Celiac And Autoimmune Thyroid Disease In Patients With Anti-Gad Positive Type-1 Diabetes Mellitus.

BACKGROUND: Type-1 diabetes mellitus (T1DM) and autoimmune thyroid disease can occur concomitantly and patients with TIDM have a high risk of other autoimmune conditions like thyroid disease and celiac disease. This study aimed to analyze the association of anti-GAD positive T1DM with anti-thyroid antibodies and celiac disease. METHODS: This cross-sectional study was conducted at the Department of Paediatric Endocrinology & Diabetes, National Institute of Child Health, Karachi Pakistan from July 2022 to December 2022. A total of 115 children of both genders aged between 1-18 years having known T1DM were analyzed. Children with chronic kidney disease or chronic liver disease were excluded. Those children were also not included whose parents/caregivers did not wish their children to be part of this research. The blood sample of each child was taken in a sterilized container and sent to an institutional laboratory for biochemical investigations. RESULTS: In a total of 115 patients, 67 (58.3%) were female and 48 (41.7%) males. The mean age was 8.87±3.43 (ranging between 1.5-17 years). The mean HbA1c was 11.86±7.31%. It was found that anti-GAD IgG was having signification association with celiac disease (p<0.001). Significant correlation of anti-GAD positive antibodies with Ttg-IgG antibodies (correlation coefficient=0.303, p=0.001), thyroid peroxidase antibodies (correlation coefficient=0.228, p=0.001). CONCLUSIONS: High proportions of children with anti-GAD positive T1DM patients were found to have thyroid disorders and celiac disease. A significant correlation was found between anti-GAD positive antibodies, celiac disease and anti-thyroglobulin antibodies.

Thiamine-Responsive Megaloblastic Anaemia With Hypothyroidism, A Puzzling Association.

Background: Thiamine-responsive megaloblastic anaemia (TRMA) is characterized by the classic trio of diabetes mellitus, sensorineural hearing loss, and megaloblastic anaemia, typically emerging subtly between infancy and adolescence. Administration of high-dose thiamine often yields improvements in anaemia and occasionally in diabetes. Uncommon manifestations include optic atrophy, congenital heart defects, short stature, and stroke. In this specific case, a 5-year-old diagnosed with insulin-dependent diabetes mellitus (IDDM) since the age of one presented with symptoms such as polyuria, fever, and vomiting, revealing an HbA1c of 10.64. Further examinations disclosed compromised hearing and vision. A negative antibody workup and a thyroid profile indicating hypothyroidism prompted additional investigations, including Brainstem Evoked Response Audiometry (BERA) and retinal examination, confirming bilateral sensorineural hearing loss and maculopathy, respectively. A comprehensive blood count unveiled megaloblastic anaemia. Genetic profiling confirmed a homozygous mutation in the SLC19A2 gene, thus diagnosing TRMA. An early diagnosis, coupled with genetic confirmation, enables timely intervention, with patients responding positively to high-dose thiamine. Genetic counselling plays a pivotal role in enlightening families about the disease and its inheritance patterns, fostering awareness and understanding.

A Rare Skeletal Dysplasia-Close Mimicker Of Juvenile Idiopathic Arthritis-Progressive Pseudorheumatoid Dysplasia.

Progressive pseudorheumatoid dysplasia or spondyloepiphyseal dysplasia tarda is caused by a mutation in Wnt1 inducible signalling pathway protein 3 (WISP3) and passes in an autosomal recessive manner. Prevalence underestimated as one per million and most of the cases remain undiagnosed or treated as Juvenile Idiopathic Arthritis (JIA). Differentiation between JIA and PPRD is really challenging however, this case is genetically confirmed from our country. 7-year-old, short stature boy, with multiple joint swellings of hands and feet, initially suspected to have JIA and had been worked up and took treatment for that for the past 2 years. He had progressive stiffness of small joints. Baseline biochemistry, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor and ANA, were within normal limits. He was moderately growth hormone deficient. Thyroid function tests and insulin-like growth factor 1 (IGF-1) were within reference ranges. Skeletal survey showed typical findings of pseudorheumatoid skeletal dysplasia. Physical therapy and genetic counselling were done.

MFS transportome of the human pathogenic yeast Candida albicans.

BACKGROUND: The major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast Candida albicans. RESULTS: Computational analysis of C. albicans genome enabled us to identify 95 potential MFS proteins which clustered into 17 families using Saier's Transport Commission (TC) system. Among these SP, DHA1, DHA2 and ACS represented major families consisting of 22, 22, 9 and 16 members, respectively. Family designations in C. albicans were validated by subjecting Saccharomyces cerevisiae genome to TC system. Based on the published available genomics/proteomics data, 87 of the putative MFS genes of C. albicans were found to express either at mRNA or protein levels. We checked the expression of the remaining 8 genes by using RT-PCR and observed that they are not expressed under basal growth conditions implying that either these 8 genes are expressed under specific growth conditions or they may be candidates for pseudogenes. CONCLUSION: The in silico characterisation of MFS transporters in Candida albicans genome revealed a large complement of MFS transporters with most of them showing expression. Considering the clinical relevance of C. albicans and role of MFS members in antifungal resistance and nutrient transport, this analysis would pave way for identifying their physiological relevance.

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans.

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.

Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system.

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.

Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans.

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.

Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis.

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.

Characterization of Cdr1p, a major multidrug efflux protein of Candida albicans: purified protein is amenable to intrinsic fluorescence analysis.

Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding.

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.