Prevalence of Autoantibodies against 3-DG-Glycated H2A Protein in Type 2 Diabetes.

Advanced glycation end-products (AGEs) have been found to be critically involved in initiation or progression of diabetes secondary complications (nephropathy, retinopathy, neuropathy, and angiopathy). Various hyper-glycating carbonyl compounds such as 3-deoxyglucosone (3-DG) are produced in pathophysiological conditions that form AGEs in high quantity both in vivo and in vitro. In the first stage of this study, we glycated histone H2A protein by 3-DG, and the results showed the formation of various intermediates and AGEs as well as structural changes in the protein. In the second stage, we studied the immunogenicity of native and 3-DG-glycated H2A protein in female rabbits. The modified H2A was highly immunogenic, eliciting high titer immunogen-specific antibodies, while the unmodified form was almost nonimmunogenic. Antibodies against standard carboxymethyllysine (CML) and pentosidine were detected in the immunized female rabbits, which demonstrates the immunogenic nature of AGEs (CML and pentosidine) as well. The results show both structural perturbation and AGEs have the capacity of triggering the immune system due to the generation of neoepitopes that render the molecule immunogenic. This study shows the presence of autoantibodies against 3-DG-modified H2A, CML, and pentosidine in the sera of type 2 diabetes patients having secondary complications. Autoantibodies against damaged H2A and AGEs may be significant in the assessment of initiation/progression of secondary complications in type 2 diabetes mellitus patients or may be used as a marker for early detection of secondary complications in diabetes.

Modified tubularized incised plate urethroplasty in distal hypospadias repair: Stepwise technique with validated functional and cosmetic outcome.

OBJECTIVE: To describe modification of the tubularized incised plate urethroplasty (TIP) for distal hypospadias, and assess its efficacy, and functional and cosmetic outcomes. METHODS: A prospective evaluation of a consecutive series of patients operated for primary distal hypospadias was conducted at a tertiary reference center. A standardized modification of the TIP (mTIP) procedure was performed on a 10 French catheter. Clinical data were collected in a dedicated database. Intraoperative variables, postoperative complications and outcomes, by means of uroflowmetries and a validated (HOPE) questionnaire, were assessed. Efficacy was evaluated with the reported complications: functional outcome was evaluated with uroflowmetries and cosmetic assessment by a validated questionnaire (HOPE). A descriptive statistical analysis was performed. RESULTS: Of the 112 boys operated between 30/09/2011 and 1/04/2014, 50 completed long-term follow-up with functional and esthetic evaluation, as required for inclusion. Median age at surgery was 25 months (range 14-156); median follow-up time was 21.5 months (range 6-48). Complications requiring re-intervention occurred in 2/50 boys. Uroflowmetry presented a bell-shaped curve in 47/50 boys, and the median HOPE score was 9.5 (range 7.6-10.0). CONCLUSION: The mTIP procedure provided satisfactory long-term functional and cosmetic outcomes, as validated by uroflowmetries and standardized questionnaire.

Molecular characterization and fluorescence analysis of HCV non-structural proteins NS3, NS3-4A and NS4A of genotype 3a.

Hepatitis C virus (HCV) chronically infects almost 2% of world's population. Chronic infection can lead to liver failure and hepatocellular carcinoma (HCC). Approximately 10% of the Pakistani population is infected with HCV and type 3 is the most prevalent genotype with 75-90% prevalence. In this study we have developed transiently expressing cell culture based system for the expression of HCV non-structural NS3, NS3-4A and NS4A proteins of genotype 3a. HCV non-structural genes NS3, NS3-4A and NS4A were cloned in to pFLAG-CMV2 and pEGFP-C1vectors. All vectors were transfected separately to Huh-7 cells and their protein expression was analyzed by Western blot and immunofluorescence. All proteins were expressed correctly and in the transfection we have obtained 42-70% efficiency for all clones. This system can be used for the development of novel antiviral strategies to inhibit the viral replication, to study apoptosis pathways induced by HCV, for the evaluation of vaccine candidates and also to study the role of HCV different signaling pathways.

FROM Qutn TO Bt COTTON: DEVELOPMENT, ADOPTION AND PROSPECTS. A REVIEW.

Cotton has unique history of domestication, diversification, and utilization. Globally it is an important cash crop that provides raw material for textile industry. The story of cotton started from human civilization and the climax arrived with the efforts of developing transgenic cotton for various traits. Though conventional breeding brought steady improvement in developing resistance against biotic stresses but recent success story of gene transferfrom Bacillus thuringiensis into cotton showed game changing effects on cotton cultivation. Amongst various families of insecticidal proteins Bt Cry-toxins received more attention because of specificity against receptors on the cell membranes of insect midgut epithelial cells. Rapid Bt cotton adoption by farmers due to its economic and environmental benefits has changed the landscape of cotton cultivation in many countries. But the variable expression of Bt transgene in the newly developed Bt cotton genotypes in tropical environment is questionable. Variability of toxin level in different plant parts at various life stage of plant is an outcome of genotypic interaction with environmental factors. Temporal gene expression of Cry1Ac is also blamed for the epigenetic background in which transgene has been inserted. The presence of genotypes with sub-lethal level of Bt toxin might create resistance in Lepidopteron insects, limiting the use of Bt cotton in future, with the opportunityfor other resistance development strategies to get more attention like gene stacking. Until the farmers get access to more recent technology, best option is to delay the development of resistance by applying Insect Resistance Management (IRM) strategies.

Glycoxidation of biological macromolecules: a critical approach to halt the menace of glycation.

Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Water-clear cell adenoma: A rare form of hyperparathyroidism.

INTRODUCTION: Primary hyperparathyroidism is a common endocrine disorder, with an incidence of 21.6 per 100,000 person-years. Asymptomatic elevated serum calcium levels on routine biochemical investigations accounts for 80% of newly diagnosed primary hyperparathyroidism. Solitary adenoma is the commonest cause of primary hyperparathyroidism and can be treated by excision of a single gland. PRESENTATION OF CASE: We present a case of primary hyperparathyroidism in a 74-year-old female was referred to our surgery endocrine outpatients for assessment of a persistently elevated calcium level, lower abdominal pain and constipation. Biochemical analysis revealed corrected serum calcium of 3.13mmol/L (reference range 2.17-2.51mmol/L) and an intact parathyroid hormone level (iPTH) of 488.9ng/L (reference range 15-65ng/L). Sestamibi scan localised a persistent increased area of activity inferior to the lower pole of the left lobe of thyroid gland. DISCUSSION: The patient underwent a minimally invasive parathyroidectomy using a 3cm incision with intra-op radionucliotide localisation. At surgery a single large parathyroid gland measuring 5.5cm was excised without complication. Grossly the parathyroid gland was an encapsulated tan mass measuring 5.5cm×2.5cm×2cm and weight 13g and histological assessment revealed a water-clear cell (WCC) adenoma. She made an uneventful post op recovery with normalisation of her serum calcium. CONCLUSION: WCC adenomas have a "low endocrinological activity" in which serum calcium levels do not elevate until the adenoma has reached considerable size. Our case supports this hypothesis and aids to the understanding of these rare tumours.

Use of fraction flow reserve to predict changes over time in management of superficial femoral artery.

BACKGROUND: Peripheral arterial disease is a condition characterized by progressive arterial narrowing, which affects patients' quality of life. The purposes of this study were to (1) establish the feasibility of obtaining peripheral fractional flow reserve (pFFR) in the peripheral vascular circulation, (2) demonstrate an association between baseline pFFR and peak systolic velocity (PSV) measured by duplex ultrasound, and (3) correlate postintervention pFFR with future restenosis using the change in PSV over time as a surrogate. METHODS: Twenty patients underwent baseline ankle brachial index (ABI) and PSV testing. Pre- and postintervention pFFR was performed. Patients were followed with three ABI and PSV recordings during the 1 year follow-up period. The association between baseline PSV, ABI, and pFFR with changes in PSV over time were explored. Predictors of postprocedural PSV over time were determined. RESULTS: The baseline translesional-resting ratio was significantly different from the pFFR using adenosine (0.79 ± 0.08 vs. 0.71 ± 0.09, P = 0.01). Baseline PSV was significantly associated with preintervention pFFR (-0.77, P < 0.001). Compared to patients with a postprocedure pFFR > 0.95, patients with a postprocedure pFFR < 0.95 had a significantly more rapid rise in PSV over time (P = 0.009). CONCLUSION: This is the first study to demonstrate that the peripheral vascular bed does respond to vasodilatation thereby supporting the use of pFFR for this procedure. In our study, postintervention pFFR < 0.95 predicted a more rapid increase in PSV over time, which is a reasonably accepted surrogate for restenosis.

Saudi Oncology Society clinical management guidelines for prostate cancer.

In this report, guidelines for the evaluation, medical and surgical management of testicular germ cell tumors is presented. It is categorized according to the stage of the disease using the tumor node metastasis staging system, 7th edition. The recommendations are presented with supporting level of evidence.

Colorectal liver metastases.

The diagnosis and management of CRLM is complex and requires a multidisciplinary team approach for optimal outcomes. Over the past several decades, the 5-year survival following resection of CRLM has increased and the criteria for resection have broadened substantially. Even patients with multiple, bilateral CRLM, previously thought unresectable, may now be candidates for resection. Two-stage hepatectomy, repeat curative-intent hepatectomy, and even selected resection of extrahepatic metastases have further increased the number of patients who may be treated with curative intent. Multiple liver-directed therapies exist to treat unresectable, incurable patients with adequate survival benefit and morbidity rates.

Screening for Y-chromosome microdeletions in a population of infertile males in the Gaza Strip.

Infertility is an extraordinary public health problem in the Arab world, as it affects about 15% of couples seeking children. The male partner is responsible for infertility in approximately half of these cases. Classic microdeletions of the Y-chromosome involving the azoospermia factor (AZF) regions are known to be associated with spermatogenic impairment, and non-obstructive azoospermia must be differentiated on the basis of endocrine evaluation and testicular biopsy. Partial AZFc deletions remain controversial because there is no clear agreement regarding their role in spermatogenic failure. In the current study, 50 fertile males (controls) and 125 patients with primary idiopathic male infertility were studied in order to describe the frequency of Y-chromosome mirodeletions among male infertility patients in the Gaza Strip-Palestine area. No Y chromosome classical microdeletions could be detected in any of the 125 infertile men, suggesting that ethnic factors, genetic background, and Y chromosome haplogroups are key factors in such deletions. On the other hand, six gr/gr and one b1/b3 AZFc partial deletions were detected in the infertile population. The gr/gr deletion was also noted in relatives of four of the six patients with this deletion, and in one of the fertile controls. In conclusion, our study shows that the incidence of Y-chromosome microdeletions in our population is rare; these data suggest that other genetic, epigenetic, nutritional and/or local factors are responsible for impairments in semen parameters observed in this Gazan population. We further hypothesise that the gr/gr deletion is not associated with male infertility, at least in this sub-group.

Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis.

Tumor-derived immune suppression is considered to be a major mechanism of tumor evasion from the immune system destruction, however, little is known regarding the induction of T-cell functional suppression by tumor-derived exosomes. Herein, we investigate tumor-derived exosomes involved in normal immunological communications as means of inhibiting an antitumor T-cell response. Exosomes derived from LNCaP, a human prostate cancer cell line, were visualized by FACS and identified based on size (80-200 nm) in comparison to marker beads. Exosomes from tumor cell line inhibited T-cell proliferation. Dose-dependent apoptosis of T cells was induced by co-culture with tumor exosomes. Addition of anti-FasL antibody blocked the apoptosis induction by tumor exosomes. This study suggests that induction of T-cell apoptosis by tumor-derived exosomes appears to be a novel mechanism of tumor immune evasion.

Normocortisolemic Cushing's syndrome initially presenting with increased glucocorticoid receptor numbers.

A girl who developed Cushingoid features in peripuberty, but was eucortisolemic, was previously reported to have markedly elevated lymphocyte glucocorticoid receptor sites per cell with normal binding affinity as a potential cause of her phenotype. Her circadian rhythm of cortisol and pituitary-adrenal axis were initially intact, but later proved to be dysregulated. The patient presented at age 10.8 yr with centripetal obesity, moon facies, buffalo hump, and purple striae, but no statural stunting, which is a cardinal sign of Cushing's syndrome. At 11.5 yr she suffered a compression fracture of the L1 vertebra. That prompted treatment with the antiprogestin drug mifepristone (RU486), which was administered at high dose to achieve an antiglucocorticoid effect. From ages 13.75 yr through 15.5 yr, RU486 was administered in various intervals to suppress her Cushingoid features. Once RU486 was introduced, however, a consistent correlation over time between the Cushingoid features and glucocorticoid receptor sites per cell was no longer observed. However, the number of glucocorticoid receptor sites per cell tended to decrease in response to administering RU486. Ultimately, her Cushingoid phenotype proved to be transient.

Cloning of a teleost fish glucocorticoid receptor shows that it contains a deoxyribonucleic acid-binding domain different from that of mammals.

In the teleost fish, physiological and biochemical studies suggest that glucocorticoids regulate both salt balance and metabolic activities. In mammals, however, these functions are divided between glucocorticoids and mineralocorticoids. In mammals, separate receptors for these two classes of steroid hormone have been cloned and sequenced. To begin to understand the regulation in fish of the vital processes ascribed to glucocorticoids, we have cloned, sequenced, expressed, and studied the steroid-binding and transcriptional activation capabilities of the rainbow trout (Onchorhynchus mykiss) glucocorticoid receptor. Northern blot analysis shows a single rainbow trout GR messenger RNA species of 7.5 kilobases expressed in gill, intestine, skeletal muscle, kidney, and liver. The trout GR 2274-nucleotide coding sequence provides for a protein of 758 amino acids, with appropriate similarities to mammalian GR, with one striking exception. As in other members of the steroid/thyroid/retinoid receptor family, the DNA-binding domain contains two putative zinc fingers. These have high homology with those of other GRs. However, between the zinc fingers in the trout GR are found 9 more amino acids than are seen in mammalian GRs, raising questions as to the functional form of the fish, as opposed to the mammalian, GR. It has been proposed that as fish appear to use glucocorticoids for both metabolic and salt control, presumably through a single GR, GR would prove to be the evolutionary precursor to mammalian GR and mineralocorticoid receptor (MR). Computer analysis of the known sequences of GRs and MRs, however, suggests that the fish GR did not give rise to the MR of higher animals, but that both subfamilies of receptor arose from some earlier gene.

Cranial fasciitis: presentation as a post-auricular mass.

Cranial fasciitis is a rare variant of nodular fasciitis. It is a benign condition with features resembling sarcoma, seen principally in young males. It involves the skull bones and grows at a rapid pace. Accurate diagnosis and surgical excision is the key to management. Prognosis is good with recurrence rare. By 1992, 17 cases had been reported in the literature. The present case is the first reported in Pakistan. It presented as a post-auricular mass.

Identification of the activation-labile gene: a single point mutation in the human glucocorticoid receptor presents as two distinct receptor phenotypes.

CCRF-CEM-C7 is a well characterized human leukemic clonal cell line which is lysed by dexamethasone (dex). Originating from the wild-type CEM-C7 cells are two dex-resistant clones which are not lysed by 1 microM dex and have functionally defective glucocorticoid receptors (GR). They are receptorless ICR27TK.3 and activation-labile 4R4 cells. ICR27TK.3 and 4R4 cells have distinct cellular phenotypes, as indicated by dissimilar numbers of dex-binding sites despite similar levels of GR mRNA and immunochemically detectable GR. We have now investigated the molecular defects in the GR of ICR27TK.3 and 4R4 cells by determining the nucleotide sequence of their GR. Our results support the biochemical evidence previously reported by others for the presence of both a normal (GR+) and a mutant (GR*) allele in CEM-C7 cells. We clearly show that the wild-type CEM-C7 cells express two alleles of GR, the normal GR+ and the abnormal GR*, which has a Leu753--> Phe753 mutation. We demonstrate that both ICR27TK.3 and 4R4 cells contain only the abnormal GR* and that the normal GR+ gene is deleted in both of these GR defective clones. Our results further show that the GR* is basically an activation-labile receptor and has diminished functional capability in a transfection assay measuring GR-driven transcription. Thus, these two phenotypically different cell lines express similar amounts of an identical GR* containing a single point mutation at amino acid 753. A single point mutation in the steroid-binding domain of the GR, therefore, may behave differently, depending on the cellular milieu in which it is expressed.

Glucocorticoid receptors in leukemias, lymphomas and myelomas of young and old.

In this paper we have briefly reviewed the nature of leukemias and lymphomas in the old and the young. We surveyed in general the ways in which lymphoid cells and other hematologic elements respond to glucocorticoids, mentioning that there may be direct or indirect effects on their growth by these ligands. We have reviewed the current general model for the action of glucocorticoids in all cells, namely the fact that the actions of these steroids are mediated to a large extent through binding with ligand-activated transcription factors, their receptors. The growing wealth of detail about the nature of the interaction of these receptors with regulatory sites in the genome is discussed. Finally, we have described our results with lines of tissue culture cells representing clones from a typical leukemia of the young, and of myeloma, a typical hematologic malignancy of the elderly. Several features of the effects of glucocorticoids on these cells point up areas that would be pertinent to explore in aging and in the relationship of hematologic diseases to survival and response to therapy in the older versus the younger patient.

Glucocorticoids in malignant lymphoid cells: gene regulation and the minimum receptor fragment for lysis.

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocorticoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocorticoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half "Zn fingers" of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, preceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.

Transoral decompression and posterior stabilisation in Morquio's disease.

A 3.5 year old boy with Morquio's disease was referred with a persisting left hemiparesis four months after a fall and was found to have craniocervical junction compression due to atlantoaxial subluxation and significant anterior soft tissue compression. Transient unconsciousness at the time of the fall was probably due to medullary concussion as a result of hyperextension, not a head injury. Spinal cord compression due to atlantoaxial subluxation at the craniovertebral junction is a major cause of disability and death in these patients. Once cervical myelopathy appears, early posterior occipitocervical fusion has been advocated in order to arrest the progression of neurological disability and this is successful in most cases. This conventional approach was considered unsafe because of the significant anterior compression. A combined anterior transoral decompression with posterior fusion to deal with this particularly difficult problem is described.

Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic (CEM) cells.

Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure. In both wild-type and highly dexamethasone(dex)-resistant clones of the human leukemic cell line CEM, exposure to cortivazol leads to cell death. It has been shown recently that in wild-type CEM cells but not in a dex-resistant, glucocorticoid receptor(GR)-defective clone ICR-27 TK-3, dex induces GR mRNA. To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there, wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied. Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones, although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction. Increased levels of GR mRNA were noticed as early as 3 h after treatment. A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found. Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells, or might serve as a marker for the process. However, the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells.