Mutant specific anti calreticulin antibody (CAL2) immunohistochemistry as a screening test for calreticulin (CALR) mutation testing.

BACKGROUND: About 50 different CALR frameshift mutations have been identified in BCR-ABL1 negative MPN, all leading to the development of common new protein C terminus. Antibody targeting this terminal epitope can be useful to identify this driver mutation using immunohistochemistry. MATERIALS AND METHODS: CALR mutation analysis was carried out in 51 JAK2V617F negative cases, PMF (n = 22) and ET (n = 29). PCR followed by fragment analysis was performed for molecular detection of CALR mutation. Bone marrow biopsy specimens of corresponding patients were subjected to IHC using mutation specific antibody CAL2. Staining pattern and intensity were observed. Staining of <2% of background nonmegakaryocytic (non- MK) cells were regarded as Pattern A, while staining of more than 2% of background nonmegakaryocytic (non-MK) was regarded as pattern B. RESULTS: CALR mutation was noted in 40.9% (9/22) and 41.4% (12/29) of JAK2V617F negative PMF and ET, respectively. All CALR mutated cases, irrespective of the mutation type, showed a positive IHC staining in the megakaryocytes with moderate to bright intensity. All CALR wild-type cases were negative on IHC. (Concordance rate- 100%). Pattern A was noted in 40% cases, while pattern B was noted in 60% cases. Pattern A staining had significantly higher chances of having type 1 mutation as compared to pattern B. In contrast, pattern B had a nonsignificant trend toward higher bone marrow cellularity and marrow fibrosis. CONCLUSION: CAL2 IHC detects all types of CALR mutation. This can act as a sensitive, specific, rapid, and cost-effective screening test for CALR mutation analysis.

Necessity to identify candida species accurately with minimum inhibitory concentration determination in each case of bloodstream infections.

BACKGROUND: Bloodstream Candida infection is a life-threatening event among ICU admitted patients. This infection is caused by a diverse range of Candida species having varied minimum inhibitory concentrations. OBJECTIVES: To identify Candida species causing bloodstream infections with their antifungal susceptibility determination. METHODS: Candida species isolated from the blood of ICU admitted patients were identified by phenotypic as well as by molecular methods including PCR-RFLP using MspI restriction enzyme and MALDI TOF MS. The minimum inhibitory concentration of fluconazole, voriconazole, amphotericin B and caspofungin was determined against isolated Candida species by CLSI M27A3 guidelines. RESULTS: A total of 119 Candida species were isolated. Among them, C. tropicalis(n=29) was the predominant isolate followed by C. parapsilosis(n=18), C. glabrata (n=12), C. krusei (n=11) and C. albicans(n=11). Uncommon Candida species isolated were; Wickerhamomyces anomalus(n=15), Kodaemia ohmeri(n=8), C. lusitaniae (n=5) and C. auris (n=2). A varied antifungal MIC values were observed. Caspofungin had the lowest MIC among the tested antifungals. Increased fluconazole MIC was observed against the isolated Candida species including C. tropicalis. All the isolated C. lusitaniae and C. auris strains have ≥1mcg/ml amphotericin B MIC. In comparison to fluconazole, voriconazole was more effective when tested in vitro. CONCLUSION: Emergence of uncommon Candida species having varied antifungal MIC warns the physicians to have a prompt, accurate identification with antifungal MIC determination in each case of bloodstream Candida infections.

Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry.

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Silk cocoon of Bombyx mori: proteins and posttranslational modifications--heavy phosphorylation and evidence for lysine-mediated cross links.

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2-DE was carried out with multi-enzyme in-gel digestion followed by MS identification of silk-peptides. High-sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys->allysine formation have been observed providing evidence for lysine-mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di-tyrosine cross links. A high degree of sequence conflicts probably representing single-nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.

Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells.

Manganese superoxide dismutase (Mn-SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn-SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2-DE, multienzyme digestions were carried out and peptides were analyzed by MALDI-TOF/TOF, Qq-TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp-186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2-oxo-histidine and a previously described intermediate (His+14). Complete sequencing of the two Mn-SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn-SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn-SOD activity.

Determinants of glaucoma awareness and knowledge in urban Chennai.

AIM: To assess the awareness and knowledge levels about glaucoma and its determinants in an urban population of Chennai in south India. MATERIALS AND METHODS: Chennai glaucoma study (CGS) was a population based prevalence study to estimate the prevalence of glaucoma in a rural and urban south Indian population. A total of 3850 subjects aged 40 years or above participated in the urban arm of CGS. A systematic random sample of 1926 (50.0%) subjects completed a questionnaire that assesses their awareness and knowledge level of glaucoma. Respondents "having heard of glaucoma" even before they were contacted/recruited for the study were defined as "aware" and respondents having some understanding of the eye disease were defined as "knowledgeable". RESULTS: Overall 13.5% were aware of glaucoma, the age-gender adjusted rate for awareness was 13.3% (95% CI: 11.57 to 15.03). Two clinicians graded knowledge on glaucoma, based on the subject's knowledge of risk factors, definitions and treatment aspects of glaucoma. Overall 8.7% had some knowledge about glaucoma. Among those who had knowledge 0.5% had good knowledge about glaucoma, 4% had fair knowledge and 4.2% had poor knowledge. We observed a very good agreement between the clinicians in grading knowledge (k =0.92). Determinants of glaucoma awareness and knowledge were higher levels of education, females, age, religion and family history of glaucoma. CONCLUSION: Awareness and knowledge about glaucoma was very low among the urban population of Chennai. We have found that younger subjects and men were less aware of glaucoma. Subjects with lower levels of education were less aware and knew less about glaucoma than their counterparts. The study findings stress the need for health education for effective prevention of blindness due to glaucoma.

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742.

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two-dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ-TOF mass spectrometry. Quantification of proteins from four groups (NaCl-treated mice, SGS742-treated mice, SGS742-treated yoked controls, and NaCl-treated yoked controls) was carried out according to an in-gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABAB antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742-treated mice (mean+/-SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19+/-0.41 for synapsin Ia, and 1.41+/-0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl-treated controls and the use of yoked controls that were ruling out swimming- and stress-mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation.

Neurotrophin 3/TrkC-regulated proteins in the human medulloblastoma cell line DAOY.

Medulloblastoma (MB) is the most common malignant childhood brain tumor and high neurotrophin (NP) receptor TrkC mRNA expression was identified as a powerful independent predictor of favorable survival outcome. In order to determine downstream effector proteins of TrkC signaling, the MB cell line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. A proteomic approach was used to search for expressional changes by two mass spectrometric methods and immunoblotting for validation of significant results. Multiple time points for up to 48 h following NP-3-induced TrkC receptor activation were chosen. Thirteen proteins from several pathways (nucleoside diphosphate kinase A, stathmin, valosin-containing protein, annexin A1, dihydropyrimidinase-related protein-3, DJ-1 protein, glutathione S-transferase P, lamin A/C, fascin, cofilin, vimentin, vinculin, and moesin) were differentially expressed and most have been shown to play a role in differentiation, migration, invasion, proliferation, apoptosis, drug resistance, or oncogenesis. Knowledge on effectors of TrkC signaling may represent a first useful step for the identification of marker candidates or reflecting probable pharmacological targets for specific treatment of MB.

Which is the optimal risk stratification system for surgically treated localized primary GIST? Comparison of three contemporary prognostic criteria in 171 tumors and a proposal for a modified Armed Forces Institute of Pathology risk criteria.

BACKGROUND: This study aims to validate and compare the performance of the National Institute of Health (NIH) criteria, Huang modified NIH criteria, and Armed Forces Institute of Pathology (AFIP) risk criteria for gastrointestinal stromal tumors (GISTs) in a large series of localized primary GISTs surgically treated at a single institution to determine the ideal risk stratification system for GIST. METHODS: The clinicopathological features of 171 consecutive patients who underwent surgical resection for GISTs were retrospectively reviewed. Statistical analyses were performed to compare the prognostic value of the three risk criteria by analyzing the discriminatory ability linear trend, homogeneity, monotonicity of gradients, and Akaike information criteria. RESULTS: The median actuarial recurrence-free survival (RFS) for all 171 patients was 70%. On multivariate analyses, size >10 cm, mitotic count >5/50 high-power field, tumor necrosis, and serosal involvement were independent prognostic factors of RFS. All three risk criteria demonstrated a statistically significant difference in the recurrence rate, median actuarial RFS, actuarial 5-year RFS, and tumor-specific death across the different stages. Comparison of the various risk-stratification systems demonstrated that our proposed modified AFIP criteria had the best independent predictive value of RFS when compared with the other systems. CONCLUSION: The NIH, modified NIH, and AFIP criteria are useful in the prognostication of GIST, and the AFIP risk criteria provided the best prognostication among the three systems for primary localized GIST. However, remarkable prognostic heterogeneity exists in the AFIP high-risk category, and with our proposed modification, this system provides the most accurate prognostic information.

Association of VEGF gene polymorphisms with diabetic retinopathy in a south Indian cohort.

BACKGROUND: Polymorphisms in vascular endothelial growth factor (VEGF) gene have been associated with diabetic retinopathy (DR) in various populations. A promoter polymorphism and a 3'UTR variation are studied for association with DR. MATERIALS AND METHODS: Type 2 diabetic patients with and without retinopathy were recruited. The -634C/G and 936C/T polymorphisms were genotyped by direct sequencing and their frequencies were analyzed using relevant statistical tests. RESULTS: No significant association was observed between genotypes, alleles and haplotypes of -634C/G and 936C/T polymorphisms and DR or its severity. However, C(-634)G genotype was found to increase the risk for DR in patients with microalbuminuria (OR: 8.9, 95% CI: 1.4, 58.3). CONCLUSION: Our study broadly suggests lack of association of VEGF gene polymorphisms with DR.

The tele-screening model for diabetic retinopathy: evaluating the influence of mydriasis on the gradability of a single-field 45 degrees digital fundus image.

The purpose of this article was to study the influence of pupillary dilatation on the gradability of a single-field 45 degrees digital fundus images taken in a telescreening model for diabetic retinopathy. Telescreening camps for diabetic retinopathy were organized in rural south India. Sixty-eight patients with type 2 diabetes were enrolled. Single-field 45 degrees digital fundus images were obtained before (group I) and after pupillary dilatation (group II). Digital fundus images were obtained using nonmydriatic fundus camera and transmitted in real time to the base hospital for grading by a retinal specialist. Various factors that could influence the gradability of images were studied, including patients' age and visual acuity, experience of the photographer, and interobserver variability. After pupillary dilatation, the nongradability of digital fundus images was reduced from 29.1% to 8.6%. With each line of improvement in Snellen's Visual acuity, the gradability improved by 12.1%; likewise, with each year of age, the gradability improved by 5.5% following mydriasis. Interobserver variation was excellent (k = 0.88). The learning curve of photographer had no effect on image gradability. Pupillary dilatation improves the gradability of a single-field 45 degrees digital fundus image during telescreening of diabetic retinopathy.

Diabetic retinopathy and IGF-1 gene polymorphic cytosine-adenine repeats in a Southern Indian cohort.

BACKGROUND/AIMS: Growth factors have been implicated in the pathogenesis of diabetic retinopathy (DR). IGF-1 is known to trigger a critical cascade of molecular events that initiate retinal angiogenesis. Increased vitreous IGF-1 levels have been correlated with the severity of ischemia-associated diabetic retinal neovascularization. In the present study, a cytosine-adenine (CA)(n) repeat in the promoter of the IGF-1 gene is studied for association with DR. METHODS: A total of 127 patients with retinopathy (cases: DR+) and 81 patients without retinopathy (controls: DR-) who had type 2 diabetes were recruited for the study. Patients underwent detailed clinical examination and DR was graded based on stereoscopic digital fundus photographs. Frequencies of alleles and genotypes between the two groups were analyzed for significance using relevant statistical tests. (CA)(17) and (CA)(18) repeats were the more frequent alleles. RESULTS: The frequency of the 18-repeat genotype was significantly higher in DR+ patients when compared to DR- patients and found to confer a 2.4 times (95% CI: 1.2-5.0) and 2.8 times (95% CI: 1.1-7.5) higher risk for developing DR and proliferative DR, respectively, when compared to <18-repeat genotypes. CONCLUSIONS: Our study suggests that the 18-repeat genotype is a susceptibility genotype for DR and its clinical severity in a Southern Indian cohort.

Population norms for frequency doubling perimetry with uncorrected refractive error.

PURPOSE: To compare the effect of uncorrected refractive error on threshold estimation using frequency doubling perimetry (FDP) full-threshold N-30 with emmetropia among normal subjects. METHODS: One thousand two hundred ninety-nine subjects were enrolled from the Chennai Glaucoma Study, a population-based glaucoma prevalence study. Subjects underwent a comprehensive eye examination including the FDP full-threshold N-30 test. Normal subjects (with spherical equivalent refractive errors within +/-7 D) with no other ocular pathology were stratified into six groups based on the degree of myopia and hyperopia and compared with age-matched emmetropic controls. A subset of 22 subjects with higher refractive errors was assessed for within-subject effect on FDP parameters. The following FDP parameters were compared: mean deviation, pattern standard deviation, central threshold (CT), mean sensitivity (MS), paracentral points (PA), and peripheral threshold. RESULTS: The one-way analysis of variance between all refractive error groups and emmetropes showed no statistically significant difference for the mean deviation (p = 0.1002) and pattern standard deviation (p = 0.4789). FDP parameters did not show a statistically significant difference for between and within-group comparisons. The variability of FDP sensitivity (derived from the 95% confidence interval range) as a proportion of the threshold range of the instrument was 31, 41, 46, and 41% for CT, MS, PA, and peripheral sensitivity, respectively, without spectacle correction and 29, 34, 36, and 35% for CT, MS, PA, and peripheral sensitivity, respectively, with correction. The mean CT, PA, and peripheral sensitivity show a decreasing trend from central to periphery in all the refractive error groups and a similar trend was noted in the emmetropic controls. CONCLUSION: Between-subject (uncorrected ammetropes and age-matched emmetropes) and within-subject comparisons showed no statistically significant differences in any of the FDP parameters or in the contrast sensitivity estimates between the central and peripheral test locations. This finding is likely due to the high within-subject variability of FDP.

Mass spectrometric studies on mouse hippocampal synapsins Ia, IIa, and IIb and identification of a novel phosphorylation site at serine-546.

Synapsins are key phosphoproteins in the mammalian brain, and structural research on synapsins is still holding center stage. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis (2-DE), and the spots were analyzed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS. Synapsins Ia, IIa, and IIb were unambiguously identified and represented by 15 individual spots on 2-DE. Several serine phosphorylation sites were confirmed, and a novel phosphorylation site was observed at Ser-546 in synapsin IIa in all gels analyzed.

Intron 4 VNTR of endothelial nitric oxide synthase (eNOS) gene and diabetic retinopathy in type 2 patients in southern India.

A 27-bp variable number tandem repeat (VNTR) in intron 4 of endothelial nitric oxide synthase (eNOS) gene has been associated with the risk for developing diabetic retinopathy (DR) in various ethnic populations. Hundred and eighty seven patients with retinopathy (cases; DR+) and 188 patients without retinopathy (controls: DR-) from southern India who had type 2 diabetes mellitus (T2DM) for more than 10 years, were included in the study. We could neither find significant allelic association with clinical severity of DR nor with macular edema. Our results suggest lack of association of intron 4 VNTR of eNOS gene with DR in southern India.

Nitric oxide and oxygen radical attack on GDP-dissociation inhibitor 2 (GDI-2) in spinal cord injury of the rat.

Protein oxidation and nitration have been described during spinal cord injury (SCI) in animal models. Herein, mass spectrometry unambiguously identified GDP-dissociation inhibitor-2 (GDI-2) in SCI with post-translational modifications of 3-aminotyrosine (8 h post-injury) and an acrolein adduct of GDI-2 (72 h post-injury). On the basis of mass spectrometry evidence, we conclude that lipid-peroxidation and protein nitration do take place on an important signalling protein that may be prevented by specific experimental therapeutic interventions.

Patient satisfaction levels during teleophthalmology consultation in rural South India.

The application of new technologies for analysis of retinal images, neural networks for automated retinopathy grading, and teleophthalmology have been shown to have great value in ophthalmology. India has only one ophthalmic surgeon for every 107,000 population. Given this situation, teleophthalmology can play a vital role in addressing to the eye care needs of the country. The objective of this study was to assess patient satisfaction levels and factors influencing it during teleophthalmology consultation in India. A patient satisfaction questionnaire was prepared to assess the prominent aspects of patient satisfaction, including teleophthalmology screening, confidence in the technology used to transfer the fundus images, comprehensiveness of information available to the doctor in the central hub, and graded the opinion regarding the teleophthalmology. A cross-sectional survey among patients who attended a teleophthalmology screening conducted across eight villages in rural Tamilnadu. Responses were then subjected to qualitative analysis and conclusions made. The number of respondents was 348. Of this, 56.4% were males. The mean age of was 50 +/- 17 years. Age ranged from 2 years to 83 years. 44.4% of the respondents were satisfied with teleophthalmology screening. (95% confidence interval [CI]: 38.58%-49.42%) No association was found between age, gender, education, and occupation, respectively, with satisfaction levels. We found that patients who asked questions during the screening were 2.18 times more likely to be satisfied with teleophthalmology than those who did not (odds ratio [OR] = 2.19, 95% CI 1.37-3.5). This study highlights sentiments of the rural subjects when they underwent teleophthalmology consultations. This study provides valuable insights about patient's preferences and satisfaction levels with this newer technology.

Diabetic retinopathy in type II diabetics detected by targeted screening versus newly diagnosed in general practice.

INTRODUCTION: The aim of this study was to compare the occurrence of diabetic retinopathy in targeted screening diabetic patients (Group I) with newly diagnosed diabetic patients in general practice (Group II). MATERIALS AND METHODS: This was an observational cross-sectional study. Data were obtained from 25,313 subjects who participated in the diabetic screening camps, and 128 newly diagnosed diabetes who presented to the diabetic retinopathy screening camps in general practice in rural and urban south India. The study variables were collected from all patients who underwent eye examination from the target screening detected diabetics [(n = 173) Group I] and those newly diagnosed in general practice [(n = 128) Group II]. The variations in prevalence of diabetic retinopathy and sight-threatening diabetic retinopathy in Group I and Group II and the factors affecting it were identified. RESULTS: The occurrence of diabetic retinopathy was 6.35% (95% CI, 2.5-9.5) in Group I and 11.71% (95% CI, 5.6-16.4) in Group II. No significant difference was observed on occurrence of diabetic retinopathy, including sightthreatening retinopathy, in rural versus urban population and in Group I versus Group II. Patients diagnosed in general practice (Group II) with systolic blood pressure (BP) >140 were more likely to have retinopathy (P = 0.02). CONCLUSIONS: Diabetic retinopathy including sightthreatening complications was found at the time of diagnosis of diabetes in the targeted screening group as well as in newly diagnosed diabetics in the general practice group.

Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure.

Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance.