnia in older people and their relationship to personality traits

A large percentage of older people suffer from chronic insomnia, affecting many aspects of life quality and well-being. Although insomnia is most often treated with medication, a growing number of studies demonstrate the efficiency of various relaxation techniques. The present study had three aims: first, to compare two relaxation techniques--music relaxation and progressive muscular relaxation--on various objective and subjective measures of sleep quality; second, to examine the effect of these techniques on anxiety and depression; and finally, to explore possible relationships between the efficiency of both techniques and personality variables. Fifteen older adults took part in the study. Following one week of base-line measurements of sleep quality, participants followed one week of music relaxation and one week of progressive muscular relaxation before going to sleep. Order of relaxation techniques was controlled. Results show music relaxation was more efficient in improving sleep. Sleep efficiency was higher after music relaxation than after progressive muscular relaxation. Moreover, anxiety was lower after music relaxation. Progressive muscular relaxation was related to deterioration of sleep quality on subjective measures. Beyond differences between the relaxation techniques, extraverts seemed to benefit more from both music and progressive muscular relaxation. The advantage of non-pharmacological means to treat insomnia, and the importance of taking individual differences into account are discussed.     

Life-threatening serotonin toxicity due to a citalopram-fluconazole drug interaction: case reports and discussion

OBJECTIVE: To discuss two cases of life-threatening serotonin toxicity due to a drug interaction between citalopram and fluconazole and to review the pertinent literature. METHODS: A Medline search without date limitation was conducted using the terms serotonin syndrome, serotonin toxicity, fluconazole and citalopram. RESULTS AND DISCUSSION: Fluconazole inhibits CYP2C19. Citalopram is a substrate for 2C19 and inhibition of its metabolism may result in serotonin toxicity. Serotonin toxicity in oncology patients may not present with the classic constellation of signs typically described in the literature. Delirium may be the only presenting feature. Current level of evidence for treatment of serotonin toxicity is level 4 or 5 (case series and expert opinion). Nevertheless, there is a strong theoretical basis for treating serotonin toxicity in medical patients with a 5H(2A) blocker such as cyproheptadine. CONCLUSIONS: Consultation-liaison psychiatrists and oncologists should be aware of this preventable and underrecognized interaction. Citalopram should be stopped or substituted prior to the concurrent administration of fluconazole, and in the event of toxicity, treatment with cyproheptadine has a favorable risk-benefit ratio despite a lack of randomized controlled data to support its use.     

14C-labeled pulegone and metabolites binding to alpha2u-globulin in kidneys of male F-344 rats

Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and menthofuran were previously studied in rodents, and higher concentrations of pulegone- and menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, alpha2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 microCi/kg) or menthofuran (60 mg/kg, 120 microCi/kg) or menthones (80 mg/kg, 120 microCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney cytosol proteins of male but not female rats. The chemicals present in the male rat kidney cytosol after dialysis were extracted with dichloromethane and characterized by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). All parent compounds were detected, and the metabolites characterized included piperitone from pulegone or menthones treatment, menthones and possibly 8-hydroxymenthones from pulegone treatment, and mintlactones (diastereomeric mintlactone and isomintlactone) and 7a-hydroxymintlactone from menthofuran treatment. Analysis of the male rat kidney cytosol by a gel filtration column demonstrated that the retention was due to reversible binding of these chemicals with the male rat-specific protein alpha2u-globulin. However, binding of pulegone and/or metabolites to alpha2u-globulin did not produce accumulation of this protein in the kidney.     

A centrifuge technique for the evaluation of the extent of water movement in wet powder masses.

A centrifuge method has been applied to the assessment of water retention in pharmaceutical powders. Five drug models and microcrystalline cellulose (MCC) were each mixed with different amounts of water and centrifuged at different speeds. The amount of water retained by the wet mass was evaluated by drying the powders to constant weight. Binary mixtures of each of the five model drugs, MCC and water were also processed in the same way. From the amount of water extracted the moisture retention capacity (MRC) was calculated. The MCC retained water more strongly than the different drug models over a wider range of initial water contents. The five drug models, although similar in their chemical structure, were divided into two groups, in terms of their MRC values. 4-HBA and propyl gallate recorded higher MRC values than methyl, propyl and butyl paraben. For the drug models mixed with MCC, the MRC values recorded were similar, though it was still possible to divide the drugs into the two subgroups. A correlation between the MRC value recorded for the different systems and the hydrogen bonding solubility component was found. The application of different centrifuge speeds indicated that within the same material there were different mechanisms of water retention.     

A CD40 Kozak sequence polymorphism and susceptibility to antibody-mediated autoimmune conditions: the role of CD40 tissue-specific expression

Previously, we and others have demonstrated the association of a C/T single nucleotide polymorphism (SNP), in the Kozak sequence of CD40, with Graves' disease (GD). Here, using an expanded data set of patients, we confirm the association of the CD40 SNP with GD (n=210, P=0.002, odds ratio (OR)=1.8). Subset analysis of patients with persistently elevated thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies (Abs), (TPO/Tg Abs), after treatment (n=126), revealed a significantly stronger association of the SNP with disease (P=5.2 x 10(-5), OR=2.5) than in GD patients who were thyroid antibody-negative. However, the CD40 SNP was not associated with TPO/Tg Abs in healthy individuals. Next, we tested the CD40 SNP for association with Myasthenia Gravis (MG), which, like GD is an antibody-mediated autoimmune condition. Analysis of 81 MG patients found no association of the SNP with disease. Functional studies revealed significant expression of CD40 mRNA and protein in the thyroid (target tissue in GD) but not in skeletal muscle (target tissue in MG). Combined, our genetic and tissue expression data suggest that the CD40 Kozak SNP is specific for thyroid antibody production involved in the etiology of GD. Increased thyroidal expression of CD40 driven by the SNP may contribute to this disease specificity.     

Molecular-based diagnosis of <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection is associated with reduced mortality

Polymerase chain reaction (PCR) for the diagnosis of Clostridium difficile infection (CDI) might result in overdiagnosis. The clinical outcomes of symptomatic CDI patients diagnosed by PCR remain uncertain. We aimed to determine whether patients whose diagnosis of CDI was based on PCR had different characteristics and clinical outcomes than those diagnosed by toxin immunoassay. Consecutive CDI patients, hospitalized at Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel, between January 2013 and January 2016, were identified retrospectively and included in the study. Diagnosis of CDI was based on PCR or diagnosis by immunoassay for C. difficile toxin. The main outcome was 30- and 90-day all-cause mortality. The PCR group included 165 patients and the immunoassay group included 157 patients. In comparison to the immunoassay group, patients in the PCR group were more likely to be younger, to be independent, to undergo previous abdominal surgery, and to use laxatives. The 30-day mortality rate in the PCR group was significantly lower than that in the immunoassay group, 29/165 (18%) vs 49/157 (31%), respectively; p?=?0.028. On multivariate analysis, PCR diagnosis was associated with reduced mortality, OR 0.48 (95% CI 0.26–0.88). PCR-based diagnosis of CDI is associated with reduced all-cause mortality rates. Further studies are needed to determine the management of patients with discrepant immunoassay and PCR diagnosis of CDI.     

The β subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD′ and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the β subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the β subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the β subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly −1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD′, UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the β subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.